InteractiveFly: GeneBrief

target of Poxn: Biological Overview | Developmental Biology | Evolutionary Homologs | References


Gene name - target of Poxn

Synonyms - biparous

Cytological map position - 74B1--2

Function - transcription factor

Keywords - CNS and PNS

Symbol - tap

FlyBase ID: FBgn0015550

Genetic map position -

Classification - bHLH, Neurogenin/NeuroD homolog

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Yuan, L., Hu, S., Okray, Z., Ren, X., De Geest, N., Claeys, A., Yan, J., Bellefroid, E., Hassan, B.A. and Quan, X.J. (2016). The Drosophila Neurogenin, Tap, functionally interacts with the Wnt-PCP pathway to regulate neuronal extension and guidance. Development [Epub ahead of print]. PubMed ID: 27385016
Summary:
The Neurogenin (Ngn) transcription factors control early neurogenesis and neurite outgrowth in mammalian cortex. In contrast to their proneural activity, their function in neurite growth is poorly understood. Drosophila has a single predicted Ngn homologue called Tap, whose function is completely unknown. This study shows that Tap is not a proneural protein in Drosophila but is required for proper axonal growth and guidance of neurons of the mushroom body (MB), a neuropile required for associative learning and memory. Genetic and expression analyses suggest that Tap inhibits excessive axonal growth by fine regulation of the levels of the Wnt signaling adaptor protein, Dishevelled.
BIOLOGICAL OVERVIEW

The Drosophila bHLH transcription factor target of poxn (tap; also termed biparous) is expressed in a small subset of neurons when they undergo differentiation. Although the sequence of tap resembles that of proneural genes, in fact it is a neural differentiation gene, acting later in the hierarchy of gene activation than the proneural genes (Bush, 1996 and Gautier, 1997). In the CNS tap is expressed in late neural precursors prior to their exit from the the mitotic cycle, but tap is not expressed in neurons (Bush, 1996). In the peripheral nervous system, tap is expressed exclusively in one of the neurons that innervate each larval chemosensory organ, possibly controlling the specific properties of that neuron. Sequence comparisons suggest that tap is most closely related to two bHLH genes, neurogenin and neuroD, identified in several vertebrate species; these genes are involved respectively in neural determination and in neuronal differentiation (Gautier, 1997). tap is also expressed at a late stage in the development of the gustatory (chemosensory) bristles of the leg, wing and proboscis. tap is expressed very early in the development of a second type of chemosensory receptors, the olfactory organs of the antenna (Ledent, 1998).

Numerous vertebrate homologs of Drosophila proneural genes of the achaete-scute complex (AS-C) and of atonal have been cloned, supporting the argument that many aspects of neurogenesis in Drosophila and vertebrates are homologous. Indeed, characteristics consistent with a role in neural precursor determination have been demonstrated for some of these genes, including Xash3 (a Xenopus AS-C homolog); Math1 and Math5 (ato homologs), and neurogenin1 (a tap homolog). Surprising was the fact that expression of many of these homologs are activated later in neural development, after the stage of neural precursor determination (Golding, 2000).

Examples of such genes that play late roles in neurogenesis included asense (ase) an aberrant member of the AS-C, and cato, a gene that bears close relationship to proneural gene atonal. Mutation of ase has no apparent effect in most of the cells in which it is expressed, which may be because the phenotype is too subtle or there is redundancy with other factors. cato is expressed widely in the developing PNS after neural precursor selection but before terminal differentiation. Consistent with this pattern, cato appears to be required for proper sensory neuron morphology. It is clear that cato is not a proneural gene, despite its close sequence relationship with ato. The available evidence suggests that instead, it is associated later in neurogenesis with correct neuronal morphology. cato therefore potentially represents a gene that functions similar to Drosophila tap and vertebrate neuronal differentiation regulators, such as neuroD (Golding, 2000).

From these observations, it has been proposed that cascades or networks of bHLH factors may function during all stages of neural development from commitment of precursors, through to proliferation and migration, and finally to postmitotic terminal differentiation. The implication of this hypothesis is that bHLH proteins control appropriate target genes for each of these stages, including the activation of the next bHLH protein in the cascade (Golding, 2000b and references therein).

tap is a potential target of Paired box neuro (Pox-n). pox-n is expressed in two clusters of cells in each segment, one dorsal and one ventral. The dorsal-most cluster is displaced laterally in the second and third thoracic segments, a pattern typical of the chemosensory organs. The expression of tap follows the same pattern, with two important differences. (1) While pox-n is expressed in the sensory mother cell (SMC) and throughout the lineage until shortly before the progeny undergo differentiation, tap is expressed only at or near the onset of differentiation. Thus tap expression is very transient, lasting probably for less than an hour. (2) While pox-n is expressed in most or all of the progeny of the SMC, tap is expressed in only one cell of each organ (Gautier, 1997).

It has been confirmed that tap depends (directly or indirectly) on pox-n by inducing the ectopic expression of pox-n early during embryogenesis. the overexpression of pox-n has been shown to result in the development of ectopic chemosensory organs, both in the larva and in the adult. Additional cells expressing tap were observed embryos were the ectopic expression of pox-n was induced at 4-6 h after egg laying. Conversely, in embryos homozygous for a deficiency removing pox-n, the expression of tap is completely abolished and largely but not completely so in the CNS. Pox-n binds to polytene chromosomes at 74B, the same location that codes for tap (Gautier, 1997).

tap is a perfect example of a neural precursor gene, silent during the early stages of neurogenesis but activated during the generation of neural sublineages. The transient expression of tap in GMCs also demonstrates that these immediate progeny of the neuroblast have their own program of gene expression, thus pointing to the necessity for more intensive investigations of GMC gene expression. Regulated gene expression in the GMC represents an additional level of complexity in the process of neurogenesis, beyond the dynamic expression of genes in the neuroblast.

To determine the expression pattern of TAP mRNA during embryogenesis, in situ hybridization experiments were performed. TAP mRNA is expressed in a dynamic and complex pattern, starting at stage 10 and lasting beyond stage 15. An important feature of Tap expression at all stages of embryogenesis is its brief duration in select groups of cells. The earliest expression is seen at stage 10 in 5 cells per segment: an unpaired cell on the midline and two pairs lateral to the midline. Based on their large size and position in the neuroblast layer, the midline cell and the pair of cells just lateral to the midline are neuroblasts. Double-labeling of embryos for the expression of the Engrailed protein and TAP mRNA reveals that the position of the cell on the midline coincides with an En stripe, suggesting that this cell is the median neuroblast. Double-labeling of embryos for the expression of the wingless-lacZ gene and TAP mRNA reveals that the more lateral of the paired cells is a row 4 neuroblast, residing one cell anterior to the row 5 neuroblasts which express wingless. Its position and lateral shape suggest that it is neuroblast 4-1. During embryogenesis, both the median neuroblast and neuroblast 4-1 generate solely neuronal progeny. During germ-band extension, there is a large burst of biparous expression in clusters of cells in the ganglion mother cell layer of the developing ventral nerve cord. The cells expressing TAP at this time are distinguishable from neuroblasts based on their location and their smaller size. Although precise quantitation is difficult, it is estimated that 12-18 cells per hemisegment in the ganglion layer express Tap between 6 and 7 hr of development. After this large burst of expression, TAP mRNA is restricted to progressively fewer cells. At embryonic stage 14, TAP mRNA is found in cells peripheral to the longitudinal axon tracts, at the lateral edge of the CNS. By stage 15, 2 large cells per hemisegment maintain tap expression (Bush, 1996).

To determine whether TAP-positive cells are neurons, embryos were double-labeled for the ELAV protein and the TAP transcript. ELAV is an RNA binding protein expressed in all postmitotic neurons. These experiments reveal that TAP mRNA is largely excluded from ELAV-positive cells. This is true even at stage 15 when the vast majority of cells in the CNS have reached the postmitotic state. Further confirmation of the absence of TAP staining from neurons was obtained using anti-HRP antiserum, which recognizes all neurons. To determine whether TAP-expressing cells express the glial-specific gene repo, embryos were double-labeled for the TAP transcript and the REPO protein. Cells express repo after they have committed to the glial cell fate. The large burst of tap expression in the ganglion mother cell layer precedes most REPO expression (stage 11 vs stage 12). Later in neurogenesis when the two genes do overlap, most TAP-positive cells are REPO negative. However, close inspection of these embryos shows that a TAP-positive cell is sometimes directly apposed to a REPO-positive cell. Therefore, the possibility that in some cases expression of the two genes might overlap for a very short time in the same cell cannot be excluded. Since biparous-positive cells express neither elav nor repo, it is possible that they are not yet postmitotic. To determine whether TAP-positive cells are synthesizing DNA, embryos were labeled with bromodeoxyuridine (BrdU). Some TAP-positive cells also incorporate BrdU. This demonstrates that TAP-positive cells are synthesizing DNA, suggesting that they are progenitor cells that give rise to neurons and/or glia (Bush, 1996).

To follow the fate of the TAP-expressing cells, a fusion gene was constructed in which 5.5 kb of the tap promoter drives expression of lacZ. It was reasoned that perdurance of beta-galactosidase would permit observation of cells that had turned on biparous even after disappearance of the transcript. The promoter fragment used in this experiment included 166 N-terminal amino acids of Tap. Staining of these embryos for beta-galactosidase revealed a recapitulation of part of the late expression pattern of endogenous tap. Specifically, beta-galactosidase immunoreactivity was observed in the position of TAP-positive cells at stages 12 through 15, with other phases of biparous expression absent. Similar to endogenous tap, the expression of this transgene is transient. This suggests that the tap promoter/lacZ transgene possesses sufficient cis-regulatory information to direct beta-galactosidase expression to TAP-positive cells at stage 12 through 15 (Bush, 1996).

To confirm that the beta-galactosidase-positive cells recapitulate the expression of tap, embryos were double-labeled for beta-galactosidase and the TAP transcript. If the fusion gene reflects the endogenous TAP pattern, one would predict that some cells would express both TAP transcript and beta-galactosidase protein as was observed. In addition, beta-galactosidase appears to persist beyond the time when the TAP transcript is no longer detectable by in situ hybridization. The beta-galactosidase signal is observed in cells that have migrated both medially and laterally (and this is probably a result of the perdurance of the beta-galactosidase protein. Alternatively, the lacZ fusion gene could be missing cis-elements necessary for turning off tap expression and therefore lacZ is still transcribed after the endogenous gene has been shut off. This possibility is thought to be unlikely since beta-galactosidase expression is only slightly more prolonged than TAP mRNA. Finally, this expression pattern has been observed with at least five independent insertions, demonstrating that the regulatory information contained within the promoter is independent of chromosomal location (Bush, 1996).

It was also observed that the anti-beta-galactosidase staining is nuclear. Since the lacZ gene used in the biparous promoter construct encodes a cytoplasmic protein, this result indicates that the N-terminal portion of Biparous is able to direct nuclear localization. Ten residues N-terminal to the fusion point is a sequence, KRFRR, which conforms well to the canonical nuclear localization sequence, KKRK, suggesting that this sequence may be responsible for nuclear localization. Since the beta-galactosidase signal persisted longer than the TAP transcript, an examination was made to see whether the beta-galactosidase-positive cells express either neuronal or glial markers. When stage 12 embryos were costained with antisera to REPO and beta-galactosidase, some cells were clearly double-labeled. In this region, repo is expressed in the segmental nerve glia, the intersegmental nerve glia, and the exit glia. These three sets of glia are all lateral to the longitudinal connectives, with the segmental and intersegmental glia associated with their respective nerve tracts and the exit glia located at the boundary between the CNS and PNS. Since the double-labeled cells may have not reached their final destination, it is not evident which of these three groups of glia are double-labeled. Nevertheless, since repo is a glial-specific gene, the colocalization provides evidence that the tap gene is transcribed in precursor cells that generate glial progeny. To determine whether beta-galactosidase protein is also expressed in neurons, embryos were double labelled for ELAV and beta-galactosidase. Some cells simultaneously express both proteins. This shows that the tap promoter is also activated in neuronal precursors (Bush, 1996).

Since tap is expressed in glial precursors, it was of interest to determine its relationship to gcm, a gene required for normal glial development. Both biparous and gcm are expressed relatively early during CNS development, with both transcription factors, turned on before many neuronal and glial progenitors have completed their final mitosis. Nevertheless, it is important to note that the large burst of TAP mRNA expression in the ganglion mother cell layer precedes most gcm expression (stage 11 vs stage 12). This sequence of expression is consistent with tap being upstream of gcm in gliogenesis. In support of this hypothesis, the pattern of biparous expression is unchanged in gcm mutant embryos. Since TAP mRNA is expressed relatively early in neurogenesis, it is also possible that it is a downstream target of the proneural genes of the achaete-scute complex. To determine the effect of these genes on biparous expression, in situ hybridization experiments were performed with achaete-scute mutant backgrounds. It was found that removing asense or achaete and scute has no effect on the pattern of TAP mRNA (Bush, 1996).

One theory suggests that bHLH genes determine primordial cell types early in development, and this is followed by the action of other genes that influence the appearance of more specialized cell types This holds true for Drosophila where the bHLH genes of the achaete-scute complex confer on neuroectodermal precursor cells the ability to become neuroblasts. Although tap is activated in neuroblasts, its expression persists until the final stages of embryogenesis, considerably after the disappearance of achaete, scute, and lethal of scute. Thus, tap may present a new function for neuronal bHLH genes, since it is expressed at a different time and in fewer cells than the achaete-scute complex genes. Given its late and more restricted expression pattern, one potential role for tap would be to specify unique characteristics of individual lineages of neurons and glia (Bush, 1996).


DEVELOPMENTAL BIOLOGY

Embryonic

In the lineage that gives rise to the external sense organs of the peripheral nervous system, the first division of the sensory mother cell gives rise to two second-order precursors, one of which will produce the outer cells that form the external structures of the organ (trichogen and tormogen cells) while the other will form the inner cells of the organ (sheath cells and neurons). In the case of the abdominal chemosensory organs, the lineage produces two types of neurons: two bipolar neurons, which extend their dendrite within the external structures of the organ, and one multidendritic neuron (md) whose dendrites extend under the epidermis and are not connected to the external structure of the organ. Outer and inner cells were distinguished by double labelling with anti-Tap and with 22C10 monoclonal antibody, which labels the membranes of all sensory neurons. The cell containing the Tap protein is localized in the clusters of neurons, and is therefore not one of the outer cells (Gautier, 1997).

Analysis was concentrated on the ventral abdominal poly-innervated organ, the papilla p6, because its two bipolar neurons and their sib md neuron form a small cluster of three cells. The multidendritic neuron is ventral to the other two and is called v'pda. The two bipolar neurons that innervate p6 are dorsal-most and have been collectively called v'es2, since there has been no way to distinguish them from one other. The results suggest that the anterior and posterior v'es2 behave differently, however, and they have been termed v'aes2 (anterior) and v'pes2 (posterior). The anti-Tap antibody appears to label v'aes2, the anterior-most of the two v'es2 neurons. The sheath cell is closely apposed to the neurons, however, and the possibility that Tap is present in the sheath cell cannot be excluded. However, anti-Prospero antibody labels the nuclei of the sheath cells, and anti-Pros and anti-Tap antibodies recognize different cells, confirming that tap is expressed in one of the neurons and not in the sheath cell (Gautier, 1997)

Larval and Pupal

Tap is expressed at a late stage in the development of one type of adult chemosensory organ, the gustatory bristles of the leg, wing and proboscis. tap is also expressed very early in the development of a second type of chemosensory receptor, the olfactory organs of the antenna. The results of behavioral experiments suggest that the ectopic expression of tap affects the response to sugar and salt. The sensitivity to sugar is impared and the sensitivity to salt is increased. It must be noted that the resonse to salt is but an inhibition of the response to sugar, so that the two effects may actually reflect the same cause (Ledent, 1998).

In the pupal wing, immunolabeling reveals expression of Tap from 16 to 22 hours after puparium formation (APF), at the time when the expression of Pox-n is subsiding. Either single cells or pairs of cells are labeled; in the latter case, one cell is usually more intensely labelled than the other cell. Tap is first expressed in a single cell, then in a pair, and finally maintained in only one. The sub-epidermal location of these cells suggests that they are neurons. These results suggests that Tap is first expressed in the neural precursor, then transiently in both daughter cells. It is thought that Tap is expressed in leg chemosensory organs around 10-14 h APF, a period when imaginal discs are refractory to immunolabeling. Tap is expressed in a few cells of the everting leg disc, at positions that suggest that they correspond to the neurons innervating the Keilin organs (Ledent, 1998).

In the case of the proboscis, the precursors of chemosensory organs appear in three waves, at respectively 0, 6, and 24 h APF. At 16 h many clusters of Pox-n-expressing cells are observed. Those clusters near the midline comprise several cells, and correspond to the first wave of precursors. Most other clusters comprise 2-3 cells, and correspond to the precursors of the second wave. This pattern is identical to that observed in the enhancer-trap line A37, which labels all cells of the sensory lineages, suggesting that all organs of the proboscis express Pox-n. The presence of Tap protein is observed from 18 h APF onwards, in single cells or pairs of cells corresponding to the organs of the first wave. Thus in the proboscis as well as in the wing, Tap is expressed in a pattern that largely overlaps the pattern of Pox-n expression, at stages that are consistent with the idea that Tap is expressed at the time chemosensory neurons are just about to, or are beginning to differentiate (Ledent, 1998).

Adult

Tap-positive cells are observed in everting antennal discs, at three sites near the base of the third antennal segment. The number of labelled cells at each site increases progressively during the first 6 h APF, from 1-3 to more than 10. This pattern parallels the appearance of precursor cells in this segment as seen in the A101 enhancer-trap line A101, suggesting that Tap is expressed in one subset of olfactory precursors. The position of the labeled cells is consistent with the idea that they may correspond to three large groups of basiconic sensilla found near the junction between the third and the second antennal segment (Ledent, 1998).


EVOLUTIONARY HOMOLOGS

Evolution of neural precursor selection: functional divergence of proneural proteins

How conserved pathways are differentially regulated to produce diverse outcomes is a fundamental question of developmental and evolutionary biology. The conserved process of neural precursor cell (NPC) selection by basic helix-loop-helix (bHLH) proneural transcription factors in the peripheral nervous system (PNS) by atonal related proteins (ARPs) presents an excellent model in which to address this issue. Proneural ARPs belong to two highly related groups: the ATONAL (ATO) group and the NEUROGENIN (NGN) group. A cross-species approach was used to demonstrate that the genetic and molecular mechanisms by which ATO proteins and NGN proteins select NPCs are different. Specifically, ATO group genes efficiently induce neurogenesis in Drosophila but very weakly in Xenopus, while the reverse is true for NGN group proteins. This divergence in proneural activity is encoded by three residues in the basic domain of ATO proteins. In NGN proteins, proneural capacity is encoded by the equivalent three residues in the basic domain and a novel motif in the second Helix (H2) domain. Differential interactions with different types of zinc (Zn)-finger proteins mediate the divergence of ATO and NGN activities: Senseless is required for ATO group activity, whereas MyT1 is required for NGN group function. These data suggest an evolutionary divergence in the mechanisms of NPC selection between protostomes and deuterostomes (Quan, 2004).

ATO and NGN proteins share 47% identity in the bHLH domain including eight out of 12 amino acid residues in the DNA binding basic domain and are expressed in both the Drosophila and vertebrate PNS. However, differences in their usage for early NPC specification in the PNS between vertebrates and invertebrates have been noted. NGN proteins do not act early in NPC specification in invertebrates, whereas ATO proteins do not act early in NPC specification in vertebrates. Does this switch in the use of proneural proteins reflect a mechanistic difference or an inert change of expression pattern of otherwise functionally equivalent genes? To address this issue, a comparative analysis was initiated using Drosophila and Xenopus as model systems. To assay the proneural activity of mouse NGN1 and fly ATO in vertebrates, the mRNA of each was injected into a single blastomere of a two cell-stage Xenopus embryo. Neuronal induction was detected at stage 15 via whole-mount in situ hybridization for N-tubulin, an early marker of neuronal differentiation. Compared with uninjected embryos, injection of Ngn1 mRNA induces a large number of ectopic neuronal precursors on the injected side. By contrast, injection of Ato mRNA does not induce a significant increase in N-tubulin expression. Similarly, the injection of mRNA for Math1, a mouse ortholog of ato does not significantly increase N-tubulin expression at stage 15 but very few scattered N-tubulin positive cells can be seen at stage 19. Similar observations have been made for Xath1 which has been shown to be a much weaker inducer of neuronal precursors than NGN1. These data suggest that the Xenopus ectoderm responds robustly to NGN group proteins, but very weakly to ATO group proteins, to induce neurogenesis (Quan, 2004).

One explanation for the weakness of ATO and MATH1 activity in Xenopus is that NGN proteins are more potent neural inducers than ATO proteins and that, in parallel, stronger induction is needed in the vertebrate neuroectoderm than in the Drosophila neuroectoderm. To test this possibility, ATO proteins and NGN proteins were misexpressed in Drosophila using the UAS/Gal4 system and neural induction was assayed by counting the number of sensory bristles produced. Consistent with the fact that ato and Math1 completely rescue each other's loss of function, the two genes show very similar phenotypes and they were used interchangeably throughout the study. Expression of ngn2 with four different wing disc Gal4 drivers (C5-Gal4, 71B-Gal4, 32B-Gal4 and dpp-Gal4) showed no neural induction. Sixteen out of 23 ngn1 transgenic lines showed no neural induction. The other seven showed very weak induction with the strongest Gal4 driver, dppGal4. Therefore, the combination of dppGal4 and the strongest uas-ngn1 transgenic line were used in the rest of this study to determine the genetic and molecular basis of the difference in activity between ATO proteins and NGN proteins. The dppGal4 driver in Drosophila is used to induce genes of interest along the anteroposterior (AP) axis of the wing disc. Wild-type flies have no external sensory bristles or chordotonal organs (CHOs) on the AP axis of the wing blade. By contrast, a large number of sensory bristles is found along the AP axis of the wing with 100% penetrance when either MATH1 or ATO is expressed using dppGal4. In addition, both ATO and MATH1 induce CHOs. Expression of the strongest NGN1 transgenic line results in very few bristles in only 70% of the flies examined and no detectable CHOs. Quantitative analysis reveals that the number of sensory bristles induced by MATH1 is sixfold more than induced by NGN1. Identical observations were made in the few surviving flies under the same conditions using strong UAS-ATO lines. In the vertebrate PNS, NGN1 and NGN2 are sometimes co-expressed, and activate the expression of NeuroD group proteins. Therefore, it is possible that the weak neural induction of mouse NGN1 is due to the lack of homologs of NeuroD proteins in flies. However, co-expression of NGN1 and NGN2 or NGN1 and MATH3, a NeuroD group protein, failed to enhance the proneural activity of NGN1 in Drosophila (Quan, 2004).

One explanation for the very small number of bristles obtained after strong expression of NGN1 may be that the protein is able to induce NPCs, but most of these NPCs fail to differentiate properly and do not give rise to sensory organs. To test this possibility, NPC formation was examined directly upon expressing NGN1, ATO and MATH1 with dppGal4 in A101-lacZ flies. A101-lacZ is an NPC specific enhancer trap. The normal pattern of NPCs is revealed by anti-ß-GAL staining in third instar larval (L3) wing discs. Misexpression of ATO along the AP axis of the wing disc results in the induction of ectopic NPCs within the domain of ATO expression. By contrast, despite high levels of NGN1 expression, no detectable increase in NPCs is observed upon expression of NGN1. Similarly, ATO, but not NGN1, induced asense expression, another marker of NPC specification (Quan, 2004).

Is the weak activity of NGN1 specific to ectopic expression in the wing disc? Wide expression of NGN1 in embryos using da-Gal4 does not result in ectopic neurons. Finally, attempts were made to rescue the loss of ato in the eye imaginal disc using Gal4-7 and uas-ngn1. Gal4-7 induces expression anterior to the morphogenetic furrow and has been used to restore photoreceptors to ato mutant eye discs using scute and Math1. Expression of NGN1 in ato mutant discs did not result in any rescue nor did it induce ectopic R8 cells when expressed in control discs. For simplicity, the number of bristles was used as a quantitative assay for NPC formation for the remainder of the study (Quan, 2004).

To explore whether the differential activities of NGN proteins and ATO proteins can be understood at the level of the proteins themselves, a comparative analysis of the amino acid sequence of the basic domain was performed. Several studies have shown that important information is encoded by the basic domain, or specific residues therein. In addition, the 12 amino acids in the basic domain are sufficient to phylogenetically delineate ATO proteins and NGN proteins, arguing that sequence differences within the basic domain are of functional significance. However, previous studies did not investigate the genetic basis or address the evolutionary implications of the variation in basic domain sequence. ATO proteins and NGN proteins share eight residues out of 12 in the basic domain. One is variable, and the other three residues show almost absolute group specificity: they are highly conserved within each group but are essentially never the same between the two groups. To investigate whether this sequence specificity can explain the species-specific activities of ATO proteins and NGN proteins, a chimeric protein was created, exchanging the three group-specific amino acids in the basic domain of NGN1 for those present in ATO, named NGNbATO. Expression of NGNbATO induces the appearance of bristles along the AP axis of the wing in all transgenic lines examined. Strong UAS-NGNbATO lines mimic strong UAS-ATO lines and result in significant lethality and more than 60 bristles per wing in the few surviving flies. Moderate UAS-NGNbATO lines behave like moderate UAS-ATO lines and induce an average of 33 bristles per fly along the AP axis when compared with an average of seven for strongest UAS-NGN1 lines. Conversely, a chimeric protein was created exchanging the three group-specific amino acids in the basic domain of ATO to NGN1, named ATObNGN. Whereas the injection of Ato mRNA in Xenopus embryos has no significant effect on the N-tubulin expression pattern, the injection of AtobNGN mRNA induces N-tubulin expression, indistinguishable from that caused by the injection of NGN1. Therefore, the NGNbATO mutant recovers the NPC inducing activity of ATO in Drosophila, and the ATObNGN mutant recovers the NPC inducing activity of NGN1 in Xenopus (Quan, 2004).

It is worthwhile to notice that only some of residues in the basic domain are directly contacting to DNA. The specific activities of ATO and NGN1 are unlikely to depend on differential DNA-binding activity, since ATO proteins and NGN proteins have identical DNA contact amino acids and can activate the NeuroD promoter via the same E-boxes in P19 cells. Interestingly, biophysical and DNA-binding studies comparing MASH1 and MyoD have shown that they display similar binding preferences leading to the conclusion that their different target specificities cannot be explained solely by differential DNA binding. Similar conclusions were made comparing ato and sc activities in neural subtype specification (Quan, 2004).

To investigate whether other functionally specific motifs exist in the bHLH domain of ARP proteins, the evolutionary trace (ET) analysis method was used. ET tracks residues whose mutations are associated with functional changes during evolution. This approach has been used to identify novel functional surfaces, and has recently been shown to be widely applicable to proteins. In practice, ET relies on the phylogenetic tree of a protein family and identifies residues of the alignment that are invariant within branches but variable between them. These positions are called 'class specific'. The smallest number of branches at which a position first becomes class specific defines its rank. The top ranked positions (1) do not vary. Very highly ranked positions (2-8) are such that they vary little and, whenever they do, there is also a major evolutionary divergence. By contrast, poorly ranked positions vary more often, and their variation does not seem to correlate with divergence. Thus, highly ranked positions tend to be functionally important, while poorly ranked ones tend not to be. When examining ARP bHLH domains, ET identified a number of positions that are jointly important in different bHLH domains, yet that undergo significant variation between them. These residues varied in rank from 2 to 7, suggesting that they can undergo non-conservative mutations that are likely to correspond to functional divergence events. These positions tend to be most conserved between NeuroDs and NGN proteins and then undergo variations in ATO proteins, suggesting that they are important for an activity shared by NGN proteins and NeuroDs, but absent in ATO proteins. The data above show that the ability to induce NPCs in vertebrates is precisely such an activity. To investigate further the role of these group-specific residues on functional specificity, a chimeric protein, named NGNH2ATO (exchanging amino acids 37, 39, 43, 44 and 46 in Helix2 of NGN1 with those present in ATO), was created and tested in Drosophila. Expression of the strongest NGNH2ATO transgenic line induces a maximum of two bristles along the AP axis of the wing per fly in 50% of the flies. Quantitative analysis shows that, unlike ATO, NGNH2ATO induces an average of 0.8 bristles along AP axis per fly. These data indicate that the group-specific motif in Helix2 of ATO does not encode proneural activity in Drosophila. Conversely, a chimeric protein, named ATOH2NGN, was generated exchanging the same five amino acids in Helix2 of ATO to those found in NGN1. Injection of ATOH2NGN mRNA causes ectopic N-tubulin expression, indistinguishable from the injection of NGN1. Therefore, ATOH2NGN recovers the activity of NGN1 in Xenopus. Taken together, the mutational analysis results agree with the predictions of the ET analysis indicating that the identified residues in Helix2 mediate the activity of NGN proteins but not of ATO proteins (Quan, 2004).

The data support the hypothesis that ATO proteins and NGN proteins act via different genetic pathways to specify NPCs in different species. What might those pathways be? One simple explanation may be that NGN1 is not able to form heterodimers with fly Daughterless (DA), a required partner protein for NPC specification. In order to test this possibility, co-IP experiments were performed, in which 35S-labeled ATO, MATH1 or NGN1 were co-precipitated with DA-Myc using anti-Myc antibodies. In the presence of DA, mouse MATH1, fly ATO and mouse NGN1 are co-precipitated. Only background levels of NGN1 are detected in the absence of DA. These results suggest that mouse NGN1 can bind physically to fly DA in vitro. To test if DA and NGN1 can interact genetically in vivo, NGN1 was expressed in the absence of one copy of da. The number of sensory bristles produced by NGN1 along the AP axis is greatly decreased in a heterozygous da background. Therefore, mouse NGN1 can physically and genetically interact with fly da in Drosophila in a dose-sensitive manner (Quan, 2004).

Next, the possibility that mouse NGN1 does not respond to the Drosophila Notch signaling pathway was examined. To test this, neural induction by NGN1 was examined in the absence of one copy of Notch (N+/–) or with the co-expression of Notch pathway genes. The proneural activity of NGN1 is enhanced in a N+/– background. Conversely, NGN1 activity is completely inhibited by co-expression of a constitutively active form, Nintra or members of the E(Spl) complex, m8 and mdelta. These data demonstrate that mouse NGN1 can be regulated by the Notch signaling pathway in Drosophila. It should be noted that overexpression of ATO in a N heterozygous background results in almost complete lethality and in extremely deformed wings, owing (in part) to a very large number of bristles in the few surviving flies. Since both ATO and NGN proteins can respond to levels of Notch signaling but only ATO proteins can efficiently specify NPCs, it is possible that ATO proteins and NGN proteins use different mechanisms to interact with the Notch signaling pathway. One possibility is that NGN proteins are more sensitive than ATO proteins to levels of transcriptional inhibitors of proneural activity encoded by the E(spl) genes because NGN1 activity, like that of ATO, can be repressed by ectopic expression of E(spl) proteins. However, in contrast to what is observed with Notch, removing a copy of the E(spl) complex does not alter NGN1 activity, suggesting that NGN1 is not more sensitive to levels of transcriptional inhibitors of proneural activity (Quan, 2004).

NPC formation in Drosophila requires the Zn-finger protein Senseless (SENS). Fly proneural proteins first induce sens expression and then synergize with it in a positive feedback loop. This appears to enhance the ability of proneural genes to downregulate Notch signaling in the presumptive NPC. In vertebrates, Senseless-like proteins appear not to act in NPC formation, although they are expressed in the PNS. To test the possibility that SENS shows group specific interactions with bHLH proteins during NPC selection, the abilities of ATO and NGN1 to induce SENS were examined. SENS expression in wild-type L3 wing discs marks NPC formation. Ectopic SENS induction is detected along the AP axis of wing discs when ATO is misexpressed. However, SENS expression is not induced by NGN1. These data suggest that unlike ATO, NGN1 does not efficiently induce SENS expression. Whether lowering endogenous levels of Notch would allow NGN1 to induce SENS was examined. Expression of NGN1 in Notch heterozygous animals, although significantly increasing the number of induced bristles, fails to induce SENS expression when compared with N+/– controls, arguing that NPCs induced by NGN proteins are specified via a different mechanism not normally used in Drosophila. Although NGN1 does not induce SENS, it is possible that synergy might occur if the requirement for SENS induction is bypassed. Therefore the ability of NGN1 and MATH1 to synergize with SENS in vivo was compared by co-expressing either NGN1 or MATH1 with SENS using a moderate scutellar Gal4 driver (C5-Gal4). Neural induction was examined by counting the ectopic bristles induced on the scutellum. Wild-type flies have four large bristles, or macrochaete, on their scutella. Expression of SENS or MATH1 alone with C5-Gal4 induces a number of ectopic microchaete, or small bristles, on the scutellum. No ectopic sensory bristles were found when NGN1 was expressed alone. Co-expression of NGN1 and SENS has the same effect on the scutellum as the misexpressing SENS alone. Co-expression of MATH1 and SENS, however, causes the appearance of a large number of both micro- and macrochaete. Finally, NGN1 or MATH1 were co-expressed in the absence of one copy of sens. No effect on NGN1 activity in a sens+/– background was observed. By contrast, the average number of sensory bristles produced by MATH1 along the AP axis was reduced by 42% if a single copy of sens was removed suggesting dose-sensitive interactions. Thus, neither by loss nor gain of function criteria does NGN1 appear to interact with SENS, thus explaining its weak proneural activity and inability to efficiently antagonize Notch signaling in Drosophila. Therefore, SENS is a key extrinsic difference in how ATO proteins and NGN proteins regulate NPC selection (Quan, 2004).

In Xenopus, the C2HC-type Zn-finger protein X-MyT1 is expressed in primary neurons and can be induced by NGN proteins. In addition X-MyT1 has been suggested to play a role in NPC formation and to synergize with NGN proteins. In order to test if X-MyT1, like SENS, shows specificity in its interaction with ARP proteins, its ability to interact with NGN1 and ATO in Xenopus was compared. X-MyT1 mRNA was injected alone or co-injected with either Ngn1 or Ato mRNA. As expected, the injection of X-MyT1 increases the number of N-tubulin-expressing cells in the neural plate domains where neurons normally form, while the injection of Ngn1 mRNA alone leads to induction of N-tubulin expression. Co-injection of Ngn1 and X-MyT1 mRNAs results in very strong N-tubulin induction, pointing to a synergistic interaction between the two proteins. By contrast, co-injection of Ato and X-MyT1 mRNAs does not cause a detectable increase in N-tubulin expression compared with the injection of X-MyT1 mRNA alone. Similarly, the few ectopic N-tubulin-expressing cells observed when Math1 mRNA is injected are not increased by co-injection of Math1 and X-MyT1. Thus, X-MyT1 interacts specifically with NGN1 and not with ATO or MATH1. The data above demonstrate that the correct combination of ARP protein and Zn-finger protein is necessary for NPC induction (Quan, 2004).

Does the coding sequence difference mediate the divergence in the genetic interactions of ARPs? To test this, whether the chimeric proteins recover the ability to interact with the respective Zn-finger proteins was examined. Indeed, expression of NGNbATO in Drosophila results in the induction of SENS, and the number of bristles induced by NGNbATO in absence of one copy of sens (sens+/–) is reduced by ~44%. In addition, strong synergy was observed by co-expression of NGNbATO and SENS using the dppGal4 driver. Therefore, NGNbATO is able to induce and interact with SENS in Drosophila. In Xenopus, just like Ngn1, co-injection of AtobNGN and X-MyT1 mRNAs results in synergy and very strong ectopic N-tubulin expression when compared with the injection of X-MyT1 or AtobNGN alone. Similarly, co-injection of ATOH2NGN and X-MyT1 mRNAs results in synergy and very strong induction of N-tubulin expression suggesting that ATOH2NGN and ATObNGN use the same mechanism of action as NGN1 (Quan, 2004).

At the developmental level, the data presented in this study can be explained by two possibilities. The first is that Drosophila and vertebrates use different bHLH proteins with divergent mechanisms for selecting similar cell types: the earliest born neural progenitors. Alternatively, NGN proteins may be involved in selecting neuronal (versus glial) rather than earliest born neural progenitors in vertebrates. This is certainly the case in the mammalian inner ear and it should be determined whether it is a more generally applicable rule, at least in the PNS. These two models for NGN function are not mutually exclusive. It is possible that in different lineages, NGN proteins select first neural, and then neuronal, precursors. This would be compatible with data from both flies and vertebrates showing that Notch signaling, in addition to having anti-neural effects, has also anti-neuronal and pro-glial effects during neural lineage development. Analysis of the fly NGN protein, TAP, may shed some light on this issue. At any rate, a comparative approach should provide a powerful tool for the systematic analysis of the pathways which program neural stem cells (Quan, 2004).

Regardless of the precise developmental step at which ATO proteins and NGN proteins act, it is clear that the genetic and molecular mechanisms by which they act are different, suggesting that the functions of ATO proteins and NGN proteins are regulated by different factors. Furthermore, it is clear that the group-specific amino acids underlie these molecular differences. At this point it is difficult to interpret the precise role of the group specific residues in molecular terms. Nonetheless, three possibilities seem reasonable. The first is that currently unknown proteins bind to these residues. The second is that these residues are sites of differential post-translational modifications which in turn influence the choice of target gene specificity. Finally, it is possible that while these residues do not bind to DNA themselves, they influence the three dimensional structure or the conformational changes which DNA binding residues assume upon contacting DNA. In this scenario, these residues do ultimately influence the choice of the binding site without themselves contacting it. The data illustrate the power of a comparative approach in identifying not only conserved, but also divergent, developmental mechanisms, and suggest a platform for screening for the genes mediating the divergence. It is noteworthy that NGN1, on the one hand, and XATH1 and MATH1, on the other, seem to have retained a type of proneural activity which is largely no longer needed in flies and vertebrates, respectively (Quan, 2004).

Finally, genes common to protostomes and deuterostomes (including atonal, ngn genes, Notch signaling genes, sens and X-MyT1) most probably derive from the last common bilaterian ancestor. This implies that such an ancestor already possessed all the tools to specify a large diversity of neural cell types and lineages, suggesting a structurally, and consequently behaviorally, complex animal (Quan, 2004).

Genomic cis-regulatory networks in the early Ciona intestinalis embryo

Precise spatiotemporal gene expression during animal development is achieved through gene regulatory networks, in which sequence-specific transcription factors (TFs) bind to cis-regulatory elements of target genes. Although numerous cis-regulatory elements have been identified in a variety of systems, their global architecture in the gene networks that regulate animal development is not well understood. This determined the structure of the core networks at the cis-regulatory level in early embryos of the chordate Ciona intestinalis by chromatin immunoprecipitation (ChIP) of 11 TFs. The regulatory systems of the 11 TF genes examined were tightly interconnected with one another. By combining analysis of the ChIP data with the results of previous comprehensive analyses of expression profiles and knockdown of regulatory genes, it was found that most of the previously determined interactions are direct. Focus was placed on cis-regulatory networks responsible for the Ciona mesodermal tissues by examining how the networks specify these tissues at the level of their cis-regulatory architecture. Many interactions were found that had not been predicted by simple gene knockdown experiments, and a significant fraction of TF-DNA interactions were found to make major contributions to the regulatory control of target gene expression (Kubo, 2010).

The developmental fates of blastomeres in the Ciona embryo have been determined by the gastrula stage. A comprehensive study has revealed that 53 TF genes are zygotically expressed and regulate one another in complex networks before gastrulation begins. To dissect the architecture of these networks at the level of protein-DNA interactions, focus was placed on 11 TF genes that play core roles in gene regulatory networks for endomesoderm specification: Brachyury, FoxA-a, FoxD, MyoD, Neurogenin, Otx, Snail, SoxC, Tbx6b, Twist-like1 and ZicL. Because the Ciona genome contains multiple copies of FoxD, Tbx6b and ZicL as gene clusters and their precise copy numbers have not yet been determined, these genes are collectively referred to FoxD, Tbx6b and ZicL in this paper. Likewise, there are two copies of Twist-like1, which are highly similar to each other, and these are collectively referred to as Twist-like1 (Kubo, 2010).

Eleven gene-fusion constructs were prepared that encode GFP-tagged TFs expressed under the control of their own promoters (e.g. a fusion gene that encodes GFP-tagged Brachyury driven by the Brachyury promoter). When these constructs were introduced into eggs, the resultant embryos expressed the fusion genes at the same time and in the same blastomeres as the endogenous genes. Exceptions were the Twist-like1 and the Snail constructs. Twist-like1 is normally expressed in three cell lineages (A7.6, B7.7 and B8.5), but the construct drove Twist-like1-GFP expression only in the B7.7 and B8.5 lines. Snail expression in the notochord lineage is normally very weak. The Snail construct did not recapitulate this expression in the notochord lineage but did drive Snail-GFP expression in the remaining lineages (Kubo, 2010).

Expression of these genes did not affect embryonic morphology at the stage when the embryos were fixed. The fixed embryos were subjected to ChIP using anti-GFP antibodies, and subsequently to microarray analysis. To define significant regions, two programs were used employing totally different algorithms. DNA segments regarded as positive by both programs were defined as significant. To confirm that this approach successfully identified TF binding sites, the sequences of ZicL and Tbx6b binding regions defined with three different false discovery rates (FDRs) were analyzed, as the consensus binding motifs of these two TFs are known. The frequencies of matches to the consensus binding sequences for ZicL and Tbx6b around peaks in 0.1% FDR were generally better than in 0.01% and 1% FDRs. As expected, the frequencies of the consensus binding sequences for ZicL and Tbx6b were markedly higher around peaks in the identified regions, suggesting that the method was able to successfully identify the TF binding regions (Kubo, 2010).

Brachyury and Ci-tropomyosin-like are the only known direct targets of ZicL and Brachyury, respectively. As an independent confirmation, the TF binding sites of these genes was expected. The ZicL ChIP profile showed a sharp peak around two known strong ZicL binding sites. The Brachyury ChIP profile also showed a peak around the known Brachyury binding site in the Ci-tropomyosin-like promoter. These peaks were included in significant regions identified with all the FDRs described above. ChIP-qPCRs were performed for these two known interactions. The ChIP-qPCR results showed excellent agreement with the ChIP-chip results (Kubo, 2010).

Next, the promoters were examined of genes that were identified in previous studies as likely direct targets of one of the 11 TFs on the basis of expression assays and gene knockdown assays. Among 29 interactions that had been found in the gene knockdown assays and for which both the source and target genes are expressed in the same cells, 28, 23 and 19 interactions were indicated to be direct under the FDRs of 1%, 0.1% and 0.01%, respectively. The remainder of the interactions were not regarded as direct. Otx expression in the A-line lineage requires a cis-regulatory module that includes Fox binding sites and is suppressed in FoxA-a morphants. The FoxA-a binding to this cis-regulatory element was counted with FDRs of 1% and 0.1%, but not with the most stringent FDR (0.01%). Similarly, several lines of evidence have suggested that MyoD is directly regulated by ZicL. First, MyoD expression is suppressed in ZicL morphants. Second, MyoD and ZicL are both expressed in presumptive muscle cells and the time windows of their expression overlap. Lastly, there is a putative ZicL binding site near to the peaks found in the MyoD upstream region. This putative binding was observed under the FDRs of 1% and 0.1%, but not under the most stringent FDR of 0.01%. On the basis of the above observations, in the following sections the results obtained at an FDR of 0.1% are generally described (Kubo, 2010).

The frequencies of the consensus sequences for ZicL and Tbx6b binding were markedly higher around peaks in the identified regions. Since the consensus binding motifs of the other nine TFs had not been determined previously, similar analyses was performed with motifs of homologs in other animals. The frequencies of the consensus binding motifs for six of the TFs, but not FoxD, SoxC or Twist-like1, were markedly higher around peaks. Because the position weight matrices (PWMs) for FoxD, SoxC and Twist-like1 gave higher background, no significant changes were seen. However, the number of matches to the motifs was markedly higher around peaks than in flanking regions and the background. These observations suggested that the method was able to successfully identify the TF binding regions (Kubo, 2010).

As has been reported in other animals, it was found that the regions bound by Brachyury, MyoD, Neurogenin, Snail, Tbx6b, Twist-like1 and ZicL, especially around the peaks, showed a marked GC bias. This bias is likely to be related to the consensus sequences, because the consensus sequences for these TFs are generally more GC-rich than those of the remaining TFs. The observed enrichment of recognition sequences was unlikely to be an artifact of GC bias because even if background sequences were picked with a base composition comparable to the averaged GC content of the bound regions (the difference between the average GC content of the bound and background regions was less than 0.8%), matches to the PWMs were enriched around peaks versus each of the GC-adjusted backgrounds (Kubo, 2010).

Next, attempts were made to discover overrepresented motifs in the regions (360 bp) around the peaks identified by each ChIP experiment using the Trawler program. It was found that overrepresented motifs were similar to the PWMs that were determined experimentally (Tbx6b and ZicL) or to those of homologs in other animals (the remaining nine TFs). This further supported the conclusion that the results of the ChIP experiments were of high quality (Kubo, 2010).

It is generally believed that TFs tend to bind near promoters, although many examples are known in which TFs bind to enhancers far from promoters. The distributions of peaks in all experiments, except Snail ChIP, were higher around transcription start sites. The reason why Snail binding sites were not enriched around transcription start sites is unclear, but this does not necessarily indicate that the results of the Snail ChIP were of low quality. Altogether, these observations support the conclusion that all of the ChIP experiments revealed in vivo occupancies of the TFs (Kubo, 2010).

TF genes were significantly enriched among the target genes of the 11 TFs. Among 670 potential TF genes in the Ciona genome, at least 607 encode proteins with known TF motifs or proteins with two or more zinc-finger motifs that potentially bind to DNA. A significantly greater number of TF genes were found among the targets than would be expected from random sampling. This enrichment indicates that the TFs examined bind targets selectively and not randomly (Kubo, 2010).

The ChIP data was compared with the results of the comprehensive gene knockdown experiments of a previously study. Among 76 interactions previously found in the early embryo, the ChIP assays indicated that 58 are direct. In addition, 251 novel interconnections were found. Among 121 (11×11) possible interconnections, 84 were observed in the present study. The data indicate that these genes are tightly interconnected with one another (Kubo, 2010).

Because the gene regulatory network model previously constructed from comprehensive expression profiles and comprehensive knockdowns of regulatory genes is of single-cell resolution, the ChIP data was interpred into this network by assuming that the examined TFs bind to the targets wherever their mRNAs are expressed. The reconstructed networks had a complex architecture (Kubo, 2010).

The reconstructed regulatory networks allow tracing of development at the single-cell level. Figs S8 and S9 in the supplementary material show the interconnections among the core 11 TFs in A-line and B-line blastomeres, which give rise to endomesodermal tissues, from the 8-cell to the early gastrula stage. Two of the three mesenchymal lineages (B-line mesenchymal cells) and 28 out of 36 muscle cells (B-line muscle cells) in the tadpole larvae are derived from B4.1 blastomeres at the 8-cell stage. Thirty-two and eight notochord cells are derived from A4.1 and B4.1 blastomeres, respectively. Previous studies demonstrated that Twist-like1, MyoD and Brachyury are essential for specification of the mesenchyme, muscle and notochord, respectively (Kubo, 2010).

Twist-like1 is expressed exclusively in the mesenchymal lineage and is regulated by FoxA-a, Otx and ZicL, as indicated by the fact that knockdown of any of these three genes results in loss or reduction of Twist-like1 expression. No direct binding was detected of FoxA-a to the Twist-like1 promoter, but it was found that FoxA-a binds to the upstream regions of Otx and ZicL, and that ZicL and Otx bind to the promoter of Twist-like1. Therefore, it is highly likely that FoxA-a mainly activates Twist-like1 indirectly through activating Otx and ZicL (Kubo, 2010).

Twist-like1 expression begins in B7.7 (the posterior B-line mesenchyme) at the 64-cell stage and in B8.5 (the anterior B-line mesenchyme) at the early gastrula stage. These two mesenchymal lines contribute to distinct adult tissues after metamorphosis. ZicL might be associated with the differences between these two lineages because the contribution of ZicL to Twist-like1 activation is weaker than that of Otx. To confirm this idea, a mutant Twist-like1 promoter was tested, from which a 150 bp segment containing the identified ZicL binding region was deleted. Because the Otx ChIP result indicated that the Otx binding region is distinct from the ZicL binding region, Otx was expected to bind to this mutant promoter. When introduced into fertilized eggs by electroporation, the wild-type promoter (1550 bp) drove reporter expression in 65% of the embryos, whereas the mutant promoter drove reporter expression in 36% of the embryos. In addition to the significant decrease in the number of embryos expressing the reporter, the overall fluorescence was weaker and the posterior B-line mesenchyme did not appear to express the reporter in the mutant construct. To confirm this observation, the experimental embryos were cleavage-arrested at the 110-cell stage. Cells in the arrested embryos cannot divide further, but the developmental programs proceed as in normal embryos. The mutant construct failed to drive reporter expression in the posterior B-line mesenchyme. These results suggest that ZicL contributes to the difference between these two lineages (Kubo, 2010).

A previous study showed that nine mesenchyme-specific non-regulatory genes are under the control of Twist-like1. None of these genes was identified as a direct target in the present study. Even when applied with an FDR of 1%, only one gene was identified as a direct target. Therefore, it is likely that Twist-like1 regulates the expression of mesenchyme-specific genes through its downstream regulatory gene circuit, although there is a possibility that Twist-like1 binds to the regulatory elements of these genes at later stages (Kubo, 2010).

The B6.2 and B6.4 cell pairs in the 32-cell embryo have the potential to give rise to mesenchyme and muscle. At the 64-cell stage, these cells divide, and one of the daughter cells becomes specified to give rise to the muscle cells. Previous functional assays showed that ZicL, Tbx6b and MyoD are essential for specification of muscle cells. Tbx6b begins to be expressed at the 16-cell stage, and cells expressing Tbx6b give rise not only to muscle cells but also to mesenchyme cells. Tbx6b expression declines to undetectable levels before the tailbud stage. ZicL starts to be expressed at the 32-cell stage in a variety of cells, including those with developmental fates of muscle, mesenchyme, notochord and neurons. ZicL expression in the muscle lineage disappears before the late gastrula stage. MyoD expression begins at the 44-cell stage exclusively in the muscle lineage under the control of Tbx6b and ZicL. The present study showed that ZicL, Tbx6b and MyoD constituted a tightly interconnected gene circuit that is responsible for this specification: (1) ZicL bound to the promoters of MyoD and Tbx6b; (2) Tbx6b bound to the promoters of MyoD and ZicL; and (3) MyoD bound to the promoter of Tbx6b and to its own promoter. All of these interactions, except MyoD binding to the Tbx6b promoter, have been confirmed by functional assays (Kubo, 2010).

To understand how this gene circuit regulates downstream muscle-specific genes, the promoters were examined of 13 muscle structural genes that are well annotated and known to be expressed in the larval tail muscle. Of these, ten were directly bound by MyoD and Tbx6, one by MyoD and ZicL, one by Tbx6b and ZicL, and one by MyoD alone (Kubo, 2010).

Both MyoD and Tbx6 bound to the promoters of more than three-quarters of the muscle genes examined. To test the action of this feed-forward loop comprising MyoD and Tbx6b in the regulation of muscle-specific gene expression, the expression patterns of genes under the control of this circuit were examined. Of the 155 genes under the direct control of MyoD and Tbx6b, 50 (including the above ten) were already known to be expressed in muscle cells. From the remaining 105 genes, 20 were randomly chosen, and 15 were found to be expressed in muscle cells, suggesting that this circuit is widely used for the regulation of genes expressed in muscle cells, and also that this circuit might not necessarily be sufficient for driving expression of the target (Kubo, 2010).

Brachyury is activated at the 64-cell stage exclusively in the notochord lineage, and this expression specifies the notochord fate. ZicL directly binds to the Brachyury promoter and activates its expression. It has also been shown that FoxD and FoxA-a are required for Brachyury expression, probably through regulating ZicL expression, and that FGF signaling is also required for Brachyury expression. The present assays showed that not only ZicL, but also FoxD binds to the Brachyury promoter. Although FoxD mRNA is not present in the notochord lineage at the 32-cell and 64-cell stages, when ZicL and Brachyury are activated, respectively (FoxD is expressed in the ancestors of cells in which ZicL and Brachyury are expressed), the ChIP assay indicated that FoxD binds to the promoters of ZicL and Brachyury. Because knockdown of FoxD eliminates ZicL and Brachyury expression and because the FoxD-GFP fusion protein exists in the notochord lineage at the 32-cell stage, it is likely that FoxD protein exists in these cells and binds to the promoters of ZicL and Brachyury when these two genes begin to be expressed (Kubo, 2010).

FoxA-a binding to the Brachyury promoter was not identified under 0.1% FDR. There was, however, a small peak that was counted as significant under 1% FDR. The possibility could not be ruled out that FoxA-a binds weakly to the Brachyury promoter. It is also possible that FoxA-a could bind weakly to a FoxD binding site because the FoxA-a binding peak coincided with that of FoxD. Even if this weak binding occurs in vivo, the regulation of Brachyury by FoxA-a would largely be achieved indirectly through FoxD and ZicL, since strong binding was found of FoxA-a to the promoters of FoxD and ZicL (Kubo, 2010).

Next, 14 non-regulatory genes were examined that are known to be expressed in the notochord under the control of Brachyury. Among them, 11 were identified here as direct targets of Brachyury. The present results suggest that the remaining three genes are regulated indirectly through a gene circuit under the control of Brachyury, although it cannot be ruled out that Brachyury binds to the regulatory elements of these three genes at later stages (Kubo, 2010).

The present study found many interactions between TFs and genomic DNA that were unexpected from preceding gene knockdown assays. Similar observations were also reported in preceding ChIP studies. To estimate what proportion of the binding makes a major contribution to gene regulation in Ciona embryos, MyoD mRNA or an MO against MyoD was injected into eggs, and their effects were analyzed on the expression of the same targets that were analyzed at the gastrula stage or at the tailbud stage, respectively. The mRNA levels of 14 targets, ten of which were expressed in muscle, were significantly increased (>2-fold) in embryos injected with MyoD mRNA, and MyoD MO injection significantly reduced the mRNA levels of three of these targets. The mRNA level of one target (KH.C12.38), which was weakly expressed in muscle at the tailbud stage, was significantly decreased in embryos injected with MyoD mRNA, whereas the mRNA level of one target (KH.C9.27), which was expressed in muscle at the gastrula stage, was significantly increased in embryos injected with the MyoD MO. In total, the mRNA levels of 16 targets were significantly altered by MyoD mRNA overexpression or gene suppression. The remaining four were not significantly affected, although three of these were expressed in muscle, implying that MyoD binding makes a relatively small contribution to activating these target genes. It was also found that eight of 15 Brachyury targets and seven of 12 Twist-like1 targets were significantly affected in the embryos by overexpression or knockdown of Brachyury or Twist-like1, respectively. Therefore, it is estimated that more than half of TF binding makes a major contribution to the regulatory control of gene expression (Kubo, 2010).

Neurogenin/NeuroD homologs in Fish

Zebrafish neurogenin1 encodes a basic helix-loop-helix protein that shares structural and functional characteristics with proneural genes in Drosophila melanogaster. neurogenin1 is expressed in the early neural plate in domains comprising more cells than the primary neurons known to develop from these regions; its expression is modulated by Delta/Notch signaling, suggesting that it is a target of lateral inhibition. Misexpression of neurogenin1 in the embryo results in development of ectopic neurons. Markers for different neuronal subtypes are not ectopically expressed in the same patterns in neurogenin1-injected embryos suggesting that the final identity of the ectopically induced neurons is modulated by local cues. Induction of ectopic motor neurons by neurogenin1 requires coexpression of a dominant negative regulatory subunit of protein kinase A, an intracellular transducer of Hedgehog signals (see Drosophila PKA). Inhibition of ngn1 expression in the lateral plate in embryos injected with constitutively active PKA suggests that PKA may act as a dominant repressor of ngn1 expression. The pattern of endogenous neurogenin1 expression in the neural plate is expanded in response to elevated levels of Hedgehog (Hh) signaling or abolished as a result of inhibition of Hh signaling. Other factors induced by Hedgehogs must be required in addition to Neurogenin1 for development of motor neurons. It is possible that ngn1 expression in the lateral neural plate is controlled by BMP4/7 and that the interplay of the two signaling centers causes the striped pattern of Ngn1 in the posterior neural plate. Together these data suggest that Hh signals regulate neurogenin1 expression and subsequently modulate the type of neurons produced by Neurogenin1 activity (Blader, 1997).

In zebrafish, cells at the lateral edge of the neural plate become Rohon-Beard primary sensory neurons or neural crest. Delta/Notch signaling is required for neural crest formation. ngn1 is expressed in primary neurons; inhibiting Ngn1 activity prevents Rohon-Beard (RB) cell formation but not formation of other primary neurons. Reducing Ngn1 activity in embryos lacking Delta/Notch signaling restores neural crest formation, indicating Delta/Notch signaling inhibits neurogenesis without actively promoting neural crest. Ngn1 activity is also required for later development of dorsal root ganglion (DRG) sensory neurons; however, RB neurons and DRG neurons are not necessarily derived from the same precursor cell. It is proposed that temporally distinct episodes of Ngn1 activity in the same precursor population specify these two different types of sensory neurons (Cornell, 2002).

Based on the activity of bHLH proneural genes in Drosophila, it seemed possible that RBs and DRG neurons could be two derivatives of a single Ngn1-dependent precursor in the lateral neural plate. In Drosophila, neuronal precursor cells transiently express PNGs and most will subsequently generate many neurons in a specific lineal order. Since RBs and DRG neurons both derive from precursors in the lateral neural plate, they might be derived from a single Ngn1-dependent precursor. However, ngn1 is expressed in the position of DRG neurons, while late derivatives of neuronal precursors in flies do not express PNGs. More significantly, dye-labeling experiment reveals that although RBs and DRG neurons can be lineally related, there is no obligate lineage relationship between these cells, which would necessarily be the case if they both derived from a single precursor. Together, these results provide evidence for two episodes of Ngn1 activity: one that directs some lateral neural plate precursors to become RBs, and a second, presumably later one that directs some neural crest cells, which are also derived from neural plate precursors, to become DRG neurons. This second episode may begin shortly after the first (Cornell, 2002).

In the postembryonic zebrafish forebrain, subpial locations of neurogenesis do exist in the early cerebellar external granular layer, and -- unusually among vertebrates -- in the primordial pretectal (M1) and preglomerular (M2) anlagen as shown with BrdU/Hu-immunocytochemistry and in situ hybridization of neuroD. Hu, related to Drosophila ELAV, is a neuronal protein expressed in proliferating neurogenic cells. An intermediate BrdU incubation time of 12-16 h reveals, in addition to proliferative ventricularly located cells, those in M1 and M2. This BrdU saturation-labeling shows, in conjunction with a Hu-assay demonstrating earliest neuronal differentiation, that proliferating cells in M1 and M2 represent neuronal progenitors. This is demonstrated by single BrdU-labeled and double BrdU-/Hu-labeled cells in these aggregates. Further, expression of NeuroD, a marker for freshly determined neuronal cells, confirms this unusual subpial postembryonic forebrain neurogenesis (Mueller, 2002).

Cells delaminate from epithelial placodes to form sensory ganglia in the vertebrate head. The formation is described of cranial neurogenic placodes in the zebrafish, Danio rerio, using bHLH transcription factors as molecular markers. A single neurogenin gene, neurogenin1 (ngn1), is required for the development of all zebrafish cranial ganglia, which contrasts with other described vertebrates. Expression of ngn1 delineates zebrafish ganglionic placodes, including trigeminal, lateral line, and epibranchial placodes. In addition, ngn1 is expressed in a subset of cells within the otic vesicle that will delaminate to form the octaval (statoacoustic) ganglion. The trigeminal placode is the first to differentiate, and forms just lateral and adjacent to the neural crest. Expression of ngn1 is transient and prefigures expression of a related bHLH transcription factor, neuroD. Interfering with ngn1 function using a specific antisense morpholino oligonucleotide blocks differentiation of all cranial ganglia but not associated glial cells. Lateral line sensory neuromasts, structures that contain mechanosensory hair cells that detect water flow, develop independently of ngn1 function, suggesting that two derivatives of lateral line placodes, ganglia and migrating primordia, are under separate genetic control (Andermann, 2002).

The homeodomain transcription factor Floating head (Flh) is required for the generation of neurons in the zebrafish epiphysis (pineal gland). Flh regulates expression of two basic helix loop helix (bHLH) transcription factor encoding genes: ash1a (achaete/scute homolog 1a) and neurogenin1 (ngn1), in epiphysial neural progenitors. ash1a and ngn1 function in parallel redundant pathways to regulate neurogenesis downstream of flh. Comparison of the epiphysial phenotypes of flh mutant and of ash1a/ngn1 double morphants reveals that reduced expression of ash1a and ngn1 can account for most of the neurogenesis defects in the flh-mutant epiphysis but also shows that Flh has additional activities. Furthermore, different cell populations show different requirements for ash1a and ngn1 within the epiphysis. These populations do not simply correspond to the two described epiphysial cell types: photoreceptors and projection neurons. These results suggest that the genetic pathways that involve ash1a and ngn1 are common to both neuronal types (Cau, 2003).

A homeobox gene, pnx, a homolog of Drosophila Slouch/S59/NK-1, is expressed in prospective posterior neurogenic regions and later in primary neurons. pnx expression is regulated by a signal from the non-axial mesendoderm and by Notch signaling. Pnx contains an Eh1 repressor domain, which interacts with Groucho and acts as a transcriptional repressor. Misexpression of pnx increases neural precursor cells and postmitotic neurons, which express neurogenin1 and elavl3/HuC, respectively. Expression of an antimorphic Pnx (VP16Pnx) or inhibition of Pnx by antisense morpholino oligonucleotide lead to the reduction in the number of a subset of primary neurons. Misexpression of pnx promotes neurogenesis independent of Notch signaling. Epistatic analyses shows that Pnx also functions downstream of the Notch signal. These data indicate that pnx is a novel repressor-type homeobox gene that regulates posterior neurogenesis (Bae, 2003).

pnx functions in the lateral inhibition mechanism. Gain and loss of Pnx function shows that pnx regulates the expression of the proneural gene ngn1. Misexpression of ngn1, which elicits an increased or ectopic expression of deltaA, deltaD and her4, leads to a reduction in pnx-expressing cells. pnx-expressing cells increase in number within the neurogenic regions in the mib mutant embryos and DN-Delta-expressing embryos at the segmentation (neurula) stages, indicating that pnx expression is negatively regulated by the Notch signal. These data support the idea that Pnx activates proneural gene (such as ngn1)-dependent lateral inhibition machinery that suppresses the expression of pnx in non-neuronal cells and restricts the numbers of neurons (Bae, 2003).

Hedgehog signal transduction is directly required in zebrafish neural crest-derived dorsal root ganglia (DRG) precursors for proper development of DRG neurons. Zebrafish mutations in the Hh signaling pathway result in the absence of DRG neurons and the loss of expression of neurogenin1 (ngn1), a gene required for determination of DRG precursors. Cell transplantation experiments demonstrate that Hh acts directly on DRG neuron precursors. Blocking Hh pathway activation at later stages of embryogenesis with the steroidal alkaloid, cyclopamine, further reveals that the requirement for a Hh signal response in DRG precursors correlates with the onset of ngn1 expression. These results suggest that Hh signaling may normally promote DRG development by regulating expression of ngn1 in DRG precursor (Ungos, 2003).

Neural crest cells that ultimately populate the DRG migrate ventrally on the medial pathway along with sympathetic and pigment cell precursors. However, at a point adjacent to the notochord, sensory precursors stop and return dorsally to the position of the DRG where they begin to express ngn1. The timing of onset of ngn1 expression suggests that Hh signals emanating from the notochord and/or neural tube may be involved in initiation of ngn1 expression in DRG precursors. Ngns are known to be sufficient for conferring neuronal identity on uncommitted precursors. Furthermore, Ngns are thought to reinforce the neuronal program by inhibiting genes necessary for gliogenesis. This difference in migration behavior between sensory precursors and autonomic and pigment cell precursors further suggests that DRG precursors are already predisposed to respond to Hh signals early in their migration. Rather than biasing neural crest cells toward a sensory fate, Hh signaling may be influencing DRG precursors to adopt a neuronal cell fate by promoting ngn1 expression (Ungos, 2003).

Neurogenesis in both vertebrates and invertebrates is tightly controlled in time and space involving both positive and negative regulators. The bHLH factor Her5 acts as a prepattern gene to prevent neurogenesis in the anlage of the midbrain/hindbrain boundary in the zebrafish neural plate. This involves selective suppression of both neurogenin1 (ngn1) and coe2 mRNA expression in a process that is independent of Notch signalling, and where inhibition of either ngn1 or coe2 expression is sufficient to prevent neuronal differentiation across the midbrain-hindbrain boundary. A ngn1 transgene faithfully responds to Her5 and deletion analysis of the transgene identifies an E-box in a ngn1 upstream enhancer to be required for repression by Her5. Together these data demonstrate a role for Her5 as a prepattern factor in the spatial definition of proneural domains in the zebrafish neural plate, in a manner similar to its Drosophila homolog Hairy (Geling, 2004).

Both Ngn1 and Coe2 functions are necessary for the progression of neurogenesis and for the early events of neuronal differentiation in the midbrain-hindbrain domain. Blocking Coe2 activity downregulates ngn1 expression throughout the neural plate, suggesting a requirement for Coe2 in all primary neurons. The absence of ngn1 function prevents deltaB expression in the anterior proneural clusters, including the presumptive motorneurons of rhombomeres 2 and 4, and the first anterior neuronal cluster (ventrocaudal cluster, vcc), and is also necessary for neuronal differentiation of vcc derivatives, which comprise at least the first differentiating populations of the reticulospinal nMLF neurons. This, together with previous reports, indicates a strict requirement for Ngn1 in spinal sensory neurons and the MH area of the embryonic zebrafish CNS. By contrast, Ngn1 is not essential for motorneuron and interneuron development in the trunk and spinal cord, and for epiphysial neurons. Differential requirements for Ngn in CNS neuronal differentiation were also observed in other vertebrates, a typical example being the complementary requirements for Ngn2 and Mash1 in the mouse embryonic neural tube. Other bHLH factors, such as Achaete-scute or Olig, may play redundant or prominent roles in neurogenic areas that differentiate normally in ngn1-deficient embryos (Geling, 2004 and references therein).

These results point to synergistic roles of Ngn1 and Coe2 in MH neurogenesis, possibly reflecting the positive cross-regulation of their expression, and a parallel activity of these factors rather than their action in a linear cascade. It is possible that the crossregulation of ngn1 and coe2 expression helps stabilize the committed state of neuronal progenitors, as described for Xenopus Xcoe2. Together, these results led to a model for the spatial control of MH neurogenesis. In this process, ngn1 and coe2 expression are crucial elements that permit neurogenesis throughout the MH, which is initially identified as a single territory competent to form neurons. At the MHB, ngn1 and coe2 expression are the targets of Her5 inhibition. This inhibition prevents the specification of a proneural cluster in this location and permits the generation of the 'intervening zone' (Geling, 2004).

Transcription factors involved in retinogenesis are co-opted by the circadian clock following photoreceptor differentiation

The circadian clock is known to regulate a wide range of physiological and cellular processes, yet remarkably little is known about its role during embryo development. Zebrafish offer a unique opportunity to explore this issue, not only because a great deal is known about key developmental events in this species, but also because the clock starts on the very first day of development. This study identified numerous rhythmic genes in zebrafish larvae, including the key transcriptional regulators neurod and cdx1b, which are involved in neuronal and intestinal differentiation, respectively. Rhythmic expression of neurod and several additional transcription factors was only observed in the developing retina. Surprisingly, these rhythms in expression commenced at a stage of development after these transcription factors are known to have played their essential role in photoreceptor differentiation. Furthermore, this circadian regulation was maintained in adult retina. Thus, once mature photoreceptors are formed, multiple retinal transcription factors fall under circadian clock control, at which point they appear to play a new and important role in regulating rhythmic elements in the phototransduction pathway (Laranjeiro, 2014).

Neurogenin/NeuroD homologs in frogs

The Xenopus Achaete-Scute homolog, XASH-3, can induce neural plate expansion or ectopic neurogenesis within the neural plate. However XASH-3 is incapable of converting epidermal cells to neurons. Moreover, XASH-3 is expressed in a very restricted subset of the neural plate. It is therefore doubtful whether XASH-3 controls early stages of neurogenesis carried out by the Drosophila proneural Achaete-Scute proteins. Xenopus NeuroD can exert a neuronal determination function when ectopically expressed, but the timing of its expression in vivo suggests it is more likely to function in differentiation (Ma, 1996 and references).

Neurogenin appears to be a better candidate as a vertebrate neuronal determination gene. Neurogenin shows 67% identity to NeuroD and is closely related to other mammalian bHLH proteins including MATH2/Nex-1 and MATH1. MATH proteins are related to the Drosophila protein Atonal, while Neurogenin is distantly related to Atonal and more closely related to Drosophila tap. Neurogenin has been cloned from both the mouse and Xenopus. Mouse neurogenin and NeuroD are sequentially expressed in overlapping regions. In the ventral spinal cord, for example, ngn mRNA is expressed throughout the ventricular zone, in regions where uncommitted progenitors are located, while NeuroD transcripts are expressed at the lateral border of the ventricular zone that contains migrating neuroblasts (Ma, 1996).

X-MyT1 is a C2HC-type zinc finger protein that is involved in the primary selection of neuronal precursor cells in Xenopus. Expression of this gene is positively regulated by the bHLH protein X-NGNR-1 and negatively regulated by the Notch/Delta signal transduction pathway. X-MyT1 is able to promote ectopic neuronal differentiation and to confer insensitivity to lateral inhibition, but only in cooperation with bHLH transcription factors. Inhibition of X-MyT1 function inhibits normal neurogenesis as well as ectopic neurogenesis caused by overexpression of X-NGNR-1. On the basis of these findings, it is suggested that X-MyT1 is a novel, essential element in the cascade of events that allows cells to escape lateral inhibition and to enter the pathway that leads to terminal neuronal differentiation (Bellefroid, 1996).

Expression of the Xenopus Neurogenin protein, Xenopus Neurogenin-related-1 (X-NGNR-1) and XNeuroD show a similar spatial overlap with temporal displacement. X-NGNR-1 induces ectopic neurogenesis and ectopic expression of X NeuroD mRNA, but not vice-versa. X-ngnr-1 expression precedes expression of X-Delta-1, and X-NGNR-1 can serve to activate expression of X-Delta-1. Expression of the intracellular domain of XNotch-1 inhibits both the expression and function of X-ngnr-1. Thus endogenous X-ngnr-1 expression becomes restricted to subsets of cells by lateral inhibition mediated by X-Delta-l and X-Notch. The properties of X-NGNR-1 are thus analogous to those of the Drosophila proneural genes, suggesting that it functions as a vertebrate neuronal determination factor (Ma, 1996).

Xath5, a Xenopus bHLH gene related to Drosophila atonal, is expressed in the developing Xenopus retina. Targeted expression of Xath5 in retinal progenitor cells biases the differentiation of these cells toward a ganglion cell fate, suggesting that Xath5 can regulate the differentiation of retinal neurons. The relationships between the three bHLH genes Xash3, NeuroD, and Xath5 were examined during retinal neurogenesis; it was found that Xash3 is expressed in early retinoblasts, followed by coexpression of Xath5 and NeuroD in differentiating cells. Evidence is provided that the expression of Xash3, NeuroD, and Xath5 is coupled; it is proposed that these bHLH genes regulate successive stages of neuronal differentiation in the developing retina (Kanekar, 1997).

XATH-1, a basic/helix-loop-helix transcription factor and a homolog of Drosophila atonal and mammalian MATH-1, is expressed specifically in the dorsal hindbrain during Xenopus neural development. In order to investigate the role of XATH-1 in the neuronal differentiation process, the effects of XATH-1 overexpression have been examined during Xenopus development. XATH-1 induces the expression of neuronal differentiation markers, such as N-tubulin, within the neural plate as well as within nonneural ectodermal progenitor populations, resulting in the appearance of process-bearing neurons within the epidermis. The related basic/helix-loop-helix genes neurogenin-related-1 and neuroD are not induced in response to XATH-1 overexpression within the embryo, suggesting that XATH-1 may activate an alternate pathway of neuronal differentiation. In further contrast to neurogenin-related-1 and neuroD, high-level expression of general neural markers expressed earlier in development, such as N-CAM, is not induced by XATH-1 overexpression. Competent ectodermal progenitors therefore respond to ectopic XATH-1 expression by initiating a distinct program of neuronal differentiation (Kim, 1997).

The neurogenins (NGNs) are neural-specific basic helix-loop-helix (bHLH) transcription factors. Mouse embryos lacking ngn1 fail to generate the proximal subset of cranial sensory neurons. ngn1 is required for the activation of a cascade of downstream bHLH factors, including NeuroD, MATH3, and NSCL1. ngn1 is expressed by placodal ectodermal cells and acts prior to neuroblast delamination. Proximal cranial sensory ganglia, including the trigeminal, jugular and superior ganglia are deleted in ngn1-/- embryos. NGN1 positively regulates the Delta homolog DLL1 and can be negatively regulated by Notch signaling. Thus, ngn1 functions similarly to the proneural genes in Drosophila. However, the initial pattern of ngn1 expression appears to be Notch independent. Expression of ngn1 is first detected in the trigeminal placode at E8.25. NeuroD mRNA is subsequently detected at E8.75, and this is followed by expression of the bHLH protein NSCL1. Taken together with the fact that ectopic ngn1 expression can convert ectodermal cells to neurons in Xenopus, these data identify ngns as vertebrate neuronal determination genes, analogous to myoD and myf5 in myogenesis (Ma, 1998).

Primary neurogenesis in Xenopus is a model for studying the control of neural cell fate decisions. The specification of primary neurons appears to be driven by transcription factors containing a basic region and a helix-loop-helix (HLH) motif: expression of Xenopus neurogenin-related-1 (X-ngnr-1, a tap homolog) defines the three prospective domains of primary neurogenesis, and expression of XNeuroD coincides with neuronal differentiation. However, the transition between neuronal competence and stable commitment to a neuronal fate remains poorly characterised. Drosophila Collier and rodent early B-cell factor/olfactory-1 are both members of a family of HLH transcription factors containing a previously unknown type of DNA-binding domain. An orthologous gene from Xenopus, Xcoe2, has been isolated and found to be expressed in precursors of primary neurons. Xcoe2 is transcribed after X-ngnr-1 and before XNeuroD. Overexpression of a dominant-negative mutant of XCoe2 prevents neuronal differentiation. Conversely, overexpressed wild-type Xcoe2 could promote ectopic differentiation of neurons, in both the neural plate and the epidermis. In contrast to studies with X-ngnr-1 or XNeuroD, the supernumerary neurons induced by Xcoe2 appear in a 'salt-and-pepper' pattern, resulting from the activation of X-Delta1 expression and feedback regulation by lateral inhibition. XCoe2 may play a pivotal role in the transcriptional cascade that specifies primary neurons in Xenopus embryos: by maintaining Delta-Notch signaling, XCoe2 stabilizes the higher neural potential of selected progenitor cells that express X-ngnr-1, ensuring the transition between neural competence and irreversible commitment to a neural fate; and it promotes neuronal differentiation by activating XNeuroD expression, directly or indirectly (Dubois, 1998).

The posteriorizing agent retinoic acid can accelerate anterior neuronal differentiation in Xenopus laevis embryos. To elucidate the role of retinoic acid in the primary neurogenesis cascade, an investigation was carried out to see whether retinoic acid treatment of whole embryos can change the spatial expression of a set of genes known to be involved in neurogenesis. Retinoic acid expands the N-tubulin, X-ngnr-1, X-MyT1, X-Delta-1 and Gli3 domains and inhibits the expression of Zic2 and sonic hedgehog in the neural ectoderm, whereas a retinoid antagonist produces the opposite changes. In contrast, sonic and banded hedgehog overexpression reduce the N-tubulin stripes, enlarge the neural plate at the expense of the neural crest, downregulate Gli3 and upregulate Zic2. Thus, retinoic acid and hedgehog signaling have opposite effects on the prepattern genes Gli3 and Zic2 and on other genes acting downstream in the neurogenesis cascade. In addition, retinoic acid cannot rescue the inhibitory effect of NotchICD, Zic2 or sonic hedgehog on primary neurogenesis. These results suggest that retinoic acid acts very early, upstream of sonic hedgehog, and a model is proposed for regulation of differentiation and proliferation in the neural plate, showing that retinoic acid might be activating primary neurogenesis by repressing sonic hedgehog expression (Franco, 1999).

Because X-Delta-1 appears to be expressed in the future primary neurons themselves, they should be the source of the inhibitory signal that activates X-Notch-1 in the neighboring cells, thus preventing them from undergoing neuronal differentiation, inhibiting their own X-Delta-1 expression and decreasing their ability to inhibit the original signaling cell. This would generate a feedback loop that reinforces contrasts between adjacent cells. Here it is shown that RA treatment enhances the density of X-Delta-1-positive cells and it is presumed that, in this way, it impairs the developing distinction between adjacent cells, allowing more precursors to become neurons. Since X-ngnr-1 overexpression leads to X-Delta-1 overproduction, RA could be activating X-Delta-1 expression through X-ngnr-1 induction (Franco, 1999).

The POU domain gene, XlPOU 2, acts as a transcriptional activator during mid-gastrulation in Xenopus. Overexpression or misexpression of VP16-POU-GR, a fusion protein consisting of the strong activator domain of VP16 and the POU domain of XlPOU 2, results in ectopic expression of the neural-specific genes, nrp-1, en-2, and beta-tubulin. In contrast, overexpressing a dominant-inhibitory form of XlPOU 2 inhibits the chordin-induced neuralization of uncommitted ectoderm, and results in a loss of nrp-1 and en-2 expression in embryos. Furthermore, in uncommitted ectoderm, XlPOU 2 regulates the developmental neural program that includes a number of pre-pattern genes and at least one proneural gene, X-ngnr-1, thus playing a key role during neural determination. Thus XlPOU 2 is upstream from X-ngnr-1, a neuronal determinant gene expressed in all primary neurons. Although XlPOU 2 induces X-ngnr-1 readily, X-ngnr-1 is not capable of inducing XlPOU 2 in an animal cap induction assay. XlPOU 2's activation of X-ngnr-1, then leads to the induction of beta-tubulin (Matsuo-Takasaki, 1999).

The activation of pre-pattern genes occurs at the beginning of gastrulation, and thus they are likely candidates to work upstream from XlPOU 2, which is not expressed in the neuroectoderm until mid-gastrulation. These data provide evidence for the potential cross-regulation that might occur between XlPOU 2 and the Zic genes during neural determination. The induction of XlPOU 2 by both Zic 3 and by Zic r1 was investigated after the overexpression of these genes in animal cap ectoderm. Both genes are capable of neuralizing animal cap ectoderm, as evidenced by the activation of nrp-1. They are also capable of weakly inducing XlPOU 2. Although XlPOU 2 cannot be responsible for the initial activation of the pre-pattern genes at the beginning of gastrulation, it is likely that XlPOU 2 may enhance pre-pattern gene expression later in gastrulation (Matsuo-Takasaki, 1999).

Xath3 encodes a Xenopus neuronal-specific basic helix-loop-helix transcription factor related to the Drosophila proneural factor Atonal. Xath3 acts downstream of X-ngnr-1 during neuronal differentiation in the neural plate and retina and its expression and activity are modulated by Notch signaling. X-ngnr-1 activates Xath3 and NeuroD by different mechanisms, and the latter two genes crossactivate each other. In the ectoderm, X-ngnr-1 and Xath3 have similar activities, inducing ectopic sensory neurons. Among the sensory-specific markers tested, only those that label cranial neurons were found to be ectopically activated. By contrast, in the retina, X-ngnr-1 and Xath3 overexpression promote the development of overlapping but distinct subtypes of retinal neurons. Together, these data suggest that X-ngnr-1 and Xath3 regulate successive stages of early neuronal differentiation and that, in addition to their general proneural properties, they may contribute, in a context-dependent manner, to some aspect of neuronal identity (Perron, 1999).

X-ngnr-1 promotes lateral inhibition by inducing X-Delta-1 expression. To determine whether Xath3 plays a role in the induction or maintenance of X-Delta-1 expression, which starts from stage 12 onwards, Xath3 was overexpressed in embryos and these embryos were analyzed for X-Delta-1 expression by in situ hybridization. In 38/41 cases, an induction of X-Delta-1 expression was seen on the injected side, indicating that Xath3 is indeed an activator of X-Delta-1. It was then asked whether Notch signaling has an inhibitory effect on Xath3. Overexpression of mRNAs encoding a dominant-active form of X-Notch-1 (X-NotchICD), which blocks X-ngnr-1 expression, also represses Xath3 expression. Coinjection of X-NotchICD mRNA together with Xath3 mRNAs prevents Xath3 from inducing ectopic expression of the neuronal marker N-tubulin: none of 32 Xath3/X-NotchICD-injected embryos shows ectopic tubulin expression, whereas 66/70 embryos injected with Xath3 alone shows ectopic N-tubulin staining. Finally, the zinc finger transcription factor X-MyT1, which starts to be expressed at about the same time as Xath3 and has been shown to synergize with the earlier bHLH factors X-ngnr-1 and Xash3 to induce neuronal differentiation and to allow cells to escape lateral inhibition, strongly increases the ability of Xath3 to induce N-tubulin expression. Thus, Xath3 can cooperate with X-MyT1 to induce neuronal differentiation, and its expression and function, like that of X-ngnr-1, are inhibited by the Notch pathway (Perron, 1999).

Xath3 and X-ngnr-1 promote the development of distinct subtypes of retinal neurons. The different cell types of the retina are produced from proliferative precursor neuroepithelial cells at different overlapping periods during retinal neurogenesis. Ganglion, cone photoreceptor, and horizontal cells are born first, followed by amacrine and rod photoreceptor cells, while bipolar and Muller cells are born last. To know whether X-ngnr-1 and Xath3 play a specific role in regulating the differentiation of these cells, their expression was targeted to the developing retina by in vivo lipofection at stage 18. Transfection of Xath3 produces about twice the number of ganglion cells and more photoreceptor cells than control transfection with GFP alone, at the expense of bipolar and Muller glia cells. Transfection of X-ngnr-1 also produced more photoreceptor cells, at the expense of bipolar and Muller cells, but no change is observed in the number of ganglion cells. It was confirmed that the Xath3 and X-ngnr-1 transfected cells observed in the ganglion and photoreceptor cell layers are indeed differentiated ganglion and photoreceptor cells by staining with anti-islet-1 (anti-ganglion cell), anti-calbindin (anti-cone), and anti-rhodopsin (anti-rod) Abs (Perron, 1999).

The fact that distinct effects are observed upon overexpression in the nonneural ectoderm and in retinal progenitor cells (for example, ectopic expression of Islet-1 by X-ngnr-1 when overexpressed in early embryos, but not when overexpressed in the retina) most probably reflects the dependence of the activity of the bHLH factors on the context in which they are active. Positional information has been shown, for example, to be important in the case of scute, which promotes the formation of different types of external sensory organs in different places, because of the local expression of genes such as poxn or the BarH genes. It will be important to discover what determines the functional differences observed between these bHLH factors, i.e., site-recognition specificity or cofactor binding, by performing domain-swapping experiments and to identify the factors that cooperate with them to give neurons their specific properties (Perron, 1999).

The development of the vertebrate nervous system depends upon striking a balance between differentiating neurons and neural progenitors in the early embryo. The Xenopus homeodomain-containing gene Xdbx (a homolog of Drosophila Homeodomain protein 2.0, zebrafish hlx1, murine Dbx, chick ChoxE, murine Dbx2 and murine Hlx) regulates this balance by maintaining neural progenitor populations within specific regions of the neuroectoderm. In posterior regions of the Xenopus embryo, Xdbx is expressed in a bilaterally symmetric stripe that lies at the middle of the mediolateral axis of the neural plate. This stripe of Xdbx expression overlaps the expression domain of the proneural basic/helix-loop-helix-containing gene, Xash3, and is juxtaposed to the expression domains of Xenopus Neurogenin related 1 and N-tubulin, markers of early neurogenesis in the embryo. Xdbx overexpression inhibits neuronal differentiation in the embryo and when co-injected with Xash3, Xdbx inhibits the ability of Xash3 to induce ectopic neurogenesis. One role of Xdbx during normal development may therefore be to restrict spatially neuronal differentiation within the neural plate, possibly by altering the neuronal differentiation function of Xash3 (Gershon, 2000).

What is the mechanism by which Xdbx mediates altered Xash3 function? Analysis indicates that Xdbx overexpression inhibits the expression of X-Ngnr1 and NeuroD, genes with direct links to neuronal differentiation in the early embryo. Thus the ability of Xdbx to alter Xash3 function may depend upon its ability to either directly or indirectly inhibit the expression of X-Ngnr1 and/or NeuroD within zones of Xash3 expression. Xash3 overexpression induces the ectopic expression of NeuroD within neural and non-neural ectodermal progenitors and thus NeuroD appears to be a downstream target of Xash3. The fact that Xash3 and NeuroD are not normally co-expressed in neural plate progenitors therefore suggests that functions such as those described for Xdbx limit Xash3 target regulation at early stages of normal development. The regulation of Xash3 function by Xdbx could result either from competition for transcriptional targets, activation of reciprocal targets and/or direct protein-protein interactions (Gershon, 2000).

Cdk5, a member of the cyclin-dependent kinase family, has been shown to play an important role in development of the central nervous system in mammals when partnered by its activator p35. The p35/Cdk5 kinase is a neuron-specific Rac effector that inhibits Pak1 activity. This paper describes the cloning and characterization of Xp35.2, a novel activator of cdk5 in Xenopus. Xp35.2 is expressed during development initially in the earliest differentiating primary neurons in the neural plate and then later in differentiating neural tissue of the brain. This is in contrast to the previously described Xenopus cdk5 activator Xp35.1, which is expressed over the entire expanse of the neural plate in both proliferating and differentiating cells. Expression of both Xp35.1 and Xp35.2 and activation of cdk5 kinase occurs when terminal neural differentiation is induced by neurogenin and neuro D overexpression but not when only the early stages of neural differentiation are induced by noggin. Moreover, blocking cdk5 kinase activity specifically results in disruption and reduction of the embryonic eye where cdk5 and its Xp35 activators are expressed. Thus, cdk5/p35 complexes function in aspects of neural differentiation and patterning in the early embryo and particularly in formation of the eye (Philpott, 1999).

The basic helix-loop-helix (bHLH) factor Xath5 promotes retinal ganglion cell differentiation when overexpressed and may do so by regulating the expression of factors involved in the differentiation of these cells. Potential candidates include the Brn3 POU-homeodomain transcription factors, which have been implicated in retinal ganglion cell development. A new member of the Brn3 gene subfamily in Xenopus, XBrn3d, has been identified. In situ hybridization analysis shows XBrn3d expression in developing sensory neurons and developing ganglion cells of the retina. Using a hormone-inducible Xath5 fusion protein, it has been shown that in animal caps Xath5 can directly regulate the expression of XBrn3d. Since XBrn3d is also expressed in sensory populations where Xath5 is not expressed, the regulation of XBrn3d expression by the bHLH factor XNeuroD was examined. A XNeuroD-hGR fusion protein is similarly able to directly induce the expression of XBrn3d in animal caps. In addition, overexpression of XBrn3d by RNA injection promotes the expression of ectopic sensory neuronal markers in the lateral ectoderm, suggesting a role in regulating neuronal development. Therefore, Xath5 and XNeuroD can directly regulate the expression of a neuronal subtype-specific factor, providing a link between neuronal differentiation and cell fate specification (Hutcheson, 2001).

Both gain-and loss-of-function analyses indicate that proneural basic/helix-loop-helix (bHLH) proteins direct not only general aspects of neuronal differentiation but also specific aspects of neuronal identity within neural progenitors. In order to better understand the function of this family of transcription factors, hormone-inducible fusion constructs were used to assay temporal patterns of downstream target regulation in response to proneural bHLH overexpression. In these studies, two distantly related Xenopus proneural bHLH genes, Xash1 and XNgnr1, were compared. Both Xash1 and XNgnr1 induce expression of the general neuronal differentiation marker, N-tubulin, with a similar time course in animal cap progenitor populations. In contrast, these genes each induce distinct patterns of early downstream target expression. Both genes induce expression of the HLH-containing gene, Xcoe2, at early time points, but only XNgnr1 induces early expression of the bHLH genes, Xath3 and XNeuroD. Structure:function analyses indicate that the distinct pattern of XNgnr1-induced downstream target activation is linked to the XNgnr1 HLH domain, demonstrating a novel role for this domain in mediating the differential function of individual members of the proneural bHLH gene family (Talikka, 2002).

The differential downstream target activation observed at early time points in response to Xash1 and XNgnr1 overexpression provides a useful means of assaying the structure:function correlates of differential bHLH activity. Previous studies have highlighted the importance of the basic domain for the unique identity functions of bHLH proteins. In contrast, these same studies suggest that the HLH domain plays an essential but more general role in bHLH function. Thus, when the scute HLH is substituted for the MyoD HLH domain, the resulting protein retains its myogenic function. In similar studies, Stonal and Scute HLH domain substitutions do not alter the specific chordotonal-vs extrasensory neuron-inducing properties of these proteins. The current studies indicate that the HLH domain also plays a role in specific aspects of bHLH function. Cell-type identity was not studied. Instead, the regulation of a subset of downstream transcriptional targets was compared. These targets are broadly expressed in neural progenitors and may play more general roles in neuronal differentiation. Thus, these findings complement previous studies of cell identity and may indicate differing roles for the basic region and the HLH domain in mediating specific bHLH response (Talikka, 2002).

The current studies indicate that the N-terminal region of the XNgnr1 HLH domain is sufficient to specify patterns of downstream target regulation. Comparison of XNgnr1 and Xash1 protein coding sequences within this region points to six nonconserved amino acid identities, defining a starting point for further delineation of residues important for guiding downstream target regulation. Studies of MyoD have defined a single amino acid within this region (K124) that functions together with basic region amino acids (A114; T115) in the myogenic conversion of the class A bHLH protein, E12. Leucine (L130) to lysine (K) substitution at this position has been shown sufficient to convert Mash1 to a myogenic differentiation factor. In contrast, substitution of the XNgnr1 encoded amino acid (N96) for the corresponding Xash1 amino acid (L96) at this position is not sufficient to alter patterns of Xash1- and XNgnr1-specific downstream target activation (Talikka, 2002).

These studies indicate that proneural HLH domain specificity plays a significant role in guiding unique patterns of downstream target regulation in neural progenitors. The HLH domain mediates protein: protein interactions between bHLH factors and several classes of regulatory molecules. These interactions have been characterized with the general role of the HLH domain in mind, but the current studies suggest that they should be reexamined in terms of potentially specific interactions with individual proneural bHLH proteins. The best-characterized HLH-mediated interaction to date is with class A bHLH proteins related to the vertebrate E2A and Drosophila daughterless genes. The widespread expression of E2A as well as the fact that E2A proteins mediate bHLH function in a number of lineages suggest that E2A heterodimer formation is a permissive rather than a specific aspect of bHLH function. However, several vertebrate homologs of E2A-related genes have been identified, raising the possibility that further investigation may uncover more specific interactions. In addition to E2A interactions, a complex has been characterized in erythroid progenitors that includes the bHLH protein, Scl/Tal1, the LIM only protein, LMO2, the Lim domain binding protein, Ldb1, and the GATA-1 transcription factor. Recent characterizations indicate that a similar complex forms in neural progenitors. Chip is the fly homolog of Ldb1, and recent studies indicate that chip binds the HLH domain of the Achaete-Scute:Daughterless complex. This interaction is essential for autoregulation of Achaete-Scute expression within proneural domains. LMO2-related proteins are also associated with neural-specific bHLH proteins. In the frog, LMO3 binds the neural-specific bHLH protein, Hen1/Nscl, and potentiates the neuronal differentiation function of Hen1/Nscl in Xenopus overexpression assays. LMO3 also binds the Xash3, XNeuroD, and XNgnr1 proteins, albeit with decreased affinity relative to Hen1/Nscl. These findings suggest that chip/Ldb1 and LMO3 may play important roles in potentiating the function of numerous proneural bHLH-containing proteins. As with class A bHLH proteins, additional vertebrate LMO (also known as Rbtn and Ttg) and Ldb homologs have been isolated, suggesting the possibility of more specific interactions with individual bHLH proteins (Talikka, 2002).

The iroquois (iro) homeobox genes participate in many developmental processes both in vertebrates and invertebrates -- among them are neural plate formation and neural patterning. The Xenopus Iro (Xiro) function in primary neurogenesis has been studied in detail. Misexpression of Xiro genes promotes the activation of the proneural gene Xngnr1 but suppresses neuronal differentiation. This is probably due to upregulation of at least two neuronal-fate repressors: XHairy2A and XZic2. Accordingly, primary neurons arise at the border of the Xiro expression domains. In addition, XGadd45-gamma has been identified as a new gene repressed by Xiro. XGadd45-gamma encodes a cell-cycle inhibitor and is expressed in territories where cells will exit mitosis, such as those where primary neurons arise. Indeed, XGadd45-gamma misexpression causes cell cycle arrest. It is concluded that during Xenopus primary neuron formation, in Xiro expressing territories neuronal differentiation is impaired, while in adjacent cells, XGadd45-gamma may help cells stop dividing and differentiate as neurons (de la Calle-Mustienes, 2002).

This may be due to redundancy between different Gadd45 proteins. The spatial and temporal patterns of expression of Gadd45-gamma and the Notch ligand XDl1 largely coincide. Moreover, both XGadd45-gamma and XDl1 are positively regulated by proneural genes and negatively controlled by Notch signaling. According to the lateral inhibition model, activation of the Notch pathway within a cell, by signaling from neighboring cells, maintains the cell's mitotic potential and prevents its differentiation. In contrast, a cell that expresses high levels of Notch ligands and signals strongly, escapes lateral inhibition, exits the cell cycle and differentiates. XGadd45-gamma may provide a link between Notch signaling, cell-cycle arrest and differentiation. Thus, in the neural plate, cells with high levels of proneural genes have also high levels of XDl1 and XGadd45-gamma. The first allows them to escape lateral inhibition, and the second to exit the cell cycle. These cells can then differentiate. Mitotic arrest mediated by XGadd45-gamma probably occurs through interaction with cyclin and inhibitors of cyclin-dependent kinases. In neighboring cells, the Notch pathway is activated, proneural genes and XGadd45-gamma are downregulated, and cell-cycle arrest and differentiation cannot occur. It is of interest that induction of Gadd45 genes in cell culture stops the cell cycle in G1 phase. This phase is compatible with exiting the cell cycle, a requirement for terminal neuronal differentiation. Cells that differentiate outside the neural plate may resort to genes different from the proneural ones to accumulate Notch ligands and XGadd45-gamma (de la Calle-Mustienes, 2002).

This study compares the effects of overexpressing either Xiro1, -2 or -3 in neural development. To make comparisons more meaningful, equivalent constructs were prepared in the pCS2MT plasmid. The overexpression of each Xiro gene causes similar effects, although Xiro3 was approximately five to ten times more potent. Paradoxically, the overexpressions activated Xngnr1 and repressed neuronal differentiation. This may be explained at least in part by the finding that Xiro upregulates the neuronal repressors XHairy2A and XZic2. Indeed, it has been shown that XZic2 antagonizes development of Xngnr1-promoted ectopic neurons. XZic2 antagonizes Xngnr1-promoted XGadd45-gamma and XDl1 expression. Consistently with these findings, in wild type embryos the intermediate stripes of expression of XHairy2A and XZic2 are within the Xiro1 domains. Also in accordance with these results, in the prospective spinal chord, the Xiro1 domain is contained within the broader Xngnr1 domain and neurons arise at the border of the Xiro1 domain. Taken together, these observations suggest that Xiro proteins simultaneously participate in the activation of Xngnr1 and of genes that antagonize primary neuron formation (de la Calle-Mustienes, 2002).

Overexpressions of Xiro genes represses both XGadd45-gamma and XDl1 in territories where primary neurons arise. Consistently, in wild type embryos, XGadd45-gamma and XDl1 are expressed at the borders of Xiro domains. Moreover, XDl1 is activated in embryos expressing a Xiro1 chimera that converts the Xiro1 repressor into an activator (HD-GR-E1A). This activation occurs even in the absence of protein synthesis. Thus, XDl1 is probably directly repressed by Xiro. However, XGadd45-gamma is repressed by HD-GR-E1A, probably because Xngnr1 is also downregulated. Indeed, coinjection of HD-GR-E1A and Xngnr1 mRNAs rescues the expression of XGadd45-gamma. Thus, Xiro-mediated repression of XGadd45-gamma is probably indirect and may take place, at least in part, by Xiro-upregulated neuronal repressors. In this case, interference with Xiro function would suppress neuronal repressors, but would also downregulate Xngnr1, which is needed for XGadd45-gamma expression (de la Calle-Mustienes, 2002).

A model is proposed for the function of Xiro in neural patterning that integrates the above data. Xiro proteins, as well as other factors, participate in the activation of Xngnr1. Within the Xiro domains, Xngnr1 does not activate XDl1 or XGadd45-gamma, and cannot promote differentiation of primary neurons due to the upregulation by Xiro of neuronal repressors, such as XHairy2A and XZic2. In addition, Xiro probably represses XDl1 directly. Outside the Xiro domains, other factors, such as the Gli proteins, activate Xngnr1, which in turn promotes the expression of XDl1 and XGadd45-gamma in those cells that will become primary neurons. XDl1 switches on the lateral inhibition mechanism by which the Notch signaling pathway is activated in neighboring cells. This pathway downregulates proneural genes, XDl1 and XGadd45-gamma. As a consequence, these cells keep dividing and do not differentiate. In contrast, cells with high levels of Xngnr1, XDl1 and XGadd45-gamma escape lateral inhibition, exit the cell cycle (in part due to the presence of XGadd45-gamma) and differentiate as primary neurons. This differentiation is triggered by a genetic program activated by Xngnr1. Thus, Xiro proteins may help coordinate cell cycle and differentiation (de la Calle-Mustienes, 2002).

The vertebrate Six3 gene (Drosophila homolog: Optix), a homeobox gene of the Six-family, plays a crucial role in early eye and forebrain development. Candidate factors have been isolate that interact with Six3 in a yeast two-hybrid screen. Among these are two basic helix loop helix (bHLH) domain containing proteins. Biochemical analysis reveals that the bHLH proteins ATH5, ATH3, NEUROD as well as ASH1 interact specifically with XSix3. By defining the interacting domains it has been shown that the bHLH domain of NEUROD interacts with the SIX domain of XSix3. The co-expression of the interacting molecules during late retina determination/differentiation suggests a new role for Six3 and the respective interaction partner also in these late steps of eye development (Tessmar, 2002).

Co-expression of XNeuroD or Xath5 and XSix3 in the eye starts around the time when cell fate determination is still in progress, but differentiation is initiated. At the end of differentiation, their coexpression vanishes, suggesting that their interaction with SIX3 is important for the determination and/or differentiation of distinct cell types in the retina. This finding is in accordance with the notion that in a conditional inactivation of murine Pax6 in retinal progenitor cells, co-expression of Six3 and NeuroD coincides with the exclusive generation of amacrine cells. Therefore, Six3 might permit amacrine cell fate in the presence of NeuroD (Tessmar, 2002).

Xath3 and Xath5 show expression patterns similar to XNeuroD in the developing neuroretina, suggesting that these proteins likewise form part of the determination/differentiation network of the eye. The interaction of SIX3 with a specific combination of ARPs thus may specify distinct cell types of the neuroretina. In contrast, differentiation requires both a stop of proliferation and the expression of cell type specific differentiation genes. Therefore, the ARP/XSIX3 interaction should initiate, directly or indirectly, a proliferation-stop signal. Since Six3 on its own stimulates proliferations, it is tempting to speculate that the interaction of SIX3 with XNEUROD, XATH3 or XATH5 (any of which abolish Six3's proliferative activity) promotes differentiation in those cells of the retina that co-express these atonal-related protein family members (Tessmar, 2002).

One further aspect of this study is the question of evolutionary conservation of these interactions. By performing a cross-species screening experiment, interaction partners have been selected that are presumably conserved between two different vertebrates. Domain-mapping experiments further support this conservation. Since the conserved bHLH domain of XNEUROD interacts with XSIX3, and XSIX3 interacts with XNEUROD via the conserved SIX domain, it is reasonable to speculate that similar interactions might take place in other, non-vertebrate organisms. However, the interaction between the N-terminus of NEUROD and SIX3 appears to be a specific feature of the NEUROD subfamily, since no conserved domain could be detected at the amino acid sequence level (Tessmar, 2002).

The interaction of XSIX3 with the non-Atonal related bHLH protein XASH1, but not with XESR1 or the more distantly related protein XMAX2, clearly indicates specific interactions with other non-atonal-related bHLH transcription factors, the relevance of which will be addressed in future experiments (Tessmar, 2002).

The HES family of bHLH repressors plays a key role in regulating the differentiation of neural precursors in the vertebrate embryo. Members of the HES gene family are expressed in neural precursors as targets of the Notch signaling pathway, but how this occurs in the context of neurogenesis is not known. This issue is addressed by identifying enhancers driving Notch-dependent gene expression of two Hes5-like genes expressed in Xenopus called Esr1 and Esr10. Using frog transgenesis, enhancer elements were identified driving expression of Esr1 and Esr10 in neural precursors or in response to ectopic expression of the proneural protein, Xngnr1. Using deletion and mutation analysis, motifs required for enhancer activity of both genes were defined, namely Notch-responsive elements and, in the case of Esr10, E-box motifs. Esr1 and Esr10 are differentially regulated both in terms of Notch input and its interaction with heterologous factors. These studies reveal inputs required for proneural expression of genes encoding bHLH repressors in the developing vertebrate nervous system (Lamar, 2005).

The data indicates that proneural bHLH input to the Esr10 enhancer is both indirect (through Notch) and direct. The Notch intracellular domain (ICD) and Xngnr1 synergistically upregulate transcription in transfection assays, Xngnr1 binds to the Esr10 downstream E-box in vitro, and the Esr10 proneural enhancer with mutant E-boxes shows marked loss of activity in vivo, which cannot be rescued by exogenous Xngnr1. These findings extend observations in Drosophila that proneural proteins synergize with Notch in activating E(spl) genes in larval discs. The data also support analysis of the Drosophila E(spl) gene m8. In that case, E boxes and Su(H) sites only in the configuration of a classical SPS enabled synergy between ICD and bHLH proteins, and enhancer activity was lost when one Su(H) site was mutant or oriented incorrectly. The SPS motif is a bipartite binding site for the Suppressor of hairless protein. The binding sites are separated by 30 or 29 nucleotides in the promoters of E(spl) genes of Drosophila melanogaster and higher vertebrates, respectively. One of the binding sites occurs in a reverse orientation to the other. Furthermore, a hexamer motif, which lies between or within the motifs, has a functional aspect. The Esr10 proneural enhancer behaves similarly in transgenics and provides the first example of such a required architecture among vertebrate Notch targets (Lamar, 2005).

By contrast, Esr1 is not directly regulated by proneural proteins. Although Esr1/RV has three E-boxes, E3 is not conserved in X. tropicalis, E1 is not conserved in the proneural enhancer of the closely related Esr7 gene, and neither E1 nor E3 fits the RCAGSTG consensus required for high-affinity binding of Drosophila proneural proteins to E-boxes. However, the CACCTG motif seen in E2 is targeted by Drosophila proneural proteins, a CACCTG E-box is required for retinal expression of Xenopus Ath5, and CACCTG binds MyoD in vitro and in vivo. Furthermore, E2 is embedded in a 13-base homology extending beyond the E-box in numerous Hes5 orthologs, although it is not seen in the Esr10 promoter. E2 was mutated using two strategies and no effect was seen on transgene expression in vivo. Further mutation may be required to evaluate the contribution of this motif to Esr1 expression. Nonetheless that E2 is contained within the Esr1 enhancer rules out the possibility that any factor binding to E2 is sufficient (with Notch acting through S1) to activate robust enhancer activity (Lamar, 2005).

Sites required for proneural Esr1 expression other than Su(H) sites have not been identified. Su(H) sites could be sufficient to activate Esr1, and tissue-specific responses to Notch might be due either to tissue-specific repressors or to the spacing of Su(H) sites providing a distinct platform for co-activators. Alternatively, Su(H) sites in the Esr1 enhancer could synergize with heterologous (non-bHLH) factors induced by Xngnr1, which, unlike direct bHLH input to either Esr10 or m8, interact with Notch through an S1-S4 configuration of Su(H) sites. Finally, enhancer activity could require input from both Notch (dependent on Xngnr1) and neural factors not dependent on Xngnr1. Although all three scenarios are possible, observation of attenuated but spatially appropriate GFP expression driven by the Esr1 enhancer argues against Su(H) site spacing as the sole determinant of specificity and suggests rather that tissue specific input to Esr1 requires sequences downstream of Hin3 (Lamar, 2005).

Transit amplification in the amniote cerebellum evolved via a heterochronic shift in NeuroD1 expression

The cerebellum has evolved elaborate foliation in the amniote lineage as a consequence of extensive Atoh1-mediated transit amplification in an external germinal layer (EGL) comprising granule cell precursors. To explore the evolutionary origin of this layer, the molecular geography of cerebellar development was examined throughout the life cycle of Xenopus laevis. At metamorphic stages Xenopus displays a superficial granule cell layer that is not proliferative and expresses both Atoh1 and NeuroD1, a marker of postmitotic cerebellar granule cells. Premature misexpression of NeuroD1 in chick partially recapitulates the amphibian condition by suppressing transit amplification. However, unlike in the amphibian, granule cells fail to enter the EGL. Furthermore, misexpression of NeuroD1 once the EGL is established both triggers radial migration and downregulates Atoh1. These results show that the evolution of transit amplification in the EGL required adaptation of NeuroD1, both in the timing of its expression and in its regulatory function, with respect to Atoh1 (Butts, 2014).

Neurogenin/NeuroD homologs in chickens

Genetic studies in Drosophila and in vertebrates have implicated basic helix-loop-helix (bHLH) transcription factors in neural determination and differentiation. The role that several bHLH proteins play in the transcriptional control of differentiation in chick retina has been examined. The experimental system exploits the properties of the promoter for the beta3 subunit of the neuronal acetylcholine receptors, important components of various phenotypes in the CNS of vertebrates. The beta3 subunit contributes to define ganglion cell identity in retina and its promoter, whose activation is an early marker of ganglion cell differentiation, is under the specific control of the chick atonal homolog ATH5. Functional analysis of the ATH5 promoter indicates that interactions between ATH5 and several other bHLH transcription factors underlie the patterning of the early retinal neuroepithelium and form a regulatory cascade leading to transcription of the gene for beta3. ATH5 appears to coordinate the transcriptional pathways that control pan-neuronal properties with those that regulate the subtype-specific features of retinal neurons (Matter-Sadzinski, 2001).

The data suggest that the patterns of ATH5/Ngn2 and ASH1 expression in the retinal neuroepithelium define two distinct cell lineage domains and that specification of the beta3 component of ganglion cell identity depends on the establishment of the ATH5 expression domain. The autoactivation of ATH5 may play an important role in initiating an autonomous program of ganglion cell differentiation, but it may not be sufficient for its long-term maintenance. As revealed by in situ hybridization, the onsets of Ngn2 and ATH5 expression coincide and Ngn2 is expressed in the majority of ATH5-positive cells. Moreover, Ngn2 positively regulates the cloned ATH5 promoter in retinal cells, and activates the endogenous gene in retinal glioblasts. It is not yet known if Ngn2 is involved in the induction of ATH5 expression but it may, at least, contribute to the maintenance of ATH5 expression in proliferating progenitors. ATH5 is transiently expressed in newborn neurons and other bHLH proteins may control its regulation at later stages of retinogenesis. In the CNS, the transient expression of NeuroM marks cells that have just left the mitotic cycle and, in keeping with this rule, newborn ganglion cells transiently express this factor. Because the capacity of NeuroM to stimulate the ATH5 promoter is restricted to postmitotic cells, NeuroM may transiently ensure ATH5 expression in newborn ganglion cells. NeuroD, whose onset in the ganglion cell layer occurs later than that of NeuroM, may exert its demonstrated ability to activate ATH5 at the ultimate stages of ganglion cell differentiation. It is unclear, however, why several different bHLH proteins should be required for the positive regulation of ATH5. One reason might be that the autostimulatory capacity of ATH5 is inhibited at some stage and needs to be relayed by other factors. Another possibility is that Ngn2, NeuroM and NeuroD cooperate with ATH5 to overcome the negative effect of ASH1 and to enhance the overall level of ATH5 expression. Still unclear are the molecular mechanisms whereby ASH1 may exert a dominant-negative effect. Heterodimers containing ASH1 and Ngn2 do not bind to E-box elements. Similarly, ASH1 and ATH5/Ngn2 may also form heterodimers that do not interact with the E-boxes in the ATH5 and beta3 promoters. Alternatively, ASH1 may bind to these E-boxes and thus prevent binding and activation by ATH5. The ATH5 promoter has a more complex organization than the beta3 promoter. At least four of the seven E-box elements in the ATH5 promoter are functional and mutational analysis indicates that a particular E-box may preferentially react with a particular bHLH protein. A rather complex interplay between these elements may thus enable the ATH5 promoter to integrate the effects of stimulatory (e.g., ATH5, Ngn2, NeuroM, NeuroD) and inhibitory (e.g., ASH1) factors. In transactivation assays, the ATH5 promoter fails to respond to bHLH factors after retinal cells have differentiated, a change in promoter properties that is remarkably congruent with the absence of ATH5 expression in the developed retina (Matter-Sadzinski, 2001).

Since ASH1 is not expressed in mature retina, the mechanism whereby ATH5 is repressed at late stages of retina development must differ from that operating during neurogenesis. Likewise, the beta3 promoter no longer responds to ATH5 after ganglion cells have completed their differentiation and it is surmised that late in development a different transcriptional code maintains beta3 expression. The proven ability of the myogenic factor MyoD to stimulate beta3 transcription in differentiated neurons suggests that the putative regulators of beta3 in mature retina share some functional properties with MyoD (Matter-Sadzinski, 2001).

The divergence of the ganglion cell lineage may represent the first of several possible branch points on a pathway along which initially multipotent progenitors progress to produce distinct retinal cell-types. Such branch points would generate neurons of different identities in proper number and order by restricting the competence of early progenitor lineages and by preserving a population of multipotent ASH1-expressing progenitors for the generation of later-born neurons. Overexpression of ATH5 in the developing Xenopus retina results in an increase in ganglion cells and a decrease in amacrine, bipolar and Muller glia cells. Overexpression of ATH5 in early retinal cells markedly stimulates transcription of the gene for beta3 and expands its expression domain, but these effects can be effectively antagonized by ASH1 overexpression. Thus, because of its dominant-negative effect upon ATH5 and beta3, ASH1 may help contain the domain of ATH5 and beta3 expression, thereby preventing the whole pool of retinal progenitors from entering the ganglion cell differentiation program. If ASH1 is part of the genetic program keeping production of ganglion cells under control, it would be expected that this population of neurons might increase in the retina of Mash1 knockout mice. Unfortunately, these mutant mice die before retina development is completed and no comparative analysis of the proportions of ganglion cells in the wild-type and Mash1-null retina explants has been reported. Other mechanisms, in conjunction with ASH1, may prevent untimely and excessive production of ganglion cells. In particular, the Delta-Notch signaling pathway is involved in the process whereby ganglion cell progenitors become newborn neurons. The beta3-expressing cells are among the first retinal precursors to leave the mitotic cycle and Delta1 is expressed in nascent beta3-positive ganglion cells, from where it may sustain expression of Notch in neighboring progenitors, thereby keeping these cells in an uncommitted state (Matter-Sadzinski, 2001).

Basic helix-loop-helix (bHLH) transcription factors such as atonal homolog 5 (ATH5) and neurogenin 2 (NGN2) determine crucial events in retinogenesis. Using chromatin immunoprecipitation, their interactions with target promoters have been demonstrated to undergo dynamic changes as development proceeds in the chick embryo. Chick ATH5 associates with its own promoter and with the promoter of the ß3 nicotinic receptor specifically in retinal ganglion cells and their precursors. NGN2 binds to the ATH5 promoter in retina but not in optic tectum, suggesting that interactions between bHLH factors and chromatin are highly tissue specific. The transcriptional activations of both promoters correlate with dimethylation of lysine 4 on histone H3. Inactivation of the ATH5 promoter in differentiated neurons is accompanied by replication-independent chromatin de-methylation. This report is one of the first demonstrations of correlation between gene expression, binding of transcription factors and chromatin modification in a developing neural tissue (Skowronska-Krawczyk, 2004).

To determine whether the correlation between promoter activity and histone H3-K4 dimethylation is a general phenomenon, it was of interest to analyze the methylation patterns of genes expressed both in the retina and in the optic tectum. NeuroM and NeuroD are good candidates for this study as they are dynamically expressed in both tissues. In the optic tectum, the transient expression of NeuroM peaks at E6, at a time when the various cell classes exit from the mitotic cycle. In the retina, NeuroM expression obeys the same principle as in the optic tectum; however, expression does not stop at the end of neurogenesis but persists in mature bipolar and horizontal cells. In the optic tectum and retina, NeuroD has a later onset than NeuroM. In early (E4-6) retina, expression of NeuroD is detected in precursor cells and may correlate at later stages with the differentiation of photoreceptors and amacrine cells. In the optic tectum, NeuroD expression is detected at around E6 and increases slowly during development of the tissue. Immunoprecipitation of chromatin from retina and optic tectum was performed using an anti dimethylated H3-K4 antibody and correlations in both tissues were observed between histone dimethylation and the known expression patterns of NeuroM and NeuroD. In retina, methylation of the NeuroM promoter is detected at E3 and reaches its highest level at E9. It remains high in the developed retina, in accordance with the sustained NeuroM mRNA expression seen in this tissue. In the optic tectum, the transient expression of this gene is at a much lower level than in the retina and no significant enhancement of methylation was detected. This could reflect the fact that the fraction of tectal cells that express NeuroM is too small to be detected in the assay, or it may suggest different histone modification requirements for brief versus continuous expression of the gene. The level of methylation of NeuroD promoter sequences remained very low during retina and optic tectum development, but was strongly enhanced in the developed retina and optic tectum. This delayed methylation of the NeuroD promoter is congruent with the late onset of NeuroD expression in both tissues. Incidentally, the ability to detect H3 methylation at the NeuroD promoter in both retina and optic tectum demonstrates that the paucity in optic tectum methylation observed for other promoters is physiologically relevant and not due to a tissue-specific bias in chromatin quality (Skowronska-Krawczyk, 2004).

Neurogenin/NeuroD homologs in mammals

NeuroD1/beta2 is a basic helix-loop-helix (bHLH) factor expressed in the endocrine cells of the pancreas and in a subset of neurons as they undergo terminal differentiation. NeuroD1 is expressed in corticotroph cells of the pituitary gland. The NeuroD1 is involved in cell-specific transcription of the proopiomelanocortin (POMC) gene. Corticotroph-specific POMC transcription depends in part on the action of cell-restricted bHLH factors that have been characterized as the CUTE (corticotroph upstream transcription element) complexes. These complexes contain NeuroD1 in association with various ubiquitous bHLH dimerization partners. The NeuroD1-containing heterodimers specifically recognize and activate transcription from the POMC promoter E box, which confers transcriptional specificity. Interestingly, the NeuroD1 heterodimers activate transcription in synergy with Ptx1, a Bicoid-related homeodomain protein, which also contributes to the corticotroph specificity of POMC transcription. In the adult pituitary gland, NeuroD1 transcripts are detected in POMC-expressing corticotroph cells. Taken together with the restricted pattern of Ptx1 expression, these results suggest that these two factors establish the basis of a combinatorial code for the program of corticotroph-specific gene expression (Poulin, 1997).

neurogenin2 encodes a neural-specific basic helix-loop-helix (bHLH) transcription factor related to the Drosophila proneural factor tap. The murine ngn2 gene is essential for development of the epibranchial placode-derived cranial sensory ganglia. An ngn2 null mutation blocks the delamination of neuronal precursors from the placodes, the first morphological sign of differentiation in these lineages. Mutant placodal cells fail to express downstream bHLH differentiation factors and the Notch ligand Delta-like 1. These data suggest that ngn2 functions like the Drosophila proneural genes in the determination of neuronal fate in distal cranial ganglia. Interestingly, the homeobox gene Phox2a is activated independent of ngn2 in epibranchial placodes, suggesting that neuronal fate and neuronal subtype identity may be specified independently in cranial sensory ganglia. Phox2a is an essential regulator of the noradrenergic phenotype in PNS and CNS neurons. Expression of Phox2a and high levels of ngn2 transcripts appear coupled in placodal precursors suggesting that a mechanism that is independent of ngn2 and therefore of Delta-Notch signaling, may single out cells fated to become neuronal precursors (Fode, 1998).

NeuroM, is a neural-specific helix-loop-helix transcription factor related to the Drosophila proneural gene atonal. The NeuroM protein most closely resembles the vertebrate NeuroD and Nex1/MATH2 factors, and is capable of transactivating an E-box promoter in vivo. NeuroM to NeuroD expression have been compared in the developing nervous system. In spinal cord and optic tectum, NeuroM expression precedes that of NeuroD. It is transient and restricted to cells lining the ventricular zone that have ceased proliferating but have not yet begun to migrate into the outer layers. In the retina, NeuroM is also transiently expressed in cells as they withdraw from the mitotic cycle, but persists in horizontal and bipolar neurons until full differentiation, assuming an expression pattern exactly complementary to NeuroD. In the peripheral nervous system, NeuroM expression closely follows cell proliferation, suggesting that it intervenes at a similar developmental juncture in all parts of the nervous system. It is proposed that availability of NeuroM defines a new stage in neurogenesis, at the transition between undifferentiated, premigratory and differentiating, migratory neural precursors (Roztocil, 1997).

Basic helix-loop-helix (bHLH) genes have emerged as important regulators of neuronal determination and differentiation in vertebrates. Three putative neuronal differentiation factors [NEX for neuronal helix-loop-helix protein-1 (mammalian atonal homolog-2), neuroD (beta-2), and NDRF for neuroD-related factor (neuroD2)] are highly homologous to each other in the bHLH region and comprise a new bHLH subfamily. To study the role of NEX, the first bHLH protein identified in this group, the NEX gene was disrupted by homologous recombination. NEX-deficient mice have no obvious developmental defect, and CNS neurons appear fully differentiated. To investigate further whether the absence of NEX is compensated for by neuroD and NDRF, the spatiotemporal expression of all three genes was compared. The transcription patterns of NEX, neuroD, and NDRF genes are highly overlapping in the developing CNS of normal rats between embryonic day 12 and adult stages but are not strictly identical. The most prominent transcription of each gene marks the dorsal neuroepithelium of the telencephalon in early development and is sustained in the adult neocortex, hippocampus, and cerebellum. In general, neuroD provides the earliest marker of neuronal differentiation in any given region, when compared with NDRF or NEX. Whereas a few CNS regions are specific for neuroD, no region is detected in which solely NEX or NDRF is expressed. This suggests that the function of the mutant NEX gene in neuronal differentiation is compensated for by neuroD and NDRF; by analogy to myogenic bHLH proteins, neuronal differentiation factors are at least in part equivalent in function (Schwab, 1998).

NeuroD1/BETA2 is a key regulator of pancreatic islet morphogenesis and insulin hormone gene transcription in islet beta cells. This factor also appears to be involved in neurogenic differentiation, because NeuroD1/BETA2 is able to induce premature differentiation of neuronal precursors and converts ectoderm into fully differentiated neurons upon ectopic expression in Xenopus embryos. Amino acid sequences in mammalian and Xenopus NeuroD1/BETA2 have been identified that are necessary for insulin gene expression and ectopic neurogenesis. These results indicate that evolutionarily conserved sequences spanning the basic helix-loop-helix (amino acids [aa] 100 to 155) and C-terminal (aa 156 to 355) regions are important for both of these processes. The transactivation domains (AD1: aa 189 to 299, and AD2: aa 300 to 355) are within the carboxy-terminal region, as analyzed by using GAL4:NeuroD1/BETA2 chimeras. Selective activation of mammalian insulin gene enhancer-driven expression and ectopic neurogenesis in Xenopus embryos is regulated by two independent and separable domains of NeuroD1/BETA2, located between aa 156 to 251 and aa 252 to 355. GAL4:NeuroD1/BETA2 constructs spanning these sequences demonstrate that only aa 252 to 355 contain activation domain function, although both aa 156 to 251 and 300 to 355 are found to interact with the p300/CREB binding protein (CBP) coactivator. These results implicate p300/CBP in NeuroD1/BETA2 function and further suggest that comparable mechanisms are utilized to direct target gene transcription during differentiation and in adult islet beta cells (Sharma, 1999).

NeuroD/BETA2, a basic helix-loop-helix transcription factor, has been shown to play a role in tissue-specific differentiation of pancreatic and enteroendocrine cells. To gain further insight into the function of neuroD/BETA2 in the nervous system development, expression pattern of neuroD/BETA2 during embryonic and postnatal development was examined by using in situ hybridization. Dynamic changes of neuroD/BETA2 expression in the central nervous system were observed during embryogenesis, especially in telencephalon, hippocampus, cerebellum, spinal cord, and olfactory epithelium. A moderate level of expression was also detected in developing pancreas in early embryogenesis. Although the neuroD/BETA2 expression in cerebellum and hippocampus increased over time, expression in cerebral cortex, spinal cord, as well as in fetal pancreas gradually decreased as embryogenesis proceeded. High level of the neuroD/BETA2 expression in developing cerebellum and hippocampus persisted throughout postnatal development and remained at a stable level in the adult brain. Interestingly, neuroD/BETA2 expression was detected not only in postmitotic but also in mitotic cells, as was evident in its expression in external granular layer of cerebellum and granule cells of the dentate gyrus during postnatal development. This observation suggests that neuroD/BETA2 may have a unique role in proliferation, differentiation, or both, of granule cells of cerebellum and dentate gyrus (Lee, 2000).

BETA2/NeuroD is a homolog of the Drosophila tap gene. BETA2/NeuroD is widely expressed during development in the mammalian brain and pancreas. Although studies in Xenopus suggest that BETA2/NeuroD is involved in cellular differentiation, its function in the mammalian nervous system is unclear. Mutant mice homozygous for a deletion at the BETA2/NeuroD locus fail to develop a granule cell layer within the dentate gyrus, one of the principal structures of the hippocampal formation. To understand the basis of this abnormality, dentate gyrus development was analyzed by using immunocytochemical markers in BETA2/NeuroD-deficient mice. The early cell populations in the dentate gyrus, including Cajal-Retzius cells and radial glia, are present and appear normally organized. The migration of dentate precursor cells and newly born granule cells from the neuroepithelium to the dentate gyrus remains intact. However, there is a dramatic defect in the proliferation of precursor cells once they reach the dentate and a significant delay in the differentiation of granule cells. This leads to malformation of the dentate granule cell layer and excess cell death. BETA2/NeuroD null mice also exhibit spontaneous limbic seizures associated with electrophysiological evidence of seizure activity in the hippocampus and cortex. These findings thus establish a critical role of BETA2/NeuroD in the development of a specific class of neurons. Furthermore, failure to express BETA2/NeuroD leads to a stereotyped pattern of pathological excitability of the adult central nervous system (Liu, 2000a).

NeuroD2 is sufficient to induce cell cycle arrest and neurogenic differentiation in nonneuronal cells. To determine whether this bHLH transcription factor is necessary for normal brain development, homologous recombination was used to replace the neuroD2 coding region with a beta-galactosidase reporter gene. The neuroD2 gene expresses the reporter in a subset of neurons in the central nervous system, including in neurons of the neocortex and hippocampus and cerebellum. neuroD2-/- mice show normal development until about day P14, when they begin exhibiting ataxia and failure to thrive. Brain areas that expressed neuroD2 are smaller than normal and show higher rates of apoptosis. Cerebella of neuroD2-null mice express reduced levels of genes encoding proteins that support cerebellar granule cell survival, including brain-derived neurotrophic factor (BDNF). Decreased levels of BDNF and higher rates of apoptosis in cerebellar granule cells of neuroD2-/- mice indicate that neuroD2 is necessary for the survival of specific populations of central nervous system neurons in addition to its known effects on cell cycle regulation and neuronal differentiation (Olson, 2001).

Transient transfection of vectors expressing neuroD2, MASH1, ngn1 or related neural bHLH proteins, with their putative dimerization partner E12, can convert mouse P19 embryonal carcinoma cells into differentiated neurons. Transfected cells express numerous neuron-specific proteins, adopt a neuronal morphology and are electrically excitable. Pan-neuronal markers such as neurofilament-M, the HuC/D RNA-binding proteins, M6 and synapsin I are all present in most or all cells with neuronal morphology. Moreover, subsets of the transfected cells are immunoreactive for the neurotransmitters GABA and glutamate, the GABA synthetic enzyme glutamatic acid decarboxylase (GAD), the neuropeptide substance P, NMDA receptor 1, and the transcription factors Islet-1 or LIM1. At present, it is not known if the subsets of cells that express these proteins overlap. The same constellation of neurotransmitters, receptors and other markers of mature neuronal subtypes have been observed regardless of which neural bHLH vector is transfected. No expression of glial fibrillary acidic protein (GFAP) or markers of radial glia have been observed in any transfected cells. Thus, the expression of neural bHLH proteins is sufficient to confer a neuronal fate on uncommitted mammalian cells. Neuronal differentiation of transfected cells is preceded by elevated expression of the cyclin-dependent kinase inhibitor p27 Kip1 and cell cycle withdrawal. This demonstrates that the bHLH proteins can link neuronal differentiation to withdrawal from the cell cycle, possibly by activating the expression of p27 Kip1. The ability to generate mammalian neurons by transient expression of neural bHLH proteins should create new opportunities for studying neurogenesis and devising neural repair strategies (Farah, 2000).

The neural bHLH genes Mash1 and Ngn2 are expressed in complementary populations of neural progenitors in the central and peripheral nervous systems. The activities of the two genes have been systematically compared during neural development by generating replacement mutations in mice in which the coding sequences of Mash1 and Ngn2 were swapped. Using this approach, it has been demonstrated that Mash1 has the capacity to respecify the identity of neuronal populations normally derived from Ngn2-expressing progenitors in the dorsal telencephalon and ventral spinal cord. In contrast, misexpression of Ngn2 in Mash1-expressing progenitors does not result in any overt change in neuronal phenotype. Taken together, these results demonstrate that Mash1 and Ngn2 have divergent functions in specification of neuronal subtype identity, with Mash1 having the characteristics of an instructive determinant whereas Ngn2 functions as a permissive factor that must act in combination with other factors to specify neuronal phenotypes. Moreover, the ectopic expression of Ngn2 can rescue the neurogenesis defects of Mash1 null mutants in the ventral telencephalon and sympathetic ganglia but not in the ventral spinal cord and the locus coeruleus, indicating that Mash1 contribution to the specification of neuronal fates varies greatly in different lineages, presumably depending on the presence of other determinants of neuronal identity (Parras, 2002).

Vertebrate epibranchial placodes give rise to visceral sensory neurons that transmit vital information such as heart rate, blood pressure and visceral distension. Despite the pivotal roles they play, the molecular program underlying their development is not well understood. The zebrafish mutation no soul, in which epibranchial placodes are defective, disrupts the forkhead-related, winged helix domain-containing protein Foxi1. Foxi1 is expressed in lateral placodal progenitor cells. In the absence of foxi1 activity, progenitor cells fail to express the basic helix-loop-helix gene neurogenin that is essential for the formation of neuronal precursors, and the paired homeodomain containing gene phox2a that is essential for neuronal differentiation and maintenance. Consequently, increased cell death is detected, indicating that the placodal progenitor cells take on an apoptotic pathway. Furthermore, ectopic expression of foxi1 is sufficient to induce phox2a-positive and neurogenin-positive cells. Taken together, these findings suggest that Foxi1 is an important determination factor for epibranchial placodal progenitor cells to acquire both neuronal fate and subtype visceral sensory identity (S. A. Lee, 2003).

Visceral sensory neurons include the geniculate, petrosal and nodose ganglia, which have distinct but also overlapping connectivity patterns. Fate mapping experiments show that they all derive from the epibranchial placodes. However, in the no soul mutant, a differential effect on these neurons is seen: whereas the geniculate and petrosal neurons fail to develop, the nodose ganglia are partially spared. Interestingly, nodose ganglia are also less affected in mice with targeted disruption of ngn 2 and phox2a. These analyses suggest that although the three distal ganglia share a developmental origin, different mechanisms may operate in their determination. Interestingly, it was observed that unlike geniculate and petrosal ganglia, which express phox2a prior to phox2b, nodose ganglia initiate phox2b expression prior to that of phox2a. Therefore, it is possible that the commitment and differentiation of at least subsets of nodose ganglion are under the control of yet unidentified regulatory hierarchies. Alternatively, other neural progenitor populations are able to compensate for the loss of epibranchial placode-derived nodose progenitor cells (S. A. Lee, 2003 and references therein).

Many lines of evidence indicate that important traits of neuronal phenotype, such as cell body position and neurotransmitter expression, are specified through complex interactions between extrinsic and intrinsic genetic determinants. However, the molecular mechanisms specifying neuronal connectivity are less well understood at the transcriptional level. The bHLH transcription factor Neurogenin2 cell autonomously specifies the projection of thalamic neurons to frontal cortical areas. Unexpectedly, Ngn2 determines the projection of thalamic neurons to specific cortical domains by specifying the responsiveness of their axons to cues encountered in an intermediate target, the ventral telencephalon. These results thus demonstrate that in parallel to their well-documented proneural function, bHLH transcription factors also contribute to the specification of neuronal connectivity in the mammalian brain (Seibt, 2003).

Insm1 promotes endocrine cell differentiation by modulating the expression of a network of genes that includes Neurog3 and Ripply3

Insulinoma associated 1 (Insm1; mammalian homolog of Drosophila Nerfin-1) plays an important role in regulating the development of cells in the central and peripheral nervous systems, olfactory epithelium and endocrine pancreas. To better define the role of Insm1 in pancreatic endocrine cell development mice were gnerated with an Insm1(GFPCre) reporter allele and were used to study Insm1-expressing and null populations. Endocrine progenitor cells lacking Insm1 were less differentiated and exhibited broad defects in hormone production, cell proliferation and cell migration. Embryos lacking Insm1 contained greater amounts of a non-coding Neurog3 mRNA splice variant and had fewer Neurog3/Insm1 co-expressing progenitor cells, suggesting that Insm1 positively regulates Neurog3. Moreover, endocrine progenitor cells that express either high or low levels of Pdx1, and thus may be biased towards the formation of specific cell lineages, exhibited cell type-specific differences in the genes regulated by Insm1. Analysis of the function of Ripply3, an Insm1-regulated gene enriched in the Pdx1-high cell population, revealed that it negatively regulates the proliferation of early endocrine cells. Taken together, these findings indicate that in developing pancreatic endocrine cells Insm1 promotes the transition from a ductal progenitor to a committed endocrine cell by repressing a progenitor cell program and activating genes essential for RNA splicing, cell migration, controlled cellular proliferation, vasculogenesis, extracellular matrix and hormone secretion (Osipovich, 2014).

Compensational regulation of bHLH transcription factors in the postnatal development of BETA2/NeuroD1-null retina

The bHLH transcriptional factor BETA2/NeuroD1 is essential for the survival of photoreceptor cells in the retina. Although this gene is expressed throughout the retina, BETA2/NeuroD1 knockout mice show photoreceptor cell degeneration only in the outer nuclear layer of the retina; other retinal neurons are not affected. Previous studies on retina explants lacking three bHLH genes revealed that retinal neurons in the inner nuclear layer require multiple bHLH genes for their differentiation and survival. However, single- or double-gene mutations show no or a lesser degree of abnormalities during eye development, likely because of compensation or cooperative regulation among those genes. Because not all null mice survive until the retina is fully organized, no direct evidence of this concept has been reported. To understand the regulatory mechanisms between bHLH factors in retinal development, a detailed analysis of BETA2/NeuroD1 knockout mice was performed. BETA2/NeuroD1 was expressed in all 3 layers of the mouse retina, including all major types of neurons. In addition, a null mutation of BETA2/NeuroD1 resulted in up-regulation of other bHLH genes, Mash1, Neurogenin2, and Math3, in the inner nuclear layer. These data suggest that compensatory and cross regulatory mechanisms exist among the bHLH factors during retinal development (Cho, 2007).

During postnatal development, BETA2/NeuroD1 expression is observed mainly in photoreceptor cells in the outer nuclear layer (ONL). However, moderate levels of expression remain in the outer half and innermost layer of the inner nuclear layer (INL) of the retina as well as in a certain population of cells in the ganglion cell layer (GCL). Therefore, the possibility that BETA2/NeuroD1 has functions in cell type specification in the INL together with other bHLH genes could not be ruled out. Both gain-of-function and loss-of-function studies have demonstrated that BETA2/NeuroD1 participates in the neuron/glia cell fate decisions, similar to other bHLH genes, including Mash1, Math3, and Math5 in retinal explants. Thus, attempts were made to identify differences in the population of major cell types in BETA2/NeuroD1-null retina compared with wild-type littermate retina. However, no differences were found. This result may be due to compensational regulation by other bHLH genes, such as Mash1, Math3, Neurogenin2, and Math5 (Cho, 2007).

Mash1, Math3, and Neurogenin2 are known to be expressed in the developing retina and act as positive regulators. Together with homeodomain factors such as Pax6 and Crx, these factors play important roles in cell type specification during early development. For example, Mash1 and Math3 are expressed predominantly in bipolar cells, and double knockouts of these genes decreases the bipolar cell population while increasing the Müller glial cell population. Neurogenin2 is also transiently expressed in all major neuron types in the mouse retina, and its expression is required for photoreceptor cells, horizontal cells, and bipolar cells. In contrast, BETA2/NeuroD1 is transiently expressed in differentiating amacrine cells. Although BETA2/NeuroD1-null mutation shows delayed amacrine cell development at earlier stages, the number of amacrine cells eventually is the same as that found in wild-type retinas. However, in double-knockout mutations with BETA2/NeuroD1 and Math3, the number of amacrine cells is decreased and that of retinal ganglionic cells is increased. Interestingly, amacrine cells adopt the ganglion cells' fate in this BETA2/NeuroD1;Math3 double-knockout mutant. In addition, the triple bHLH knockouts Mash1;Neurogenin2;Math3 and Math3;Neurogenin2; BETA2/NeuroD1 have been shown to have fewer horizontal cells, but any combination of double mutations of Mash1 or Math3 or BETA2/NeuroD1 with Neurogenin2 display abnormalities in retina development. Furthermore, single-knockout mutations of the genes barely affect the neuronal cell population in the INL and show no retinal abnormalities. Taken together, these results suggest that the bHLH factors cross regulate each others' expression and can specify neuronal subtypes cooperatively during late retinogenesis, especially in progenitor cells in the INL, to generate various subtypes of retinal neurons. Although the precise mechanism for retina cell type specification remains to be determined, these results provide further support for cooperative and compensational regulatory specification during postnatal retinogenesis (Cho, 2007).

Conserved regulatory sequences in Atoh7 mediate non-conserved regulatory responses in retina ontogenesis

The characterisation of interspecies differences in gene regulation is crucial to understanding the molecular basis of phenotypic diversity and evolution. The atonal homologue Atoh7 participates in the ontogenesis of the vertebrate retina. This study reveals how evolutionarily conserved, non-coding DNA sequences mediate both the conserved and the species-specific transcriptional features of the Atoh7 gene. In the mouse and chick retina, species-related variations in the chromatin-binding profiles of bHLH transcription factors correlate with distinct features of the Atoh7 promoters and underlie variations in the transcriptional rates of the Atoh7 genes. The different expression kinetics of the Atoh7 genes generate differences in the expression patterns of a set of genes that are regulated by Atoh7 in a dose-dependent manner, including those involved in neurite outgrowth and growth cone migration. In summary, this study shows how highly conserved regulatory elements are put to use in mediating non-conserved functions and creating interspecies neuronal diversity (Skowronska-Krawczyk, 2009).

Given the crucial role of Atoh7 in the ontogenesis of the vertebrate retina, understanding how its gene is regulated should provide key insights into the transcriptional networks that specify and integrate the RGC lineage within the retina developmental programme. This study shows how evolutionarily conserved non-coding sequences mediate both the conserved and species-specific transcriptional features of the Atoh7 gene. The Ngn2 protein maintains the ability to initiate the retina-specific expression of Atoh7 across distant species, but diverges in its binding profile to evolutionarily conserved regulatory elements. This study reveals how such interspecies variations in transcription factor binding cause variations in the activity of the Atoh7 promoter and how these variations may underlie phenotypic differences between species (Skowronska-Krawczyk, 2009).

The onset of Ngn2 and Atoh7 expression in overlapping domains coincide in the chick and mouse retinas, consistent with the co-expression of Ngn2 and Atoh7 in individual chick and mouse progenitor cells. The downregulation of Atoh7 expression in Ngn2GFP/GFP mice and its upregulation in chick retina overexpressing Ngn2 reveal that the positive regulation of Atoh7 by Ngn2 is evolutionarily conserved. This regulation correlates with the variable in vivo occupancy by Ngn2 of the Atoh7 promoter as a function of developmental stage. Although Ngn2 is expressed in many regions of the nervous system anlage, it does not bind the promoter in tissues that do not express Atoh7. In the chick retina, the K4 di- and tri-methylation of histone H3, a modification known to reflect transcriptional competence, increases in exact register with the kinetics of Atoh7 promoter activity. Likewise, the binding of Ngn2 and its positive effect are associated with chromatin remodelling of the Atoh7 promoter in the early retina. Surprisingly, despite conservation of the proximal and distal elements, Ngn2 binds the distal sequences in the mouse and the proximal sequences in the chick. In yeast, sequence conservation does not readily predict transcription factor binding sites across related species. This study extends to vertebrates the notion that gene regulation resulting from the pattern of species-specific transcription factor binding to highly conserved regulatory elements may be a cause of divergence between species (Skowronska-Krawczyk, 2009).

Although the interplay of bHLH proteins at the proximal E-boxes E1, E2 and E4 determines the spatio-temporal specificity of Atoh7 expression in the chick retina, cooperation between E1, E2, E4 and the conserved distal E-box E9 is required for full-strength promoter activity. Consistent with this notion, mutation of E9 in chick does not alter cell specificity despite a much decreased promoter strength. The finding that in the mouse retina, E9 sets both the strength of the promoter and its specificity, highlights how the activity of conserved elements depends on the cellular context and may vary between species. In the chick, both Ngn2 and Atoh7 bind the proximal E-boxes and Atoh7 also binds E9. Although the possibility cannot be excluded that E9 could mediate competition between Ngn2 and Atoh7, the binding profiles of Atoh7 and Ngn2 proteins suggest that in the early retina, Ngn2 activates transcription of Atoh7 through the proximal promoter, whereas Atoh7 mediates a positive feedback through E9. This feedback by Atoh7 is moderated by the negative effect that the Hes1 protein exerts upon the proximal promoter, thus keeping the rate of Atoh7 transcription at a low level in proliferating progenitors. The downregulation of Hes1 in Atoh7-expressing cells that exit the cell cycle coincides with the rapid upregulation of Atoh7. This upregulation is mediated by Atoh7 and Ngn2, which drive the promoter at peak activity through the combinatorial use of E2, E4 and E9. In Hes1-/- mice, precocious peripheral expansion of the Atoh7 expression domain takes place in the retina (Skowronska-Krawczyk, 2009).

Consistent with the dominant effect of E9, Ngn2 exclusively binds the distal region in mouse. The striking difference in the functional properties of E9 between mouse and chicken does not result from sequence variations; it might reflect epigenetic differences, as chromatin modifications correlate with the binding of bHLH proteins. The fact that Ngn2 expression is low in the mouse retina, whereas it is strongly upregulated in the chick retina, suggests the interesting possibility that the proximal and distal promoter regions are selected by Ngn2 in a dose-dependent manner. Recruitment of the protein on both the distal and proximal promoter regions in mouse P19 cells that overexpress Ngn2 is consistent with this notion. The distal and proximal E-boxes have different sequence identities and their affinity for Ngn2 may thus be sequence dependent, as shown for Atoh7. The finding that the chick Atoh7 promoter in the mouse, or the mouse Atoh7 promoter in the chick, displays the activity features of the host suggests, however, that the cellular context can influence transcription in the developing nervous system (Skowronska-Krawczyk, 2009).

Consistent with the role of E9 ascertained in this report, a 0.6 kb sequence encompassing the proximal E-boxes has no promoter activity in transgenic mice, whereas a 2.3 kb sequence that includes E8 and E9 is sufficient to recapitulate in full the endogenous Atoh7 expression in the E11.5 mouse retina (Skowronska-Krawczyk, 2009).

The maintenance of Atoh7 expression in Math5-/- mice rules out a positive feedback in this species. Thus, the interplay of Ngn2 and Atoh7 at the proximal promoter seen in the chick might not occur in the mouse retina. Instead, the Ngn2 protein, acting through the distal promoter, mediates the low expression of Atoh7 seen in mouse during the period of development when RGCs are produced. The residual, but significant, expression of Atoh7 in Ngn2GFP/GFP mice suggests that other transcription factors might intervene in the regulation of this gene. Pax6 is necessary for Atoh7 expression in the mouse, but cannot by itself control the spatio-temporal and cell cycle phase expression of Atoh7 (Riesenberg, 2009). In Xenopus, Pax6 binding sites within a distal enhancer are required for its enhancer activity (Willardsen, 2009). However, Pax6 alone is not sufficient to induce ectopic expression of Xenopus Atoh7. The fact that the distal enhancer in Xenopus does not require E8 and E9 [respectively E3 and E4 in Willardsen (2009) extends to lower vertebrates the notion that conserved E-boxes assume different roles in different species (Skowronska-Krawczyk, 2009).

There is no obvious abnormality of the GCL in Ngn2GFP/GFP mice, despite the downregulation of Atoh7. However, the possibility cannot be excluded that the GCL might be populated with other cell types, such as amacrine cells, or that different ratios of the numerous RGC subclasses might be produced in response to different levels of Atoh7 (Skowronska-Krawczyk, 2009).

This study reveals that highly conserved non-coding sequences mediate non-conserved interplays of bHLH proteins at the Atoh7 promoter. The proximal E-boxes E1, E2 and E4 mediate activation by Ngn2 and, in addition, the positive feedback by Atoh7 acting upon E9 reinforces Atoh7 expression during the first phase of chick retina development. Expression of Atoh7 is at least 10-fold higher in chick than in mouse early retina, where mouse Ngn2 effects a weak activation through the distal promoter. RGCs are massively produced in the chick retina and the ratio of RGCs to photoreceptors is ~25-fold higher in the avian than in the mouse retina. It is suggested that the much-enhanced expression of Atoh7 in the chick promotes the recruitment to the RGC lineage of a larger set of retinal progenitors (Skowronska-Krawczyk, 2009).

Later, at the time when the majority of RGCs exit the cell cycle and differentiate, the interaction of the Atoh7 protein at E2, E4 and E9 mediates a strong positive feedback. This occurs in the chick but not in the mouse retina, raising the question of why such a transient Atoh7 upregulation is needed to produce RGCs in the chick. Part of the answer might reside in the coherent set of genes that are expressed in newborn RGCs and are regulated by Atoh7 in a dose-dependent manner. Proteins encoded by Stmn2, Snap25, Robo2 and Ptn are protagonists in signalling pathways that link external stimuli to processes such as growth cone protrusion, axonal pathfinding and initial formation of synaptic contacts. The stathmin proteins regulate microtubule dynamics and Stmn2 is highly expressed during neuronal development and is enriched in growth cones. The Snap25 proteins are components of the synaptic vesicle exocytotic machinery and participate in neurite outgrowth. Robo2 plays a role in the axonal pathfinding of RGCs. The regulation of the corresponding genes by Atoh7 suggests that the protein might directly control the development of dendritic arbors and axons in newborn RGCs (Skowronska-Krawczyk, 2009).

Ramon y Cajal noted early on that the avian retina is the most complicated with respect to the morphology of the RGCs. Screens of the dendritic patterns of RGCs have revealed that whereas the large majority of RGCs are monostratified in the mouse retina, bi- and tristratification patterns predominate in the chicken retina. A recent report brought into focus a novel mechanism whereby dendritic stratification of RGCs is achieved (Mumm, 2006). In that study, in vivo time-lapse imaging was used to show that zebrafish RGCs display early patterns of dendritic outgrowth that are predictive of their final lamination, rather than lamination resulting from the pruning of initially exuberant arbors, as generally accepted. It is proposed that mouse RGCs might develop simpler dendritic patterns as a result of the low expression of neurite outgrowth-associated proteins (Skowronska-Krawczyk, 2009).

Proper differentiation of photoreceptors and amacrine cells depends on a regulatory loop between NeuroD and Six6

Timely generation of distinct neural cell types in appropriate numbers is fundamental for the generation of a functional retina. In vertebrates, the transcription factor Six6 is initially expressed in multipotent retina progenitors and then becomes restricted to differentiated retinal ganglion and amacrine cells. How Six6 expression in the retina is controlled and what are its precise functions are still unclear. To address this issue, bioinformatic searches and transgenic approaches were used in medaka fish (Oryzias latipes) to characterise highly conserved regulatory enhancers responsible for Six6 expression. One of the enhancers drove gene expression in the differentiating and adult retina. A search for transcription factor binding sites, together with luciferase, ChIP assays and gain-of-function studies, indicated that NeuroD, a bHLH transcription factor, directly binds an 'E-box' sequence present in this enhancer and specifically regulates Six6 expression in the retina. NeuroD-induced Six6 overexpression in medaka embryos promoted unorganized retinal progenitor proliferation and, most notably, impaired photoreceptor differentiation, with no apparent changes in other retinal cell types. Conversely, Six6 gain- and loss-of-function changed NeuroD expression levels and altered the expression of the photoreceptor differentiation marker Rhodopsin. In addition, knockdown of Six6 interfered with amacrine cell generation. Together, these results indicate that Six6 and NeuroD control the expression of each other and their functions coordinate amacrine cell generation and photoreceptor terminal differentiation (Conte, 2010).

Neurogenin and pancreas development

The notch signaling pathway is essential for the endocrine cell fate in various tissues including the enteroendocrine system of the gastrointestinal tract. Enteroendocrine cells are one of the four major cell types found in the gastric epithelium of the glandular stomach. To understand the molecular basis of enteroendocrine cell development, gene targeting in mouse embryonic stem cells has been used to derive an EGFP-marked null allele of the bHLH transcription factor, neurogenin 3 (ngn3). In ngn3-/- mice, glucagon secreting A-cells, somatostatin secreting D-cells, and gastrin secreting G-cells are absent from the epithelium of the glandular stomach, whereas the number of serotonin-expressing enterochromaffin (EC) cells is decreased dramatically. In addition, ngn3-/- mice display intestinal metaplasia of the gastric epithelium. Thus, ngn3 is required for the differentiation of enteroendocrine cells in the stomach and the maintenance of gastric epithelial cell identity (Lee, 2002).

The analysis in ngn3-/- mice suggests the existence of both ngn3-dependent and independent enteroendocrine cell lineages. Two models are proposed to illustrate how ngn3 might specify gastric enteroendocrine development. The first model suggests that ngn3 is needed initially for the proliferation of all endocrine cells, and subsequently required for the terminal differentiation of A cells (marked by glucagon), D cells (marked by somatostatin), and G cells (marked by glucagon), but not EC cells (marked by enterochromaffin). Thus, ngn3 deficiency leads to a smaller pool of enteroendocrine precursors from which EC-cells can differentiate, resulting in the observed reduction in the frequency of this cell type. The second model suggests that ngn3 is absolutely required for the specification of A-, D-, and G-cells, but not EC-cells, because the specification of EC-cells can also be orchestrated by factor X. Factor X could be regulated by effectors in the Notch-signaling pathway or by factors in other signaling pathways. Other neurogenin family members such as ngn1 and ngn2 are potential candidates for factor X that might also be involved in governing enteroendocrine cell specification. In conclusion, ngn3 has been shown to be essential for the specification of enteroendocrine cells in the stomach and for the maintenance of gastric epithelial cell identity (Lee, 2002).

Endocrine cells of the pancreas and the gastrointestinal tract derive from multipotent endodermal stem cells. The basic helix-loop-helix (bHLH) transcription factor neurogenin3 (ngn3) is required for the specification of the endocrine lineage in uncommitted progenitors in the developing pancreas. Expression and the function of ngn3 in the control of endocrine cell development has been examined in the intestinal and gastric epithelium. As in the pancreas, gastrointestinal endocrine cells derive from ngn3-expressing progenitors. Mice homozygous for a null mutation in ngn3 fail to generate any intestinal endocrine cells, and endocrine progenitor cells are lacking. The other main intestinal epithelial cell types differentiate properly. In contrast, in the glandular stomach, the differentiation of the gastrin- (G cells) and somatostatin (D cells)-secreting cells is impaired whereas serotonin- (enterochromaffin EC cells), histamine- (enterochromaffin-like ECL cells) and ghrelin (X/A cells)-expressing cells are still present. Thus, ngn3 is strictly required for endocrine cell fate specification in multipotent intestinal progenitor cells, whereas gastric endocrine development is both ngn3 dependent and independent (Jenny, 2002).

All pancreatic endocrine cells, producing glucagon, insulin, somatostatin, or PP, differentiate from Pdx1+ progenitors that transiently express Neurogenin3. To understand whether the competence of pancreatic progenitors changes over time, transgenic mice were generated expressing a tamoxifen-inducible Ngn3 fusion protein (Ngn3-ERTM) under the control of the pdx1 promoter and the transgene was backcrossed into the ngn3-/- background, devoid of endogenous endocrine cells. Early activation of Ngn3-ERTM almost exclusively induced glucagon+ cells, while depleting the pool of pancreas progenitors. As from E11.5, Pdx1+ progenitors became competent to differentiate into insulin+ and PP+ cells. Somatostatin+ cells were generated from E14.5, while the competence to make glucagon+ cells was dramatically decreased. Hence, pancreas progenitors, similar to retinal or cortical progenitors, go through competence states that each allow the generation of a subset of cell types. The progenitors acquire competence to generate late-born cells in a mechanism that is intrinsic to the epithelium. Thus, temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types (Johansson, 2007).

Neurogenin3 is sufficient for transdetermination of hepatic progenitor cells into neo-islets in vivo but not transdifferentiation of hepatocytes

The transcription factor Neurogenin3 (Ngn3) is required for islet-cell type specification. This study shows that hepatic gene transfer of Ngn3 transiently induces insulin in terminally differentiated hepatocytes but fails to transdifferentiate them, i.e., switch their lineage into islet cells. However, Ngn3 leads to long-term diabetes reversal in mice due to the emergence of periportal islet-like cell clusters. These neo-islets display glycemia-regulated insulin, beta-cell-specific transcripts, and an islet-specific transcription cascade, and they produce all four major islet hormones. They appear to arise from hepatic progenitor cells, most likely endoderm-derived oval cells. Thus, transfer of a single lineage-defining transcription factor, Ngn3, is sufficient to induce cell-lineage switching from a hepatic to an islet lineage in these progenitor cells, a process consistent with transdetermination, i.e, lineage switching in lineage-determined, but not terminally differentiated, cells. This paradigm of induced transdetermination of receptive progenitor cells in vivo may be generally applicable to therapeutic organogenesis for multiple diseases, including diabetes (Yechoor, 2009).

The SWI/SNF chromatin remodeling protein Brg1 is required for vertebrate neurogenesis and mediates transactivation of Ngn and NeuroD

Chromatin remodeling complexes play crucial roles in transcription and are implicated in processes including cell proliferation, differentiation and embryonic patterning. Brg1 is the catalytic subunit of the SWI/SNF chromatin remodeling complex and shows neural-enriched expression. Although early lethality of Brg1-null mice reflects its importance in embryogenesis, this phenotype has precluded further study of specific Brg1-dependent developmental processes. A requirement of Brg1 has been identified for both Xenopus primary neurogenesis and neuronal differentiation of mammalian P19 embryonic carcinoma cells. In Xenopus, loss of Brg1 function does not affect neural induction or neural cell fate determination. However, the Sox2-positive, proliferating neural progenitor cell population is expanded, and expression of a terminally differentiated neuronal marker, N-tubulin, is diminished upon loss of Brg1 activity, suggesting that Brg1 is required for neuronal differentiation. The ability of the bHLH transcription factors Ngnr1 and NeuroD to drive neuronal differentiation was also abolished by loss of Brg1 function, indicating that Brg1 is essential for the proneural activities of Ngnr1 and NeuroD. Consistent with this, dominant-negative interference with Brg1 function in P19 cells suppresses neuronal differentiation promoted by NeuroD2, showing the requirement of Brg1 for neuronal differentiation is conserved in mammalian cells. Finally, Brg1 physically associates with both Ngnr1 and NeuroD and interference with Brg1 function blocks Neurogenin3- and NeuroD2-mediated reporter gene transactivation. Together, these results demonstrate that Brg1 (and by inference the SWI/SNF complex) is required for neuronal differentiation by mediating the transcriptional activities of proneural bHLH proteins (Seo, 2005a).

Geminin regulates neuronal differentiation by antagonizing Brg1 activity: Gem blocks Ngn/NeuroD and Brg1 interaction

Precise control of cell proliferation and differentiation is critical for organogenesis. Geminin (Gem) has been proposed to link cell cycle exit and differentiation as a prodifferentiation factor and plays a role in neural cell fate acquisition. The SWI/SNF chromatin-remodeling protein Brg1 has been identified as an interacting partner of Gem. Brg1 has been implicated in cell cycle withdrawal and cellular differentiation. Surprisingly, Gem was found to antagonize Brg1 activity during neurogenesis to maintain the undifferentiated cell state. Down-regulation of Gem expression normally precedes neuronal differentiation, and gain- and loss-of-function experiments in Xenopus embryos and mouse P19 cells demonstrate that Gem is essential to prevent premature neurogenesis. Misexpression of Gem also suppresses ectopic neurogenesis driven by Ngn and NeuroD. Gem's activity to block differentiation depends upon its ability to bind Brg1 and could be mediated by Gem's inhibition of proneural basic helix-loop-helix (bHLH) Brg1 interactions required for bHLH target gene activation. The data demonstrate a novel mechanism of Gem activity, through regulation of SWI/SNF chromatin-remodeling proteins, and indicate that Gem is an essential regulator of neurogenesis that can control the timing of neural progenitor differentiation and maintain the undifferentiated cell state (Seo, 2005b).

Ngn and NeuroD proteins interact directly with Brg1 and require Brg1 activity to activate target gene transcription. Since Gem interacts with Brg1, whether Gem could form a higher-order complex with Brg1 and bHLH proteins or could compete with bHLH proteins for Brg1 binding was examined. In transfection and co-IP experiments, no bHLH-Gem interactions were found, while association of Brg1 and Ngn/NeuroD was observed. Therefore, it is unlikely that Gem forms a complex together with bHLH factors and Brg1. Instead, overexpression of wild-type Gem can inhibit the association of Ngn and NeuroD with Brg1. The ability of Gem to block Ngn/NeuroD and Brg1 interaction is strongly reduced for GemDelta(BD), indicating that this activity requires an intact Brg1-binding motif. In addition, while wild-type Gem can suppress the ability of Ngn3 to activate target gene transcription, GemDelta(BD) cannot. These data suggest that Gem can suppress neuronal differentiation, at least partly, by blocking association of proneural bHLHs and Brg1, and thus preventing transcriptional activation of target genes (Seo, 2005b).

Pancreatic islet endocrine cells arise during development from precursors expressing neurogenin 3 (Ngn3). As a population, Ngn3+ cells produce all islet cell types, but the potential of individual Ngn3+ cells, an issue central to organogenesis in general and to in vitro differentiation towards cell-based therapies, has not been addressed. In vivo clonal analyses in mice was performed to study the proliferation and differentiation of very large numbers of single Ngn3+ cells using MADM, a genetic system in which a Cre-dependent chromosomal translocation labels, at extremely low mosaic efficiency, a small number of Ngn3+ cells. Large numbers of progeny were scored, arising from single Ngn3+ cells. In newborns, labeled islets frequently contained just a single tagged endocrine cell, indicating for the first time that each Ngn3+ cell is the precursor of a single endocrine cell. In adults, small clusters of two to three Ngn3+ progeny were detected, but all expressed the same hormone, indicating a low rate of replication from birth to adult stages. A model is proposed whereby Ngn3+ cells are monotypic (i.e. unipotent) precursors, and this paradigm was used to refocus ideas on how cell number and type must be regulated in building complete islets of Langerhans (Desgraz, 2009).

Periventricular notch activation and asymmetric Ngn2 and Tbr2 expression in pair-generated neocortical daughter cells

To understand the cellular and molecular mechanisms regulating cytogenesis within the neocortical ventricular zone, the spatiotemporal expression patterns of Ngn2 and Tbr2 were examined at high resolution. Individually DiI-labeled daughter cells were tracked from their birth in slice cultures and immunostained for Ngn2 and Tbr2. Both proteins were initially absent from daughter cells during the first 2 h. Ngn2 expression was then initiated asymmetrically in one of the daughter cells, with a bias towards the apical cell, followed by a similar pattern of expression for Tbr2, which was found to be a direct target of Ngn2. How this asymmetric Ngn2 expression is achieved is unclear, but gamma-secretase inhibition at the birth of daughter cells resulted in premature Ngn2 expression, suggesting that Notch signaling in nascent daughter cells suppresses a Ngn2-Tbr2 cascade. Many of the nascent cells exhibited pin-like morphology with a short ventricular process, suggesting periventricular presentation of Notch ligands to these cells (Ochiai, 2009).

The gene cascade Fezf1/Fezf2 -> Hes5 -> neurogenin 2 regulates the expression of Tbr2 and controls differentiation of the neural stem cells into the intermediate progenitors

Precise control of neuronal differentiation is necessary for generation of a variety of neurons in the forebrain. However, little is known about transcriptional cascades, which initiate forebrain neurogenesis. This study shows that zinc finger genes Fezf1 and Fezf2, homologs of Drosophila Earmuff, that encode transcriptional repressors, are expressed in the early neural stem (progenitor) cells and control neurogenesis in mouse dorsal telencephalon. Fezf1- and Fezf2-deficient forebrains display upregulation of Hes5 and downregulation of neurogenin 2, which is known to be negatively regulated by Hes5. FEZF1 and FEZF2 bind to and directly repress the promoter activity of Hes5. In Fezf1- and Fezf2-deficient telencephalon, the differentiation of neural stem cells into early-born cortical neurons and intermediate progenitors is impaired. Loss of Hes5 suppresses neurogenesis defects in Fezf1- and Fezf2-deficient telencephalon. These findings reveal that Fezf1 and Fezf2 control differentiation of neural stem cells by repressing Hes5 and, in turn, by derepressing neurogenin 2 in the forebrain (Shimizu, 2010).

An important question about neural development is how the differentiation of neural stem cells is precisely controlled in the forebrain. Asymmetric cell division of neural stem cells is thought to contribute to the differentiation of neural stem cells (radial glial cells) into either neurons or intermediate progenitors. Recent reports suggest that the orientation of stem cell division in the VZ might not directly control which of the two asymmetrically divided cells becomes a stem cell and which of the two becomes a differentiated cell. Although asymmetric centrosome inheritance during the asymmetric cell divisions was reported to play a role in the maintenance of the neural stem cells, it is not clear what factors determine cell fate. It is known that oscillation of Hes1 and neurogenin 2 expression in the telencephalic VZ plays an important role in maintenance of the neural stem cells and that stabilization of neurogenin 2 expression supports differentiation of the neural stem cells. However, it is still not understood what factor(s) control stabilization of neurogenin 2 expression and what factor(s) induce their differentiation. These reports imply that, besides asymmetric distribution of cell-fate determinants, extrinsic and intrinsic factors might bias the neural stem cells toward differentiation. Notch signaling plays an essential role in maintenance of the neural stem cells. Thus, regulators of Notch signaling and its downstream effectors might be involved in the decision as to whether to be a stem cell or a differentiated cell. This report demonstrates that Fezf1 and Fezf2, which are expressed in the neural stem cells at the beginning of mouse cortical development, inhibit the expression of the Notch effector Hes5 and promote differentiation of the neural stem cells. The findings suggest that Fezf1 and Fezf2 function as intrinsic factors to bias the neural stem cells toward differentiation (Shimizu, 2010).

Expression of fezf2 takes place in the radial glial cells of the telencephalic VZ of adult zebrafish (Berberoglu, 2009). fezf2 is also expressed in the neural progenitors and neurons in the pre-optic region and hypothalamus of the adult zebrafish brains (Berberoglu, 2009). In zebrafish, neurogenesis continuously takes place in adult brains. It is possible that fezf2 might control differentiation of the neural stem cells in the adult zebrafish forebrain as Fezf1 and Fezf2 do during early mouse cortical development (Shimizu, 2010).

Expression of Fezf1 or Fezf2 repressed both NOTCH1-dependent and NOTCH1-independent Hes5 promoter activity, but did not repress the Hes1 promoter or the artificial CBS-dependent promoter. Hes1 expression was not upregulated in the telencephalon of Fezf1-/-Fezf2-/- mice. Furthermore, FEZF1 and FEZF2 bound to the Hes5 promoter in vivo in the mouse forebrain. All of these data indicate that FEZF1 and FEZF2, rather than inhibit Notch cytoplasmic signaling, specifically bind to and directly repress the Hes5 promoter. FEZF1 and FEZF2 have an EH1 repressor motif. The data support the assertion that FEZF1 and FEZF2 function as transcriptional repressors and repress the Hes5 promoter at least during early cortical development. Hes5 deficiency suppressed neurogenesis defects in Fezf1-/-Fezf2-/- telencephalon, supporting the hypothesis that Fezf1 and Fezf2 suppress the expression of Hes5 and thereby control differentiation of the neural stem cells (Shimizu, 2010).

FEZF1 and FEZF2 repress only Hes5. Hes1 and Hes5 function redundantly in the maintenance of neural stem cells in the mouse central nervous system, whereas only Hes1 is reported to exhibit oscillatory expression in the neural stem cells, suggesting that Hes1 and Hes5 might have distinct roles in neurogenesis. Previous research has revealed that oscillation of Hes1 is involved in the maintenance of neural stem cells and, in the current study, it is speculated that Hes5 plays a different role in neurogenesis; specifically, it is proposed that Hes5, in contrast to Hes1, sets up the overall expression levels of Hes genes and neurogenin 2 in the forebrain. Once Fezf1 and Fezf2 expression exceeds a threshold, FEZF1 and FEZF2 might repress Hes5 expression, stabilize neurogenin 2 expression and thereby bias the neural stem cells toward differentiation (Shimizu, 2010).

The Drosophila homolog of Fezf1/2 (dFezf or Earmuff) has been shown to restrict the developmental potential of intermediate progenitors by negatively regulating Notch signaling. Although the mechanism by which dFezf represses Notch signaling is unknown, Fezf family genes function to negatively regulate Notch signaling in both vertebrates and invertebrates (Shimizu, 2010).

Fezf1 and Fezf2 function to repress the caudal diencephalon fate and their function is involved in proper rostro-caudal patterning of the forebrain (see Jeong, 2007). The prospective telencephalon domain is already smaller in Fezf1-/-Fezf2-/- mouse embryos than in the wild type at E9.5, before neurogenesis is initiated in the telencephalon. Therefore, the defect in rostro-caudal patterning is attributable to reduction of the telencephalon domain. In addition, Fezf2-/- or Fezf1-/-Fezf2-/- telencephalon lacks layer-V subcerebral projection neurons. Hes5 deficiency did not suppress the defects in rostro-caudal patterning of the forebrain or specification of layer-V neurons in Fezf1-/-Fezf2-/- forebrains. Therefore, Fezf1/2-mediated downregulation of Hes5 is not involved in the rostro-caudal patterning of the forebrain and the specification of layer-V neurons. Fezf1 and/or Fezf2 probably control genes other than Hes5 to elicit these functions (Shimizu, 2010).

Fezf1-/-Fezf2-/- telencephalon exhibited reduced formation of early-born neurons such as SP neurons and CR cells. A birthdate analysis revealed that the reduction of SP neurons and CR cells was not due to mis-specification of these neurons to other types of neurons. The data suggest that generation of the neural stem cells into SP neurons and CR cells is impaired in Fezf1-/-Fezf2-/- telencephalon. This finding is consistent with a reduction of differentiated (anti-neuron-specific βIII tubulin antibody TUJ1+) neurons in the Fezf1-/-Fezf2-/- telencephalon at E10.5, when subplate (SP) neurons and Cajal-Retzius (CR) cells were born in the VZ. Hes5 deficiency rescued neurogenin 2 expression at E10.5 and the generation of SP neurons and CR cells in Fezf1-/-Fezf2-/- telencephalon, indicating that Fezf1- and/or Fezf2-mediated repression of Hes5 plays an important role in the generation of these early-born cortical neurons. It is reported that formation of CR cells in the choroid plexus region, near the cortical hem, is controlled by a Hes-neurogenin cascade but that the Notch signal-mediated lateral inhibition is not involved in regulation of the Hes-neurogenin cascade in the CR cell development. Fezf1 and Fezf2 are expressed in the dorsomedial telencephalon. The current data suggest that Fezf1 and Fezf2 might control the development of CR cells by regulating Hes5 and neurogenin 2 expression in the choroid plexus domain (Shimizu, 2010). Fezf1-/-Fezf2-/- telencephalon had normal upper-layer (layer II, III) neurons but displayed a reduction of layer-IV neurons. There are two plausible explanations for this finding: Fezf1 and Fezf2 regulate the specification of layer-IV neurons or Fezf1 and Fezf2 control the generation of layer-IV neurons. Neither Fezf1 nor Fezf2 is expressed in differentiated layer-IV neurons, but both are expressed in their progenitors (neural stem cells or intermediate progenitors). Layer-IV neurons are normally born (differentiated) from E13.5 through E15.5. Birthdate analysis indicated that Fezf1-/-Fezf2-/- telencephalon contained a reduced number of Rorβ-positive neurons that were born at E13.5, suggesting that Fezf1 and Fezf2 control the generation of layer-IV neurons either from the neural stem cells or the intermediate progenitors. In Fezf1-/-Fezf2-/- telencephalon, differentiation of the neural stem cells into the TBR2+ intermediate progenitors was impaired. Tbr2 is an essential regulator of the intermediate progenitors and is directly regulated by neurogenin 2. These data suggest that the gene cascade Fezf1/Fezf2 -> Hes5 -> neurogenin 2 regulates the expression of Tbr2 and controls differentiation of the neural stem cells into the intermediate progenitors. The reduction of the TBR2+ intermediate progenitors in the Fezf1-/-Fezf2-/- telencephalon might contribute to a reduction of layer-IV neurons. Consistent with this idea, Hes5 deficiency rescued the development of TBR2+ intermediate progenitors as well as layer-IV neurons in Fezf1-/-Fezf2-/- telencephalon. It is reported that TBR1+ layer-VI neurons are increased in Fezf2-/- telencephalon, suggesting the transfate of layer-V to layer-VI neurons. However, they were not increased in Fezf1-/-Fezf2-/- telencephalon, implying that the gene cascade Fezf1/Fezf2 -> Hes5 ->neurogenin 2 controls the generation of layer-VI neurons. Future studies will clarify these issues (Shimizu, 2010).

In summary, FEZF1 and FEZF2 are transcriptional repressors that repress Hes5 expression and subsequently activate neurogenin expression. The Fezf1/Fezf2 -> Hes5 -> neurogenin 2 gene cascade controls differentiation of the neural stem cells into neurons or intermediate progenitors and contributes to the generation of a variety of neurons in the forebrain (Shimizu, 2010).

Regulation of Neurogenin transcription

The basic helix-loop-helix transcription factor Neurogenin2 (NGN2) is expressed in distinct populations of neural progenitor cells within the developing central and peripheral nervous systems. Transgenic mice containing ngn2/lacZ reporter constructs were used to study the regulation of ngn2 in the developing spinal cord. ngn2/lacZ transgenic embryos containing sequence found 5' or 3' to the ngn2 coding region express lacZ in domains that reflect the spatial and temporal expression profile of endogenous ngn2. A 4.4-kb fragment 5' of ngn2 is sufficient to drive lacZ expression in the ventral neural tube, whereas a 1.0-kb fragment located 3' of ngn2 directs expression to both dorsal and ventral domains. Persistent beta-gal activity reveals that the NGN2 progenitor cells in the dorsal domain give rise to a subset of interneurons that send their axons to the floor plate, and the NGN2 progenitors in the ventral domain give rise to a subset of motor neurons. A discrete element has been identified that is required for the activity of the ngn2 enhancer specifically in the ventral neural tube. Thus, separable regulatory elements that direct ngn2 expression to distinct neural progenitor populations have been defined (Simmons, 2001).

This study examined how genetic pathways that specify neuronal identity and regulate neurogenesis interface in the vertebrate neural tube. Expression of the proneural gene Neurogenin2 (Ngn2) in the ventral spinal cord results from the modular activity of three enhancers active in distinct progenitor domains, suggesting that Ngn2 expression is controlled by dorsoventral patterning signals. Consistent with this hypothesis, Ngn2 enhancer activity is dependent on the function of Pax6, a homeodomain factor involved in specifying the identity of ventral spinal cord progenitors. Moreover, Ngn2 is required for the correct expression of Pax6 and several homeodomain proteins expressed in defined neuronal populations. Thus, neuronal differentiation involves crossregulatory interactions between a bHLH-driven program of neurogenesis and genetic pathways specifying progenitor and neuronal identity in the spinal cord (Scardigli, 2001).

Ngn2 is involved in crossregulatory interactions with homeodomain proteins involved in neuronal fate specification in the ventral spinal cord. In one direction, Ngn2 expression is driven by distinct enhancers that are active at different dorsoventral levels and that depend to various degrees on Pax6 function. In the other direction, Ngn2 activity is itself required for the proper expression of homeodomain proteins in progenitor domains and neuronal populations throughout the ventral spinal cord. The Ngn2 enhancers characterized in this study are active in progenitor domains that are restricted along the DV axis of the spinal cord, suggesting that Ngn2 expression may be regulated by Shh-dependent pathways that establish the DV positional identity of ventral progenitors. In support of this idea, the three Ngn2 enhancers examined are dependent to various degrees on the function of Pax6, a gene repressed by Shh in the ventral spinal cord, for the establishment of their distinct DV domains of activity. In the absence of Pax6, the activity of these enhancers is reduced or abolished in their normal domains, and is expanded to ectopic sites. Loss of Ngn2 expression and loss of E3 enhancer activity in the dorsal spinal cord of Sey embryos mutant for Pax6 is likely to reflect a function for Pax6 in this region of the neural tube, where it is normally expressed at low levels. Together, these data raise the intriguing possibility that Pax6 itself defines the DV position of Ngn2 enhancer activity (Scardigli, 2001).

The specification of neural progenitors involves the parallel activation of several genetic programs -- a program of neurogenesis that controls the selection of neural progenitors and their commitment to differentiation, and a program specifying the identity of progenitors and their neuronal phenotype. A great deal of evidence supports the idea that these genetic programs do not run independently. In particular, bHLH genes have been shown to participate in both the determination of a generic neural fate and the specification of neuronal identities. Ngn2 is required for the correct expression of a number of homeodomain proteins expressed in ventral spinal cord progenitors (Pax6, Nkx2.2) and neurons (Lim1/2, En1, Chx10, Hb9, Isl1). The phenotype of MNs has been examined to gain further insights into Ngn2 function in the spinal cord. The MN defect in Ngn2 mutants differs from the respecification of MNs observed in mutations previously shown to interfere with MN development. For example, mutations in Pax6 and Nkx6.1 lead to a respecification of MNs into V3 and V1 interneurons, respectively, while a mutation in Hb9 results in the transient acquisition by MNs of features of V2 interneurons. In contrast, there is no evidence that Ngn2 mutant MN progenitors, which do give rise to postmitotic neurons as evidenced by the expression of the neuronal markers SCG10 and neurofilament, acquire an alternative interneuron fate, as evidenced by the observations that expression levels of markers for interneurons of the V3 (Nkx2.2), V2 (Chx10), and V1 (En1) classes are reduced rather than ectopically expressed in the absence of Ngn2. Other observations strongly suggest that the MN fate is maintained in the absence of Ngn2. In particular, Ngn2 mutant MNs project their axons to ventral roots, and HB9 and Isl1 are expressed, albeit at very reduced levels, in many Ngn2 mutant MNs at E10.5. Thus, although the Ngn2 mutation results in an abnormal expression of MN-specific regulatory genes, this mutant phenotype argues against an instructive role for Ngn2 in the specification of MN identity. This idea is further supported by the observation that Ngn2 is expressed broadly in the ventral spinal cord, and its mutation affects other neuronal populations in addition to MNs, including Lim1/2+ dorsal interneurons and En1+ and Chx10+ ventral interneurons (Scardigli, 2001).

Therefore, rather than having a specific role in MN or interneuron fate specification, Ngn2 appears to be more generally required in different progenitor domains for the expression of homeodomain proteins participating in various subtype-specific programs of differentiation. This suggests that Ngn2 may act as a necessary but only permissive component of pathways specifying neuronal fates in the spinal cord. This function is clearly different from that of bHLH genes in other tissues such as the retina, where mutations in Mash1, NeuroD, Math3, and Math5 all result in changes in ratios of the different retinal cell types, suggestive of instructive roles for these genes in specification of neuronal identities (Scardigli, 2001).

Expression of the proneural gene Neurogenin2 is controlled by several enhancer elements, with the E1 element active in restricted progenitor domains in the embryonic spinal cord and telencephalon that express the homeodomain protein Pax6. Pax6 function is both required and sufficient to activate this enhancer, and one evolutionary conserved sequence in the E1 element is identified with high similarity to a consensus Pax6 binding site. This conserved sequence binds Pax6 protein with low affinity both in vitro and in vivo, and its disruption results in a severe decrease in E1 activity in the spinal cord and in its abolition in the cerebral cortex. The regulation of Neurogenin2 by Pax6 is thus direct. Pax6 is expressed in concentration gradients in both spinal cord and telencephalon. The E1 element is activated only by high concentrations of Pax6 protein, and this requirement explains the restriction of E1 enhancer activity to domains of high Pax6 expression levels in the medioventral spinal cord and lateral cortex. By modifying the E1 enhancer sequence, it is also shown that the spatial pattern of enhancer activity is determined by the affinity of its binding site for Pax6. Together, these data demonstrate that direct transcriptional regulation accounts for the coordination between mechanisms of patterning and neurogenesis. They also provide evidence that Pax6 expression gradients are involved in establishing borders of gene expression domains in different regions of the nervous system (Scardigli, 2003).

A striking finding of this study is that the same mechanism is employed to control the expression of Ngn2 in progenitor domains located in two distant regions of the embryonic CNS, the ventral spinal cord and the dorsal telencephalon. Similarities in the molecular mechanisms that pattern the spinal cord and telencephalon along their dorsoventral axis have been noted before, and include common inductive signals such as Sonic Hedgehog and bone morphogenetic proteins, related intrinsic determinants, including HD proteins of the Pax and Nkx families, and bHLH proteins of the Mash and Ngn families, and in particular the establishment by Pax6 of boundaries between adjacent progenitor domains, through cross-regulatory interactions with the HD proteins Nkx2.2 in the spinal cord, and Nkx2.1 and Gsh2 in the telencephalon. The activity of the E1 enhancer in both spinal cord and telencephalon thus probably reflects a common role of Pax6 in these two territories. It must be noted however, that E1 is not active in all domains of high Pax6 expression [e.g., the retina), suggesting that regional determinants may act as co-factors to constrain Pax6 function and restrict E1 activity along the anteroposterior axis of the neural tube (Scardigli, 2003).

The generation of neurons by progenitors in the embryonic nervous system involves two distinct processes: the commitment of multipotent progenitors to a neuronal fate, resulting in their differentiation into neurons, and the specification of progenitor identity, resulting in the differentiation of neurons of a particular subtype. A number of studies suggest that these two processes are coupled at several levels. (1) Proneural bHLH genes, the major regulators of neuronal commitment in multipotent progenitors, are also involved in the specification of neuronal identity. In particular, proneural genes have been shown to control some aspects of the neuronal phenotype, such as the neurotransmission profile, through the regulation of downstream HD genes that directly activate genes encoding biosynthetic enzymes for neurotransmitters. (2) The regulation of the proneural genes themselves appears to be intimately linked with the regionalization of the neural tube, as these genes are expressed in restricted neuroepithelial domains with well-defined dorsoventral borders. Some of the genes that are involved in partitioning the neuroepithelium in dorsoventral progenitor domains have been shown to control the expression of proneural genes in these territories. For example, the HD protein Phox2b acts as a patterning gene to specify the identity of branchiomotor neuron progenitors in the hindbrain, and it simultaneously promotes the neuronal differentiation of these progenitors by upregulating the expression of the proneural genes Ngn2 and Mash1. A control of proneural gene expression by neural patterning genes has also been reported in Drosophila where the selector-like gene pannier regulates the notal pattern, and is the only factor to directly activate AS-C genes. Thus instances in which patterning genes control the expression of proneural genes are likely to be a general feature of neural development in both invertebrates and vertebrates (Scardigli, 2003).

This work provides the first demonstration that a proneural gene is directly regulated by a patterning gene in vertebrates, suggesting that neural patterning and neurogenesis may generally be tightly linked. It is likely that multiple patterning genes are involved in the generation of the complex expression patterns of proneural genes. Indeed, Pax6 is essential for the regulation of only one of the four known enhancer elements of Ngn2. Recent work suggests that in Drosophila, regulators of proneural genes act hierarchically rather than in a combinatorial manner, so that the number of direct transcriptional activators is actually very small. Further studies are necessary to determine whether this holds true for vertebrate proneural genes (Scardigli, 2003).

The basic Helix-Loop-Helix gene neurogenin1 (ngn1) is expressed in a complex pattern in the neural plate of zebrafish embryos, demarcating the sites of primary neurogenesis. The ngn1 locus was dissected to identify cis-regulatory regions that control this expression. Two upstream elements have been isolated that drive expression in precursors of Rohon-Beard sensory neurons and hindbrain interneurons and in clusters of neuronal precursors in the anterior neural plate, respectively. A third regulatory region mediates later expression. Thus, regulatory sequences with temporally and spatially distinct activities control ngn1 expression in primary neurons of the zebrafish embryo. These regions are highly similar to 5' sequences in the mouse and human ngn1 gene, suggesting that amniote embryos, despite lacking primary neurons, utilize related mechanism to control ngn1 expression (Blader, 2003).

Two regulatory sequences in the zebrafish ngn1 locus have been identified that mediate expression in the neural plate in patterns similar to that of the endogenous ngn1 gene. An upstream element drives reporter gene expression in the lateral stripes of the posterior neural plate (lateral stripe element, LSE) including the Rohon Beard sensory and hindbrain reticulospinal neuron precursors. The second element (anterior neural plate element, ANPE) controls expression in the anterior neural plate (Blader, 2003).

Differentiation of Rohon Beard neurons requires BMP2b (Swirl) signaling. It has not been clear whether BMPs act at the level of ngn1 expression, or at a later step of Rohon Beard cell differentiation. The current data suggest that the LSE could be a target of BMP2b (Swirl) signaling. LSE-dependent transgene expression in interneurons of the hindbrain is not abolished but rather expands in the mutant. Hence, the two LSE-dependent expression domains are affected differently by lack of BMP2b (Swirl) signaling. Analysis of mutants with varying levels of BMP signaling suggests that BMPs act on both sensory neurons and interneurons. Depending on the residual levels of BMP signaling, however, differentiation of either cell type can be abolished or expanded. The spatial and temporal activity of the LSE in the gastrula and early neurula of wildtype embryos and the dependence of its activity on BMP2b (Swirl) signaling are consistent with the LSE being a target of BMP signaling that senses and integrates differing levels of BMPs (Blader, 2003).

her3 encodes a zebrafish bHLH protein of the Hairy-E(Spl) family. During embryogenesis, the gene is transcribed exclusively in the developing central nervous system, according to a fairly simple pattern that includes territories in the mesencephalon/rhombencephalon and the spinal cord. In all territories, the her3 transcription domain encompasses regions in which neurogenin 1 (neurog1) is not transcribed, suggesting regulatory interactions between the two genes. Indeed, injection of her3 mRNA leads to repression of neurog1 and to a reduction in the number of primary neurons, whereas her3 morpholino oligonucleotides cause ectopic expression of neurog1 in the rhombencephalon. Fusions of Her3 to the transactivation domain of VP16 and to the repression domain of Engrailed show that Her3 is indeed a transcriptional repressor. Dissection of the Her3 protein reveals two possible mechanisms for transcriptional repression: one mediated by the bHLH domain and the C-terminal WRPW tetrapeptide; and the other involving the N-terminal domain and the orange domain. Gel retardation assays suggest that the repression of neurog1 transcription occurs by binding of Her3 to specific DNA sequences in the neurog1 promoter. Interrelationships of her3 with members of the Notch signalling pathway have been examined by the Gal4-UAS technique and mRNA injections. The results indicate that Her3 represses neurog1 and, probably as a consequence of the neurog1 repression, deltaA, deltaD and her4. Moreover, Her3 represses its own transcription as well. Surprisingly, and in sharp contrast to other members of the E(spl) gene family, transcription of her3 is repressed rather than activated by Notch signalling (Hans, 2004).

During vertebrate retinogenesis, seven classes of cells are specified from multipotent progenitors. To date, the mechanisms underlying multipotent cell fate determination by retinal progenitors remain poorly understood. The Foxn4 winged helix/forkhead transcription factor is shown to be expressed in a subset of mitotic progenitors during mouse retinogenesis. Targeted disruption of Foxn4 largely eliminates amacrine neurons and completely abolishes horizontal cells, while overexpression of Foxn4 strongly promotes an amacrine cell fate. These results indicate that Foxn4 is both necessary and sufficient for commitment to the amacrine cell fate and is nonredundantly required for the genesis of horizontal cells. Furthermore, evidence is provided that Foxn4 controls the formation of amacrine and horizontal cells by activating the expression of the retinogenic factors Math3, NeuroD1, and the Prospero-like transcription factor Prox1. These data suggest a model in which Foxn4 cooperates with other key retinogenic factors to mediate the multipotent differentiation of retinal progenitors (Li, 2004).

The molecular programs that specify progenitors in the dorsal spinal cord remain poorly defined. The homeodomain transcription factor Gsh2 is expressed in the progenitors of three dorsal interneuron subtypes, dI3, dI4 and dI5 neurons, whereas Gsh1 is expressed only in dI4 and dI5 progenitors. Mice lacking Gsh2 exhibit a selective loss of dI3 interneurons that is accompanied by an expansion of the dI2 progenitor domain. In Gsh2 mutant embryos, expression of the proneural bHLH protein Mash1 is downregulated in dI3 neural progenitors, with Mash1 mutants exhibiting a concordant reduction in dI3 neurons. Conversely, overexpression of Gsh2 and Mash1 leads to the ectopic production of dI3 neurons and a concomitant repression of Ngn1 expression. These results provide evidence that genetic interactions involving repression of Ngn1 by Gsh2 promote the differentiation of dI3 neurons from class A progenitors (Kirks, 2005).

Neurogenin 2 (Ngn2) is a proneural gene involved in neuronal differentiation and subtype specification in various regions of the nervous system. In the ventral midbrain, Ngn2 is expressed in a spatiotemporal pattern that correlates with the generation of mesencephalic dopaminergic (mesDA) neurons. Lack of Ngn2 impairs the development of mesDA neurons, such that less than half of the normal mesDA neuron number remain in Ngn2 mutant mice at postnatal stages. Analysis of Ngn2 mutant mice during mesDA neurogenesis show that medially located precursors are formed but are arrested in their differentiation at a stage when they have not yet acquired the characteristics of mesDA neuron precursors. Loss of Ngn2 function appears to specifically affect the generation of DA neurons; the development of other types of neurons within the ventral midbrain is unaltered. Ngn2 is the first example of a gene expressed in progenitors in the ventricular zone of the mesDA neuron domain that is essential for proper mesDA neuron differentiation, and whose loss of function causes impaired mesDA neurogenesis without other major abnormalities in the ventral midbrain (Andersson, 2006a).

The immediate-early (IE) genes Fos, Egr1 and Egr2 have been identified as transcriptional targets of brain derived neurotrophic factor (BDNF)/TrkB signaling in primary cortical neurons; the Fos serum response element area responds to BDNF/TrkB in a manner dependent on a combined C/EBP-Ebox element. The Egr1 and Egr2 promoters contain homologous regulatory elements. C/EBPα/β and NeuroD formed complexes in vitro and in vivo, and are recruited to all three homologous promoter regions. C/EBPα and NeuroD co-operatively activated the Fos promoter in transfection assays. Genetic depletion of Trk receptors led to impaired recruitment of C/EBPs and NeuroD in vivo, and elimination of Cebpa and Cebpb alleles reduced BDNF induction of Fos, Egr1 and Egr2 in primary neurons. Finally, defective differentiation of cortical dendrites, as measured by MAP2 staining, was observed in both compound Cebp and Ntrk knockout mice. Therefore this study identifed three IE genes as targets for BDNF/TrkB signaling, shows that C/EBPα and -β are recruited along with NeuroD to target promoters, and that C/EBPs are essential mediators of Trk signaling in cortical neurons. C/EBPs and Trks are required for cortical dendrite differentiation, consistent with Trks regulating dendritic differentiation via a C/EBP-dependent mechanism. Finally, this study indicates that BDNF induction of IE genes important for neuronal function depends on transcription factors (C/EBP, NeuroD) up-regulated during neuronal development, thereby coupling the functional competence of the neuronal cells to their differentiation (Calella, 2007).

Neurogenesis requires the coordination of neural progenitor proliferation and differentiation with cell-cycle regulation. However, the mechanisms coordinating these distinct cellular activities are poorly understood. This study demonstrates that a Cut-like homeodomain transcription factor family member, Cux2 (Cutl2), regulates cell-cycle progression and development of neural progenitors. Cux2 loss-of-function mouse mutants exhibit smaller spinal cords with deficits in neural progenitor development as well as in neuroblast and interneuron differentiation. These defects correlate with reduced cell-cycle progression of neural progenitors coupled with diminished NeuroD and p27Kip1 activity. Conversely, in Cux2 gain-of-function transgenic mice, the spinal cord is enlarged in association with enhanced neuroblast formation and neuronal differentiation, particularly with respect to interneurons. Furthermore, Cux2 overexpression induces high levels of NeuroD and p27Kip1. Mechanistically, it was discovered through chromatin immunoprecipitation assays that Cux2 binds both the NeuroD and p27Kip1 promoters in vivo, indicating that these interactions are direct. These results therefore show that Cux2 functions at multiple levels during spinal cord neurogenesis. Cux2 initially influences cell-cycle progression in neural progenitors but subsequently makes additional inputs through NeuroD and p27Kip1 to regulate neuroblast formation, cell-cycle exit and cell-fate determination. Thus this work defines novel roles for Cux2 as a transcription factor that integrates cell-cycle progression with neural progenitor development during spinal cord neurogenesis (Iulianella, 2008).

Pancreatic endocrine cells originate from precursors that express the transcription factor Neurogenin3 (Ngn3). Ngn3 expression is repressed by active Notch signaling. Accordingly, mice with Notch signaling pathway mutations display increased Ngn3 expression and endocrine cell lineage allocation. To determine how the Notch ligand Jagged1 (Jag1) functions during pancreas development, Jag1 was deleted in foregut endoderm and postnatal and embryonic endocrine cells and precursors were examined. Postnatal Jag1 mutants display increased Ngn3 expression, α-cell mass, and endocrine cell percentage, similar to the early embryonic phenotype of Dll1 and Rbpj mutants. However, in sharp contrast to postnatal animals, Jag1-deficient embryos display increased expression of Notch transcriptional targets and decreased Ngn3 expression, resulting in reduced endocrine lineage allocation. Jag1 acts as an inhibitor of Notch signaling during embryonic pancreas development but an activator of Notch signaling postnatally. Expression of the Notch modifier Manic Fringe (Mfng) is limited to endocrine precursors, providing a possible explanation for the inhibition of Notch signaling by Jag1 during mid-gestation embryonic pancreas development (Golson, 2009).

The proneural protein neurogenin 2 (NGN2) is a key transcription factor in regulating both neurogenesis and neuronal radial migration in the embryonic cerebral cortex. However, the co-factors that support the action of NGN2 in the cortex remain unclear. This study shows that the LIM-only protein LMO4 functions as a novel co-factor of NGN2 in the developing cortex. LMO4 and its binding partner nuclear LIM interactor (NLI/LDB1/CLIM2) interact with NGN2 simultaneously, forming a multi-protein transcription complex. This complex is recruited to the E-box containing enhancers of NGN2-target genes, which regulate various aspects of cortical development, and activates NGN2-mediated transcription. Correspondingly, analysis of Lmo4-null embryos shows that the loss of LMO4 leads to impairments of neuronal differentiation in the cortex. In addition, expression of LMO4 facilitates NGN2-mediated radial migration of cortical neurons in the embryonic cortex. These results indicate that LMO4 promotes the acquisition of cortical neuronal identities by forming a complex with NGN2 and subsequently activating NGN2-dependent gene expression (Asprer, 2011).

A feedback loop mediated by degradation of an inhibitor is required to initiate neuronal differentiation

Neuronal differentiation is regulated by proneural genes that promote neurogenesis and inhibitory mechanisms that maintain progenitors. This raises the question of how the up-regulation of proneural genes required to initiate neurogenesis occurs in the presence of such inhibition. Loss and gain of gene function, an interaction screen for binding partners, and biochemical analyses were carried out to uncover the regulation, developmental role, and mechanism of action of a ubiquitination adaptor protein, Btbd6a (BTB domain containing 6a). It was found that the proneural gene neurog1 up-regulates btbd6a, which in turn is required for up-regulation of neurog1. Btbd6a is an adaptor for the Cul3 ubiquitin ligase complex, and it binds to the transcriptional repressor Plzf (promyelocytic leukemia zinc finger). Btbd6a promotes the relocation of Plzf from nucleus to cytoplasm and targets Plzf for ubiquitination and degradation. plzfa is expressed widely in the neural epithelium; when overexpressed, it inhibits neurogenesis, and this inhibition is reversed by btbd6a. The antagonism of endogenous plzfa by btbd6a is required for neurogenesis, since the block in neuronal differentiation caused by btbd6a knockdown is alleviated by plzfa knockdown. These findings reveal a feedback loop mediated by degradation of an inhibitor that is essential for progenitors to undergo the transition to neuronal differentiation (Sobieszczuk, 2010).

The correct balance between the initiation of neurogenesis versus maintenance of neural progenitors is achieved by inhibitory mechanisms that limit the up-regulation of proneural gene expression. This study uncovered a novel feedback loop required for primary neurogenesis that is mediated by a ubiquitin adaptor protein, Btbd6a. It was found that the proneural gene neurog1 up-regulates expression of btbd6a, that Btbd6a decreases nuclear levels and promotes degradation of the transcriptional repressor Plzf, and that plzfa is widely expressed in the neural epithelium and inhibits neurog1 gene expression. Whereas knockdown of btbd6a leads to a major decrease in neurogenesis, its overexpression is sufficient to increase the amount of neurogenesis and to overcome the inhibition of neurogenesis by plzfa. The functional antagonism of plzfa by btbd6a is essential for neuronal differentiation, since plzfa knockdown alleviates the block in neurogenesis that occurs following knockdown of btbd6a. The up-regulation of neurog1 gene expression required for primary neurogenesis is thus enabled by positive feedback in which a widely expressed inhibitor is targeted for degradation (Sobieszczuk, 2010).

Previous studies have revealed other feedback loops that promote neurogenesis. One type of mechanism involves the inhibition of a repressor through binding; for example, in the vertebrate CNS, where proneural genes up-regulate expression of Hes6, which by heterodimerizing with Hes1 prevents it from inhibiting the expression and activity of proneural proteins. The regulatory logic of the role of Btbd6a and Plzfa is similar to this cascade, except that it involves targeted degradation of an inhibitor rather than formation of an inactive complex. As Btbd6a overexpression leads to a greater increase in neurogenesis than plzfa knockdown, Btbd6a may also target the degradation of another inhibitory factor. The widespread expression of an inhibitor of proneural gene expression that is itself inhibited or degraded downstream from proneural genes has two consequences. First, it sets a threshold to ensure that the initiation of differentiation is confined to cells in which sufficient proneural activity has been achieved. Second, once there is enough proneural activity to achieve positive feedback, this will underlie a discrete switch from a progenitor to neuronal differentiation. The degradation of an inhibitor may make such a progression less reversible than mechanisms involving binding and competition of activators and inhibitors (Sobieszczuk, 2010).

Although the genes involved are not homologous, the roles of plzfa and btbd6a in primary neurogenesis are similar to Tramtrack and Phyllopod in Drosophila. Tramtrack encodes a BTB zinc finger transcriptional repressor that inhibits specific fates of photoreceptor and sensory organ cells. This inhibition is relieved by up-regulation of Phyllopod, which acts as an adaptor to bring Tramtrack to the Sina ubiquitin ligase, thus targeting Tramtrack for degradation. In the eye, phyllopod expression is regulated upstream of proneural genes by activation of Raf and Ras1. However, in sensory organ progenitor cells, phyllopod expression is up-regulated downstream from achaete-scute proneural genes, and thus acts in a feedback loop analogous to that mediated by btbd6a in primary neurogenesis (Sobieszczuk, 2010).

In contrast to the current findings, a recent study has concluded that a Xenopus laevis homolog of btbd6 is required for late steps of neuronal differentiation, since knockdown led to decreased expression of late but not early markers. Although this difference could be due to species-specific functions, a similar decrease was observed in late and not early neuronal markers following knockdown of either of the alternative btbd6a transcripts, whereas knockdown of both blocks the onset of neurogenesis. The knockdown of one transcript in Xenopus may thus inhibit late but not early differentiation steps due to a partial blocking of btbd6 function. These observations beg the question of why partial knockdown of Btbd6a has a stronger effect on late than on early markers of differentiation. One potential explanation is that Btbd6a targets the degradation of an inhibitor acting at multiple steps in the transcriptional cascade of neuronal differentiation; consequently, partial blocking of btbd6a function would have a cumulative inhibitory effect on late markers (Sobieszczuk, 2010).

These findings raise the question of the relationship between btbd6a, plzf, and the selection process that occurs due to Notch-mediated lateral inhibition. During lateral inhibition, high Notch activity in progenitors adjacent to differentiating neurons up-regulates hes genes, leading to inhibition of the expression of proneural genes and downstream Notch ligands. In contrast, Notch activity is low in cells that become selected to differentiate, thus alleviating the inhibition of proneural gene expression by Notch-dependent hes genes. The finding that btbd6a function is required for primary neurogenesis reveals that the decrease in Notch activity is not sufficient to enable neuronal differentiation. Rather, it is required that Btbd6a is up-regulated in order to promote degradation of Plzf that otherwise would inhibit neurogenesis. Consistent with this, endogenous plzfa contributes to the inhibition of neurogenesis under conditions of low Notch activity, and knockdown of plzfa alleviates the block in differentiation that occurs following btbd6a knockdown. It is therefore concluded that the feedback loop mediated by Btbd6a is essential in the progression from the selection of progenitors for differentiation to the initiation of neurogenesis (Sobieszczuk, 2010).

PLZF was discovered as a cause of specific forms of acute promyelocytic leukemia in which chromosomal translocation generates an abnormal fusion between PLZF and Retinoic Acid Receptor α (RARα) protein. The PLZF-RARα fusion protein acts as a dominant-negative retinoic acid receptor and blocks retinoid signaling, whereas the reciprocal RARα-PLZF fusion interferes with PLZF repressor activity. The induction of leukemia by these fusion proteins is in part due to PLZF being required to inhibit the growth and differentiation of myeloid precursors. Furthermore, PLZF is required for maintenance and self-renewal of spermatagonial stem cells. The findings suggest that, in parallel with other mechanisms, plzfa inhibits the differentiation of progenitor cells during primary neurogenesis. These findings raise the prospect that PLZF contributes to the maintenance of progenitors in diverse cell lineages, perhaps by the direct or indirect repression of genes that promote differentiation (Sobieszczuk, 2010).

The finding that Btbd6a overexpression leads to decreased nuclear and increased cytoplasmic levels of Plzf protein is consistent with studies showing that ubiquitination of Plzf correlates with a shift in its distribution from nucleus to cytoplasm. This raises the question of the relationship between subcellular localization and ubiquitination. An attractive model is suggested by studies of the Keap1 adaptor protein that regulates the nucleocytoplasmic location and ubiquitination of the Nrf2 transcription factor. Keap1 affects Nrf2 localization in part by sequestering it with cytoplasmic Cul3, leading to Nrf2 degradation. In addition, binding of Keap1 to Nrf2 in the nucleus promotes export of the complex to the cytoplasm, where binding to Cul3 and degradation then occur. Similarly, Btbd6a could decrease the amount of Plzf available in the nucleus for transcriptional repression by promoting its nuclear export and/or by sequestering Plzf with Cul3 in the cytoplasm leading to ubiquitination and degradation (Sobieszczuk, 2010).

Whereas the BTB domain of a number of adaptor proteins is capable of binding Cul3, the finding that Btbd6aδBTB binds Cul3 indicates that the remaining region of Btbd6a, which is comprised of a BACK and PHR domain, is sufficient to mediate the interaction. Similarly, the BTB domain of Keap1 is not required for its interaction with Cul3. Furthermore, it was found that the BTB domain of Btbd6a is required for binding to Plzf, consistent with it providing the specificity to recruit a substrate to the Cul3 complex. It will therefore be interesting to elucidate at the structural level how different ubiquitination adaptors mediate specific binding to Cul3 and to the proteins targeted for degradation (Sobieszczuk, 2010).

Inhibitors of cell differentiation are widely used during development to regulate the maintenance of progenitors versus initiation of differentiation. The finding of a pathway mediating the targeted degradation of an inhibitor of primary neuronal differentiation raises the question of whether analogous mechanisms operate elsewhere in the nervous system and other tissues. It will be interesting to determine whether other ubiquitination adaptors act downstream from transcription factors that regulate the onset of cell differentiation (Sobieszczuk, 2010).

NeuroD/Neurogenin targets

Although serine-arginine rich (SR) proteins have often been implicated in the positive regulation of splicing, recent studies have shown that one unusual SR protein, SRp38, serves (in contrast) as a splicing repressor during mitosis and stress response. A novel developmental role for SRp38 has been identified in the regulation of neural differentiation. SRp38 is expressed in the neural plate during embryogenesis and is transcriptionally induced by the neurogenic bHLH protein neuroD. Overexpression of SRp38 inhibits primary neuronal differentiation at a step between neurogenin and neuroD activity. This repression of neuronal differentiation requires activation of the Notch pathway. Conversely, depletion of SRp38 activity results in a dysregulation of neurogenesis. Finally, SRp38 can interact with the peptidyltransferase center of 28S rRNA, suggesting that SRp38 activity may act, in part, via regulation of ribosome biogenesis or function. Strikingly, recent studies of several cell cycle regulators during primary neurogenesis have also revealed a crucial control step between neurogenin and neuroD. SRp38 may mediate one component of this control by maintaining splicing and translational silencing in undifferentiated neural cells (Liu, 2005).

Transcriptional dysregulation has emerged as a potentially important pathogenic mechanism in Huntington's disease, a neurodegenerative disorder associated with polyglutamine expansion in the huntingtin (htt) protein. This study reports the development of a biochemically defined in vitro transcription assay that is responsive to mutant htt. Both gene-specific activator protein Sp1 and selective components of the core transcription apparatus, including TFIID and TFIIF, are direct targets inhibited by mutant htt in a polyglutamine-dependent manner. The RAP30 subunit of TFIIF specifically interacts with mutant htt both in vitro and in vivo to interfere with formation of the RAP30-RAP74 native complex. Importantly, overexpression of RAP30 in cultured primary striatal cells protects neurons from mutant htt-induced cellular toxicity and alleviates the transcriptional inhibition of the dopamine D2 receptor gene by mutant htt. These results suggest a mutant htt-directed repression mechanism involving multiple specific components of the basal transcription apparatus (Zhai, 2005).

This study developed an in vitro transcription assay to dissect the potential molecular mechanisms employed by mutant htt to repress transcription of specific promoters (e.g., Sp1-dependent). Taking advantage of this well-defined in vitro transcription system, it was demonstrate that specific components (TFIID and TFIIF) of the transcriptional machinery are directly targeted by mutant htt. Importantly, these in vitro results correlate very well with the in vivo effects of mutant htt, such as the previously reported disruption of Sp1 and TAF4 interaction by mutant htt at the D2 promoter (versus NR1 promoter) in primary neurons. Bearing this principle in mind, it may be possible, in the future, to take advantage of this in vitro system to identify other potential direct targets and mechanisms of transcriptional dysregulation associated with other transcription pathways in HD. Secondly, this study demonstrates that soluble rather than aggregated forms of mutant htt may directly dysregulate transcription by interfering with specific components of the transcriptional preinitiation complex. The data suggest that transcriptional dysfunction may occur as a result of interference by the soluble forms of mutant htt early in disease before any aggregation is seen. In addition, this work suggests that mutant htt may act as a special class of transcriptional repressor or corepressor. This is a potentially important point because it suggests that one of the primary and direct effects of mutant htt on transcription is via specific repressor mechanisms, whereas other documented effects of htt such as activation of transcription may be compensatory or secondary. Finally, this work demonstrates that transcriptional repression by mutant htt is polyQ length dependent. This strongly confirms the observed toxic gain of function for mutant htt. Progressive expansion of polyQ in mutant htt appears to lead to more severe repression while little or no repression is seen with wt htt both in vitro and in vivo. The strong correlation between polyQ length and the efficiency of repression observed in vitro fits well with the documented timing and severity of HD onset. This striking finding further suggests that direct disruption of transcription integrity via aberrant interactions between mutant htt, Sp1, TFIID, and TFIIF are specific and may be significant for orchestrating the pathogenesis of HD (Zhai, 2005).

In this work, a variety of different htt N-terminal fragment constructs were used to take advantage of the various systems established by other HD researchers. Although truncated htt proteins might behave somewhat different from the intact protein, it is nevertheless believed that these in vitro and in vivo studies should be quite informative. Indeed, in vitro studies were inspired by previous findings showing that various truncated versions of mutant htt bearing different lengths of polyQ expansions are produced by proteolytic cleavage in vivo, resulting in fragments that can readily enter the nucleus. Thus, these in vitro studies largely attempt to recapitulate the situation that is thought to occur in vivo (Zhai, 2005).

The most striking finding from the in vitro studies was the identification of TFIIF as a novel direct target in mutant htt-mediated transcriptional repression. Although there have been reports linking TFIIF to the function of transcription activators and repressors, this study provides the first direct connection between TFIIF and transcriptional repression induced by a polyQ expansion protein. RAP30, a subunit of TFIIF, appears to consist of three functional domains. The N-terminal domain of RAP30 is thought to bind RAP74, the central region binds RNA Pol II, and the C-terminal domain binds DNA. In this study, it was found that mutant htt has a strong affinity for RAP30. Because RAP30 lacks a Q-rich domain, its interaction with mutant htt is likely mediated through an alternative interface. Crystal structure of the N-terminal fragments of RAP30 and RAP74 have been shown to adopt a triple-barrel structure with multiple β sheets. Since mutant htt favors the formation of an intramolecular β sheet structure, it is possible that the RAP30 mutant htt interaction involves contact between β sheet structures. Such a structure-based interference mechanism is consistent with the finding that expansion of glutamines in mutant htt enhanced its affinity for RAP30. Thus, mutant htt may target not only polyQ-containing proteins, but also non-polyQ proteins with specific β sheet structures. It should be noted that addition of Congo red, a β sheet-reactive reagent, to the in vitro system did not prevent mutant htt-mediated transcriptional repression, possibly due to its inability to prevent mutant htt from forming protofibrils in vitro (Zhai, 2005).

An important aspect revealed by this study is that mutant htt has a higher affinity for RAP30 than wt htt and may compete with RAP74 for interaction with RAP30. Because an intact TFIIF complex is required for efficient initiation and elongation of transcription at least for some promoters, it is hypothesized that TFIIF dissociation will contribute to transcriptional dysregulation by mutant htt. It is conceivable that mutant htt, which has a higher affinity for RAP30, when it accumulates in both the cytoplasm and nucleus could cause less TFIIF to be formed in the cytoplasm and more TFIIF to be disrupted in the nucleus. Such a scenario will likely result in a general decrease of transcription in HD cells, as has been observed. In several DNA microarray studies, the level of RNA Pol II large subunit has been shown to increase in mutant HD brain. Since the role of TFIIF in transcription is dependent on its interaction with RNA Pol II, it is speculate that elevated levels of RNA Pol II subunits in HD cells may arise as a compensatory mechanism triggered by decreased levels of TFIIF. However, in vitro, adding excess RNA Pol II did not rescue the htt-mediated repression (Zhai, 2005).

By contrast, the findings showed that overexpression of RAP30 is able to abrogate transcriptional repression and rescue the cellular toxicity induced by mutant htt in primary striatal neurons. There are two potential explanations. One possibility is for RAP30 to interact with mutant htt and compete it away from other htt-interacting partners. Another possibility is for RAP30 to drive the formation of more TFIIF complexes, thereby potentiating transcription of important genes involved in neuronal survival. An intriguing observation that was made is that overexpression of RAP74 alone can induce significant cellular toxicity in striatal neurons. This suggests that the chronic release of free RAP74 from TFIIF may contribute to the progressive nature of HD pathogenesis. Thus, the data favor the mechanism in which RAP30 can protect the striatal neurons by promoting TFIIF complex formation. To better understand how much the TFIIF-mediated mechanism contributes to the selective neuronal death during HD pathogenesis, it will be important to identify those genes whose transcription in striatal neurons is particularly sensitive to both mutant htt and RAP74 in future investigations (Zhai, 2005).

Taking the in vitro and in vivo observations together with previous studies, the following model is proposed for how mutant htt represses Sp1-dependent gene expression in neurons. In normal cells, Sp1 is recruited to GC-box-containing promoters through its DNA binding domain. Once bound to DNA, Sp1 utilizes its multiple glutamine-rich activation domains to target components of the basal transcription machinery, one of which is TAF4, a subunit of TFIID. In a multistep recruiting process involving TFIIA, TFIID, TFIIB, TFIIE, TFIIF, TFIIH, RNA Pol II, and CRSP, the preinitiation complex is then formed on activated promoters to potentiate transcription. In HD cells, soluble nuclear mutant htt fragment is free to bind Sp1 through direct protein interactions, thus sequestering this key transcriptional activator from binding to its cognate GC boxes. Furthermore, mutant htt can also prevent Sp1-mediated recruitment of TFIID through its interaction with TAF4. In the case where there is already an Sp1-TFIID complex formed at the promoter, mutant htt could subsequently disrupt the stepwise PIC assembly by targeting TFIIF, an essential transcription factor important for initiation, promoter escape, and elongation at certain promoters. It is anticipated that for different potential target genes, mutant htt will have differential effects because these multiple transcription factor targets may be differentially required for critical functions and rate-limiting transactions at specific gene promoters. In summary, this simple model describes one potential mechanism by which mutant htt can selectively target an activator (Sp1) and multiple components of the core machinery (TFIID and TFIIF) to interfere with various stages of the transcription process. It is anticipated that this model will undergo further refinements as more gene regulatory targets for mutant htt are identified and their molecular consequences determined (Zhai, 2005).

Neurogenin 3 (Ngn3) is key for endocrine cell specification in the embryonic pancreas and induction of a neuroendocrine cell differentiation program by misexpression in adult pancreatic duct cells. The gene encoding IA1 (Drosophila homolog: Nerfin-1), a zinc-finger transcription factor, as a direct target of Ngn3 and it forms a novel branch in the Ngn3-dependent endocrinogenic transcription factor network. During embryonic development of the pancreas, IA1 and Ngn3 exhibit nearly identical spatio-temporal expression patterns. However, embryos lacking Ngn3 fail to express IA1 in the pancreas. Upon ectopic expression in adult pancreatic duct cells Ngn3 binds to chromatin in the IA1 promoter region and activates transcription. Consistent with this direct effect, IA1 expression is normal in embryos mutant for NeuroD1, Arx, Pax4 and Pax6, regulators operating downstream of Ngn3. IA1 is an effector of Ngn3 function as inhibition of IA1 expression in embryonic pancreas decreases the formation of insulin- and glucagon-positive cells by 40%, while its ectopic expression amplifies neuroendocrine cell differentiation by Ngn3 in adult duct cells. IA1 is therefore a novel Ngn3-regulated factor required for normal differentiation of pancreatic endocrine cells (Mellitzer, 2006).

Proneural genes function upstream of Ebf expression: Ebf gene function is required for coupling neuronal differentiation and cell cycle exit

Helix-loop-helix transcription factors of the Ebf/Olf1 family have been implicated in the control of neurogenesis in the central nervous system in both Xenopus laevis and the mouse, but their precise roles have remained unclear. Two family members have been characterized in the chick, and a functional analysis was performed by gain- and loss-of-function experiments. This study reveals several specific roles for Ebf genes in the spinal cord and hindbrain regions of higher vertebrates, and enables their precise positioning along the neurogenic cascade. During neurogenesis, cell cycle exit appears to be tightly coupled to migration to the mantle layer and to neuronal differentiation. Antagonizing Ebf gene activity allows the uncoupling of these processes. Ebf gene function is necessary to initiate neuronal differentiation and migration toward the mantle layer in neuroepithelial progenitors, but it is not required for cell cycle exit. Ebf genes therefore appear to be master controllers of neuronal differentiation and migration, coupling them to cell cycle exit and earlier steps of neurogenesis. Mutual activation between proneural and Ebf genes suggests that besides their involvement in the engagement of differentiation, Ebf genes may also participate in the stabilization of the committed state. Finally, gain-of-function data raise the possibility that, in addition to these general roles, Ebf genes may be involved in neuronal subtype specification in particular regions of the CNS (Garcia-Dominguez, 2003).

Analysis of Ebf1 and Ebf3 mRNAs in the chick neural tube indicated that their accumulation is coincidental with the onset of neurogenesis and that they are detected within the entire mantle layer. This is in agreement with the expression pattern of the mouse orthologs, and shows that these genes are expressed at a high level in early post-mitotic neurons and that their expression is maintained during neuronal differentiation. Low level, scattered expression has also been observed in the neuroepithelium for mouse Ebf2 and Ebf3, presumably corresponding to cells en route to the mantle layer. Forced expression of both Ngn2, a proneural gene, and NeuroM, an early neuronal differentiation regulator, promotes Ebf1 and Ebf3 expression, indicating that the latter genes are downstream of the former in the neurogenic cascade, consistent with Ebf gene expression pattern. Expression of a dominant-negative molecule, which presumably antagonizes all Ebf activities, does not affect cell cycle exit, but prevents neuroepithelial precursor migration towards the mantle layer and expression of differentiation markers. Furthermore, the dominant-negative Ebf is also able to prevent neuronal differentiation and migration induced by the forced expression of Ngn2, but it does not affect the endogenous expression of this latter gene. Together, these observations suggest that Ebf genes play an essential role in cell engagement into neuronal differentiation and migration towards the mantle layer, coupling these processes to cell cycle exit. In agreement with a role of Ebf genes in the control of neuronal differentiation and migration, misexpression of Ebf1 in neuroepithelial progenitors promotes these processes, which indicates that Ebf genes are both necessary and sufficient. However, surprisingly, forced expression of Ebf1 also leads to exit from the cell cycle. This is correlated with a transient reinforcement of Ngn1 and Ngn2, and NeuroM expression. Induction of the complete neurogenic program by Ebf1 is largely dependent on bHLH proteins, presumably including proneural gene products, as shown by Ebf1 inhibition by the bHLH antagonist Id2. At this stage, the possibility that forced high level expression of Ebf1 in neuroepithelial progenitors may lead to non-physiological proneural gene activation, subsequently resulting in the activation of the complete program cannot be excluded. An alternative explanation, involving a second function of Ebf genes, can nevertheless be envisaged. Ebf1 misexpression also leads to changes in the balance of neuronal subtypes. This suggests the existence of a third level of intervention of Ebf genes in the neurogenic cascade. Ebf genes are considered to be downstream from proneural genes and cell cycle exit, but are absolutely required for neuronal differentiation and migration towards the mantle layer (Garcia-Dominguez, 2003).

Neurogenin and the development of motoneuron properties

Distinct classes of neurons are generated at defined times and positions during development of the nervous system. A fuller understanding of how specification of neuronal identity coordinates with acquisition of pan-neuronal properties remains elusive. Basic helix-loop-helix (bHLH) transcription factors Olig2 and Neurogenin2 (Ngn2) play vital roles in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Olig2 and Ngn2 are specifically coexpressed in motoneuron progenitors. Misexpression studies in chick demonstrate the specific, combinatorial actions of Olig2 and Ngn2 in motoneuron generation. These results further reveal crossregulatory interactions between bHLH and homeodomain transcription factors in the specification of motoneurons. It is suggested that distinct classes of transcription factors collaborate to generate motoneurons in the ventral neural tube (Mizuguchi, 2001).

Olig2 was originally identified as a bHLH factor expressed in oligodendrocytes and their progenitors. However, its expression has been detected during early development, earlier than the timing of oligodendrocyte differentiation. This early expression of Olig2 was examined in comparison with that of Ngn2 and Mash1. In the developing rat spinal cord, Olig2-positive (Olig2+) and Ngn2+ cells are first detected at embryonic (E) day 10.5, and occupy overlapping ventral domains at E11.5. At this stage, a small number of Mash1+ cells emerge in the ventral-most domain, but few motoneurons or ventral interneurons have been generated. Distinct progenitor domains have been defined by differential expression of a group of homeodomain factors along the dorso-ventral axis of the neural tube. The expression of Nkx2.2 and Irx3 limits the domain for motoneuron progenitors. Nkx6.1 is expressed in progenitors for both motoneurons and ventral interneurons, whereas Pax6 occupies a broad intermediate domain, where its expression is low in ventral motoneuron progenitors. At E12.5, the ventral and dorsal boundaries of the Olig2+ domain are closely adjacent to those of Nkx2.2 and Irx3, respectively. The dorsal boundary of the Olig2+ domain is ventral to that of the Nkx6.1+ domain, and closely matches that of the Pax6low domain. At this stage, Ngn2 is coexpressed in many, but not all, of Olig2+ cells dorsal to the Nkx2.2+ domain. Some Ngn2+ cells are also scattered more dorsally, but remain ventral to Pax7+ cells that have been defined as dorsal progenitors. The distribution pattern of such cells expressing Ngn2 proteins is consistent with that of Ngn2 mRNA (Mizuguchi, 2001).

It is notable here that Olig2 and Ngn2 can induce both pan-neuronal and motoneuron-specific genes not only in the neural tube, but also in some non-neural tissues, such as the surface ectoderm and otic vesicle, where motoneurons never arise in normal development. This observation suggests that Olig2 and Ngn2 are not simply permissive for motoneuron differentiation, but that they can instruct at least some aspects of a motoneuron fate in various cellular contexts. Not all cells that expressed ectopic Olig2 and Ngn2 differentiate into motoneurons. Moreover, endogenous Olig2 and Ngn2 are expressed in various regions of the developing CNS, including the forebrain where motoneurons do not arise. Thus, certain conditions presumably exist that restrict the activities of Olig2 and Ngn2 to induce motoneurons (Mizuguchi, 2001).

Among various bHLH factors, Olig1 and Ngn1 mimic the actions of Olig2 and Ngn2, respectively. In contrast, a structurally distant bHLH factor, Mash1, either alone or in combination with Olig2 or Ngn2, does not induce motoneuron markers. Olig1/Olig2, and Ngn1/Ngn2 constitute distinct subfamilies among bHLH factors, and their bHLH domains are highly conserved within each subfamily, but divergent from each other. The difference in structures of the bHLH domains has been implicated for distinct functions of bHLH factors. Thus, the activity to induce motoneurons appears to be specific for the Olig and Ngn subfamilies. Unlike Olig2, however, Olig1 is not expressed in the neural tube at the stage when motoneurons are generated. Preliminary data also indicate that the expression of Ngn1 begins later than that of Ngn2, and is not detected in Olig2+ progenitors. Thus, Olig2 and Ngn2, but not Olig1 or Ngn1, likely play major roles in generating motoneurons in normal development. However, the results do not exclude the possibility that other bHLH factors are also involved in motoneuron generation since some Isl1+ motoneuron-like cells still differentiate in the Ngn1/Ngn2 double mutant, albeit in a much lower number than in the wild-type (Mizuguchi, 2001).

Within the developing vertebrate nervous system, the mechanisms that coordinate neuronal subtype identity with generic features of neuronal differentiation are poorly defined. A bHLH protein, Olig2, is expressed selectively by motor neuron progenitors and has a key role in specifying the subtype identity and pan-neuronal properties of developing motor neurons. The role of Olig2 in the specification of motor neuron subtype identity depends on regulatory interactions with progenitor homeodomain proteins, whereas its role in promoting pan-neuronal properties is associated with expression of another bHLH protein, Ngn2. Both aspects of Olig2 function appear to depend on its activity as a transcriptional repressor. Together, these studies show that Olig2 has a critical role in integrating diverse features of motor neuron differentiation in the developing spinal cord (Novitch, 2001).

How does Olig2 promote neurogenesis? The ectopic expression of Olig2 during the period of motor neuron generation markedly expands the dorsoventral domain of expression of Ngn2. Ngns have been shown to promote the expression of generic neuronal markers in a wide variety of settings, and it has been found that ectopic expression of Ngn2 effectively directs the expression of such pan-neuronal markers in the spinal cord. One line of evidence that favors the idea that Olig2 promotes generic neuronal character through the activity of Ngn2 comes from the analysis of the later function of Olig2 in oligodendrocyte differentiation. Here, Olig2 expression in oligodendrocyte progenitors is preceded by the extinction of Ngn expression, and in the absence of Ngns, cells that express Olig2 retain their proliferative capacity. Moreover, in mice lacking Ngn2, the number of ventral Lim3+ and Isl1+ neurons is reduced, and in Ngn1; Ngn2 double mutants, pan-neuronal marker expression is lost. Together, these results suggest that Ngns participate downstream of Olig2 in promoting the efficient expression of motor neuron subtype properties, and in imposing pan-neuronal character (Novitch, 2001).

During neural tube development, the expression of Olig2 is likely to underlie the prominent early expression of Ngn2 within the pMN domain, in contrast to the sparse expression of Ngn2 evident in other progenitor domains at this stage. Thus, even though Ngn2 is widely expressed by progenitor cells in the spinal cord and has a general role in neurogenesis, its expression may be controlled independently in individual progenitor domains. In support of this idea, analysis of the Ngn2 promoter in the developing spinal cord has revealed distinct domain-specific regulatory elements. The early Olig2-dependent onset of Ngn2 expression within the pMN domain may therefore account for the predominance of motor neuron rather than interneuron generation during the initial stages of ventral spinal cord neurogenesis. But if Olig2 and Ngn2 are expressed at high levels within the pMN domain as early as stages 10 to 12, how are motor neuron progenitors amplified, and why do the first post-mitotic motor neurons not appear until stages 14 to 15? One possible reason could be that pMN domain progenitors transiently express inhibitory bHLH proteins, and a temporal decay in the expression of these inhibitors may be needed to unleash the neurogenic activities of Olig2 and Ngn2 (Novitch, 2001).

Within the developing vertebrate nervous system, specific subclasses of neurons are produced in vastly different numbers at defined times and locations. This implies the concomitant activation of a program that controls pan-neuronal differentiation and of a program that specifies neuronal subtype identity, but how these programs are coordinated in time and space is not well understood. Loss- and gain-of-function studies have defined Phox2b as a homeodomain transcription factor that coordinately regulates generic and type-specific neuronal properties. It is necessary and sufficient to impose differentiation towards a branchio- and viscero-motoneuronal phenotype and at the same time promote generic neuronal differentiation. The underlying genetic interactions have been examined. Phox2b has a dual action on pan-neuronal differentiation. It upregulates the expression of proneural genes (Ngn2) when expressed alone and upregulates the expression of Mash1 when expressed in combination with Nkx2.2. By a separate pathway, Phox2b represses expression of the inhibitors of neurogenesis Hes5 and Id2. The role of Phox2b in the specification of neuronal subtype identity appears to depend in part on its capacity to act as a patterning gene in the progenitor domain. Phox2b misexpression represses the Pax6 and Olig2 genes, which should inhibit a branchiomotor fate, and induces Nkx6.1 and Nkx6.2, which are expressed in branchiomotor progenitors. Phox2b behaves like a transcriptional activator in the promotion of both, generic neuronal differentiation and expression of the motoneuronal marker Islet1. These results provide insights into the mechanisms by which a homeodomain transcription factor through interaction with other factors controls both generic and type-specific features of neuronal differentiation (Dubreuil, 2002).

The underlying transcriptional mechanisms that establish the proper spatial and temporal pattern of gene expression required for specifying neuronal fate are poorly defined. This study characterizes how the Hb9 gene is expressed in developing motoneurons in order to understand how transcription is directed to specific cells within the developing CNS. Non-specific general-activator proteins such as E2F and Sp1 are capable of driving widespread low level transcription of Hb9 in many cell types throughout the neural tube; however, their activity is modulated by specific repressor and activator complexes. The general-activators of Hb9 are suppressed from triggering inappropriate transcription by repressor proteins Irx3 and Nkx2.2. High level motoneuron expression is achieved by assembling an enhancesome on a compact evolutionarily-conserved segment of Hb9 located from -7096 to -6896. The ensemble of LIM-HD and bHLH proteins that interact with this enhancer change as motoneuron development progresses, facilitating both the activation and maintenance of Hb9 expression in developing and mature motoneurons. These findings provide direct support for the derepression model of gene regulation and cell fate specification in the neural tube, as well as establishing a role for enhancers in targeting gene expression to a single neuronal subtype in the spinal cord (Lee, 2004).

Developing motoneurons sequentially express several bHLH proteins, including Ngn2 in the progenitor cells followed by NeuroM in the early postmitotic motoneurons and NeuroD in the more mature cells. Ngn2 and NeuroM have been shown to contribute to the activation of Hb9 during the initial stages of motoneuron development, but it remained unclear whether NeuroD in the mature cells could also stimulate Hb9 expression. To compare the activity of these transcription factors, P19 cells were transfected with expression constructs encoding bHLH proteins together with a luciferase reporter containing seven E box elements. Under these conditions Ngn2 activated the reporter much more than either NeuroM or NeuroD. Despite this inherent difference in transactivation, Ngn2, NeuroM, and NeuroD each synergized in a similar way with the LIM factors Isl1 and Lhx3 to trigger Hb9 expression. Likewise, each bHLH factor dimerizes with E47 and binds to the M50 and M100 E box elements in a sequence-specific manner, and exhibits a similar ability to promote motoneuron differentiation from transfected P19 embryonic carcinoma cells when expressed with Isl1 and Lhx3. Taken together, these findings suggest that the initial activation of Hb9 expression is dependent on Ngn2 and NeuroM as motoneurons become postmitotic, and that NeuroD contributes to the maintenance of Hb9 expression in mature motoneurons (Lee, 2004).

Nkx2.2, Nkx6.1, Pax6 and Irx3 control progenitor cell fate by repressing transcription. Since the deletion analysis of Hb9 indicated that repressor proteins might interact with the 2.5 kb distal segment from -8129 to -5575, tests were performed to see whether constructs with this DNA segment were repressed by Nkx2.2, Nkx6.1, Pax6 and/or Irx3 using 293 cell transfections. The Hb9 promoter was repressed ~50-500 fold by Nkx2.2 and Irx3, whereas Pax6 and Nkx6.1 were significantly less active. These findings suggest that progenitor cell factors such as Nkx2.2 and Irx3 expressed by non-motoneuron cells suppress the expression of Hb9 (Lee, 2004).

Genetic studies have shown that Hb9 feeds back negatively to modulate its own expression. Whether Hb9 could suppress the activity of its enhancer when LIM and bHLH factors synergize to activate transcription was tested. The native Hb9 protein and the EnR-Hb9 repressor (Hb9 homeodomain linked to eh1 engrailed repressor domain) both inhibited transcription under these conditions, whereas the Hb9-HD and a fusion of Hb9 to the VP16 activation domain (VP16-Hb9) lacked this activity. Thus, in developing motoneurons where Hb9 transcription is synergistically activated, co-repressors such as those recruited by the engrailed fusion (EnR) appear to be involved in negative feedback regulation. Consistent with these findings, Hb9 protein binds in a sequence-specific manner to the ATTA motifs in the enhancer (Lee, 2004).

Inductive signaling leads to the coactivation of regulatory pathways for specifying general neuronal traits in parallel with instructions for neuronal subtype specification. Nevertheless, the mechanisms that ensure that these pathways are synchronized have not been defined. To address this, how bHLH proteins Ngn2 and NeuroM controlling neurogenesis functionally converge with LIM-homeodomain (LIM-HD) factors Isl1 and Lhx3 involved in motor neuron subtype specification was investigated. Ngn2 and NeuroM transcriptionally synergize with Isl1 and Lhx3 to specify motor neurons in the embryonic spinal cord and in P19 stem cells. The mechanism underlying this cooperativity is based on interactions that directly couple the activity of the bHLH and LIM-HD proteins, mediated by the adaptor protein NLI. This functional link acts to synchronize neuronal subtype specification with neurogenesis (S. K. Lee, 2003).

Spinal motor neurons and oligodendrocytes are generated sequentially from a common pool of progenitors termed pMN cells. Olig2 is a bHLH-class transcription factor in pMN cells, but it has remained unclear how its transcriptional activity is modulated to first produce motor neurons and then oligodendrocytes. Previous studies have shown that Olig2 primes pMN cells to become motor neurons by triggering the expression of Ngn2 and Lhx3. Olig2 also antagonizes the premature expression of post-mitotic motor neuron genes in pMN cells. This blockade is counteracted by Ngn2, which accumulates heterogeneously in pMN cells, thereby releasing a subset of the progenitors to differentiate and activate expression of post-mitotic motor neuron genes. The antagonistic relationship between Ngn2 and Olig2 is mediated by protein interactions that squelch activity as well as competition for shared DNA-binding sites. The data support a model in which the Olig2/Ngn2 ratio in progenitor cells serves as a gate for timing proper gene expression during the development of pMN cells: Olig2high maintains the pMN state, thereby holding cells in reserve for oligodendrocyte generation, whereas Ngn2high favors the conversion of pMN cells into post-mitotic motor neurons (Lee, 2005).

Retinoid signaling and neurogenin2 function are coupled for the specification of spinal motor neurons through a chromatin modifier CBP

Extracellular signals and cell-intrinsic transcription factors cooperatively instruct generation of diverse neurons. However, little is known about how neural progenitors integrate both cues and orchestrate chromatin changes for neuronal specification. This paper reports that extrinsic signal retinoic acid (RA) and intrinsic transcription factor Neurogenin2 (Ngn2) collaboratively trigger transcriptionally active chromatin in spinal motor neuron genes during development. Retinoic acid receptor (RAR) binds Ngn2 and is thereby recruited to motor neuron genes targeted by Ngn2. RA then facilitates the recruitment of a histone acetyltransferase CBP to the Ngn2/RAR-complex, markedly inducing histone H3/H4-acetylation. Correspondingly, timely inactivation of CBP and its paralog p300 results in profound defects in motor neuron specification and motor axonal projection, accompanied by significantly reduced histone H3-acetylation of the motor neuron enhancer. This study uncovers the mechanism by which extrinsic RA-signal and intrinsic transcription factor Ngn2 cooperate for cell fate specification through their synergistic activity to trigger transcriptionally active chromatin (Lee, 2009).

Neurogenin and the development of interneurons

Distinct classes of neurons are generated from progenitor cells distributed in characteristic dorsoventral patterns in the developing spinal neural tube. Restricted neural progenitor populations are defined by the discrete, nonoverlapping expression of Ngn1, Math1, and Mash1. Crossinhibition between these bHLH factors is demonstrated and provides a mechanism for the generation of discrete bHLH expression domains. This precise control of bHLH factor expression is essential for proper neural development since as demonstrated in both loss- and gain-of-function experiments, expression of Math1 or Ngn1 in dorsal progenitor cells determines whether LH2A/B- or dorsal Lim1/2-expressing interneurons will develop. Together, the data suggest that although Math1 and Ngn1 appear to be redundant with respect to neurogenesis, they have distinct functions in specifying neuronal subtype in the dorsal neural tube (Gowan, 2001).

Five distinct classes of dorsal interneurons have been described by their expression of LIM homeodomain factors. These interneuron populations, D1A, D1B, D2, D3, and D4, express LH2A, LH2B, Islet1, Lim1/2, and Lmx1b, respectively. Defined here is a subclass of the D3 population, termed D3A, and characterized as the most dorsal Lim1/2-expressing cells that coexpress Brn3a (Gowan, 2001).

Expression in the dorsal neural tube of the bHLH transcription factors, Ngn1 and Math1, defines progenitor populations destined to be distinct types of neurons. Math1 marks progenitors that generate LH2A/B-expressing interneurons, and Math1 is required for these interneurons to form. Furthermore, ectopic expression of Math1 in chick can induce LH2 expression in the dorsal neural tube; however, it is not sufficient to induce LH2 expression in ventral regions. This suggests that other factors, specific to dorsal neural tube regions, work together with Math1 to form LH2 interneurons. Another phenotype in the Math1 mutant demonstrated here is the transition of the Math1 progenitors to Ngn1/Ngn2-expressing cells that give rise to D3A interneurons at the expense of LH2 interneurons. In contrast, examination of the lacZ-expressing cells in a lacZ knockin of Math1 reveals increased overlap with Msx1/2, and thus suggests that at least some Math1-expressing cells are becoming roof plate cells. These data taken together suggest that in the absence of Math1, expression of markers normally marking cells found at either border/roof plate (dorsally) and Ngn1/2 (ventrally) expand to fill the domain (Gowan, 2001).

A newly defined cell type (D3A), which expresses Lim1/2;Brn3a and accounts for the most dorsal Lim1/2 domain, arises from progenitor cells expressing Ngn1 or Ngn2, and either Ngn1 or Ngn2 is required for these cells to form. The correlation seen between the expansion of Ngn1- and Ngn2-expressing progenitors and the increase in the D3A interneuron population in the Math1 mutant support the idea that they are sufficient for Lim1/2 expression in the dorsal neural tube. It is surprising that the dorsal Islet1 population appears to be unaffected in the Ngn1 and Ngn2 double mutants since Islet1-expressing cells are completely absent in these mutants in the DRG, and are diminished in the ventral neural tube (J. E. J., unpublished). This suggests that Ngn1 or Ngn2, combined with other factors with differential expression in the dorsoventral axis of the neural tube, generate multiple cell types. This is consistent with conclusions from studies of Math1, and with the fact that loss of a specific bHLH results in the loss of different types of neurons in different regions of the nervous system (Gowan, 2001).

Another way to classify neurons, other than by expression of homeodomain markers, is by a description of their axonal projections. Using the Math1 and Ngn1 enhancers to drive reporter gene expression in transgenic mice, two nonoverlapping populations of commissural interneurons can be distinguished. Additional commissural interneurons have been seen in transgenic animals with a Ngn2 enhancer directing lacZ expression. Although the eventual function of the different neuronal populations from Ngn1 and Ngn2 progenitors is unknown, the dorsal commissural interneurons from Math1 progenitors have recently been defined as those carrying proprioceptive information in the spinocerebellar and cuneocerebellar pathways (Gowan, 2001).

Combining the results from loss- and gain-of-function studies of Ngn1, Ngn2, and Math1 with the timing of their expression, it is clear that the bHLH factors play a vital role in the transition from proliferating neural progenitor cells to differentiated neurons in multiple discrete neuronal lineages. The expansion of distinct bHLH factors in the different mutant backgrounds in the absence of cell death, taken with the wild-type appearance of the dorsal neural tube by morphology and general neural markers, suggests that Ngn1, Ngn2, and Math1 may compensate for each other's role in inducing neurons from stem cells in the dorsal neural tube. The redundant function in neurogenesis between different bHLH factors suggested here was recently suggested in studies of Mash1;Math3 or Mash1;Ngn2 double mutants. In these studies, neurogenesis was not significantly altered in the single mutants, but in the absence of both factors in different regions of the developing nervous system, there was excess gliogenesis at the expense of neurogenesis. Thus, the neural bHLH factors appear to share the function of inducing neurogenesis (Gowan, 2001).

A separate aspect of the function of bHLH factors is their role in specifying neuronal subtype. This was first investigated in Drosophila where atonal was shown to preferentially produce chordotonal neurons and achaete preferentially produced external sense organs when ectopically expressed. Furthermore, in vertebrates, forced expression of Ngn1 preferentially drives neural crest cells into the sensory neuron fate. These experiments clearly suggest a role for the bHLH factors in neuronal specification; however, the fact that each bHLH factor is present and required for multiple distinct lineages means that other factors and/or pathways modulate this activity. By demonstrating that Ngn1, Math1, and Mash1 are expressed in distinct progenitors, and are required for the formation of definable interneuron populations within a relatively discrete environment, it has been demonstrated that they function in specifying neuronal subtype in the dorsal spinal cord. Thus, although their roles in inducing neurogenesis appears to be a shared function, their roles in specifying neuronal cell type are distinct (Gowan, 2001).

Crossinhibitory regulation plays a role in boundary formation between the Class I and Class II homeodomain factors in the ventral neural tube. The first comparisons of Ngn1, Ngn2, and Mash1 mRNA expression domains demonstrate sharp boundaries in multiple regions of the developing nervous system, giving rise to the suggestion that they negatively regulate each other's expression. Mash1 expression in the dorsal telencephalon is increased in Ngn1;Ngn2 mutant embryos, and this ectopic Mash1 expression results in ventral telencephalon markers being expressed ectopically in the dorsal regions. In addition, in the absence of the roof plate, Math1 and Ngn1 expression are lost and Mash1 is found to extend to the dorsal edge of the neural tube. At the single cell level, at least Ngn1, Math1, and Mash1 define distinct progenitors in the spinal neural tube with only a single factor expressed in each progenitor cell. Indeed, even in the ventral neural tube where Ngn1 predominates, there are scattered cells that express only Mash1. This pattern of expression is at least in part due to an active inhibition between this set of bHLH factors. This is supported by two separate experimental paradigms. In the first paradigm, in mouse embryos lacking Math1, Ngn1- and Ngn2-expressing cells increase in number with no increase in apoptosis in this region of the neural tube. The converse is also true, in the absence of Ngn1 and Ngn2, Math1-expressing cells increase. In a second set of experiments using ectopic expression in chick neural tubes, Math1 is sufficient to inhibit the endogenous chick bHLH factors c-Ngn1 and Cash1, and Ngn1 is sufficient to inhibit the endogenous factors Cath1 and Cash1. The inhibition of Cath1 by Ngn1 appears to be cell autonomous because when examined at a single cell level, cells misexpressing Ngn1 do not coexpress Cath1. Notably, these crossinhibitory phenomena appear to be between different subclasses within the bHLH family. Mash1 is of the achaete/scute subclass; Math1 is of the atonal subclass, and Ngn1/Ngn2 are of the biparous/tap subclass (Gowan, 2001).

The crossinhibitory regulation within the early neural bHLH factor family shown here suggests a mechanism for how a progenitor ends up expressing only one factor, but does not address how the overall pattern of bHLH expression is initially set up. In the dorsal neural tube, BMP signaling likely plays a major role in setting up the bHLH pattern. Loss of the BMP GDF7 results in loss of a subset of Math1-expressing cells, while BMP7 induces Math1 expression in explanted intermediate regions of the neural tube. In a roof plate ablation paradigm, where BMP signaling is dramatically disrupted, both Math1 and Ngn1 are lost in the dorsal neural tube. Math1 is induced in chick neural tubes with a high level of expression of a constitutively active BMP receptor, BMPR-Ib. In contrast, at lower levels of ectopic expression of BMPR-Ib, c-Ngn1 is induced. Taken together, these data suggest that different levels of BMP signaling have different effects on bHLH expression and this pathway may set up the initial pattern of bHLH expression in the dorsal neural tube. Subsequently, other regulatory mechanisms such as the crossinhibition presented here, and differential autoregulation of the bHLH factors, modify and refine this initial pattern (Gowan, 2001).

The dorsal spinal cord contains a diverse array of neurons that connect sensory input from the periphery to spinal cord motoneurons and brain. During development, six dorsal neuronal populations (dI1-dI6) have been defined by expression of homeodomain factors and position in the dorsoventral axis. The bHLH transcription factors Mash1 and Ngn2 have distinct roles in specification of these neurons. Mash1 is necessary and sufficient for generation of most dI3 and all dI5 neurons. Unexpectedly, dI4 neurons are derived from cells expressing low levels or no Mash1, and this population increases in the Mash1 mutant. Ngn2 is not required for any specific neuronal cell type but appears to modulate the composition of neurons that form. In the absence of Ngn2, there is an increase in the number of dI3 and dI5 neurons, in contrast to the effects produced by activity of Mash1. Mash1 is epistatic to Ngn2, and, unlike the relationship between other neural bHLH factors, cross-repression of expression is not detected. Thus, bHLH factors, particularly Mash1 and related family members Math1 and Ngn1, provide a code for generating neuronal diversity in the dorsal spinal cord with Ngn2 serving to modulate the number of neurons in each population formed (Helms, 2005).

Development of the mesencephalic dopaminergic neuron system is compromised in the absence of neurogenin 2

Neurogenin 2 (Ngn2) is a proneural gene involved in neuronal differentiation and subtype specification in various regions of the nervous system. In the ventral midbrain, Ngn2 is expressed in a spatiotemporal pattern that correlates with the generation of mesencephalic dopaminergic (mesDA) neurons. Lack of Ngn2 impairs the development of mesDA neurons, such that less than half of the normal mesDA neuron number remain in Ngn2 mutant mice at postnatal stages. Analysis of Ngn2 mutant mice during mesDA neurogenesis show that medially located precursors are formed but are arrested in their differentiation at a stage when they have not yet acquired the characteristics of mesDA neuron precursors. Loss of Ngn2 function appears to specifically affect the generation of DA neurons; the development of other types of neurons within the ventral midbrain is unaltered. Ngn2 is the first example of a gene expressed in progenitors in the ventricular zone of the mesDA neuron domain that is essential for proper mesDA neuron differentiation, and whose loss of function causes impaired mesDA neurogenesis without other major abnormalities in the ventral midbrain (Andersson, 2006a).

Phosophorylation of NeuroD and Neurogenin effect cell migration properties and the dendritic morphology

The elaboration of dendrites is fundamental to the establishment of neuronal polarity and connectivity, but the mechanisms that underlie dendritic morphogenesis are poorly understood. The genetic knockdown of the transcription factor NeuroD in primary granule neurons including in organotypic cerebellar slices profoundly impairs the generation and maintenance of dendrites while sparing the development of axons. NeuroD mediates neuronal activity-dependent dendritogenesis. The activity-induced protein kinase CaMKII catalyzes the phosphorylation of NeuroD at distinct sites, including endogenous NeuroD at Ser336 in primary neurons, and thereby stimulates dendritic growth. These findings uncover an essential function for NeuroD in granule neuron dendritic morphogenesis. This study also defines the CaMKII-NeuroD signaling pathway as a novel mechanism underlying activity-regulated dendritic growth that may play important roles in the developing and mature brain (Gaudillière, 2004).

NeuroD is expressed in differentiated neurons in a number of brain regions and is particularly high in cerebellar granule neurons. Focus was placed on the potential role of NeuroD in granule neuron morphogenesis. Granule neurons are generated within the external granular layer (EGL) of the cerebellar cortex, where they extend axons that continue to grow even as these neurons migrate to the internal granular layer (IGL). Well after the birth of axons and the formation of parallel fibers, granule neurons elaborate dendrites within the IGL that form the receiving end of synapses with the axon terminals of mossy fibers. Importantly, the sequential generation of granule neuron axons and dendrites in vivo is faithfully recapitulated in primary cultures of rat or mouse cerebellar granule neurons. For this reason and because granule neurons in primary culture display distinct and easily identifiable axons and dendrites, these neurons provide a robust system for the study of axonal and dendritic development (Gaudillière, 2004).

To determine the role of NeuroD in the specification of neuronal morphogenesis, a DNA template-based RNA interference method was used to acutely knock down the expression of NeuroD in differentiated cerebellar granule neurons. A major advantage of the genetic knockdown method is that it allows the assessment of NeuroD function in granule neurons past the stages of granule cell precursor differentiation, in which NeuroD appears to play an essential prosurvival role. Primary cerebellar granule neurons, isolated from postnatal day 6 rat pups and cultured for 2 days (P6+2DIV), were transfected with the U6/nd1 plasmid encoding NeuroD hairpin RNAs (hpRNAs) or the control U6 plasmid, together with a plasmid encoding ß-galactosidase or a plasmid encoding green fluorescent protein (GFP). Granule neurons were fixed four days after transfection and subjected to immunocytochemistry using a mouse monoclonal antibody to ß-galactosidase or a rabbit antibody to GFP. Axons and dendrites of the ß-galactosidase- or GFP-positive transfected neurons were identified based on their morphology and by the expression of the axonal marker Tau and the somato-dendritic marker MAP2. Strikingly, although the NeuroD hpRNA-expressing neurons displayed robust axons, these neurons harbor either profoundly deficient or no dendrites, with the MAP2 signal only evident in the cell body of the NeuroD hpRNA-expressing neurons. By contrast, the control U6-transfected granule neurons exhibited a normal morphological appearance with both robust axons and MAP2-positive dendrites. NeuroD RNAi did not lead to the redistribution of the dendritic and axonal proteins (MAP2 and Tau) in granule neurons which were analyzed each day for 6 days following transfection. Together, these results reveal that the genetic knockdown of NeuroD in granule neurons triggers the loss of dendrites (Gaudillière, 2004).

The characterization of the CaMKII-NeuroD signaling pathway in dendritic growth points to the versatility of CaMKII in the control of neuronal morphogenesis. CaMKII is thought to act locally within dendrites to mediate activity regulation of growth and stabilization of dendrites and dendritic spines. The findings of this study suggest that CaMKIIalpha or a CaMKII heteromer containing the alpha isoform is also employed in neurons in the propagation of a signal from L-type voltage-sensitive calcium channels to NeuroD, thereby orchestrating a program of gene expression leading to dendritic growth and maintenance. Taken together, these studies suggest that CaMKII represents a nodal point in the regulation of both local and transcription-mediated mechanisms of activity-dependent dendritic development (Gaudillière, 2004).

While the CaMKII-induced phosphorylation of NeuroD is required for activity-dependent dendritic growth, it remains to be determined if the phosphorylation of NeuroD at the CaMKII sites is sufficient to induce dendritic growth. The expression of NeuroD or ND-RES (NeuroD designed to be resistant to RNA interference), respectively, in granule neurons deprived of neuronal activity or in membrane-depolarized granule neurons, in which CaMKIIalpha RNAi was triggered, failed to induce dendritic growth. These results suggest that NeuroD expression solely is not sufficient to promote the generation of dendrites, but leave open the possibility that the phosphorylation of NeuroD at the CaMKII sites might be sufficient to induce dendritic growth. Another remaining question for future studies is how the CaMKII-induced phosphorylation of NeuroD regulates the transcriptional function of NeuroD in neurons. Mutation of serines 290 and 336 has little effect on the ability of the transactivation domain of NeuroD to activate transcription of the G4-luc reporter gene. These results raise the possibility that CaMKII-induced phosphorylation regulates aspects of NeuroD function other than transactivation per se, such as the subcellular localization of NeuroD or the binding of NeuroD to DNA. An alternative possibility is that the CaMKII sites of phosphorylation are critical for transcriptional activation, but their importance can only be revealed in the context of an endogenous target gene with surrounding chromatin rather than in the context of a G4-luc reporter gene (Gaudillière, 2004).

Although NeuroD is highly expressed in specific regions of the brain, the NeuroD subfamily of proneural bHLH transcription factor that includes NeuroD2 has a wider pattern of expression including the hippocampus and cerebral cortex. Therefore, an attractive hypothesis is that NeuroD and NeuroD-related factors may generally regulate dendritogenesis in other regions of the mammalian brain beyond the cerebellum. Consistent with the idea that neuronal activity might regulate NeuroD-related proteins, a novel consensus sequence of CaMKII phosphorylation was identified that by database screening is found in a number of brain-enriched transcriptional regulators including NeuroD2. It will be interesting to determine the functional consequences of potential CaMKII-regulation of these transcription factors and in particular that of NeuroD2 (Gaudillière, 2004).

The expression of the NeuroD subfamily in the brain persists well into maturity, suggesting that these proteins may continue to regulate the growth and refinement of dendritic morphology in the adult brain. In light of these observations, the identification of NeuroD as a calcium-regulated transcription factor raises the interesting possibility that NeuroD might serve roles in such processes as synaptic remodeling and plasticity that are critical for the adaptive functions of the brain (Gaudillière, 2004).

The molecular mechanisms specifying the dendritic morphology of different neuronal subtypes are poorly understood. This study demonstrates that the bHLH transcription factor Neurogenin2 (Ngn2) is both necessary and sufficient for specifying the dendritic morphology of pyramidal neurons in vivo by specifying the polarity of its leading process during the initiation of radial migration. The ability of Ngn2 to promote a polarized leading process outgrowth requires the phosphorylation of a single tyrosine residue at position 241, an event that is neither involved in Ngn2 direct transactivation properties nor its proneural function. Interestingly, the migration defect observed in the Ngn2 knockout mouse and in progenitors expressing the Ngn2Y241F mutation can be rescued by inhibiting the activity of the small-GTPase RhoA in cortical progenitors. These results demonstrate that Ngn2 coordinates the acquisition of the radial migration properties and the unipolar dendritic morphology characterizing pyramidal neurons through molecular mechanisms distinct from those mediating its proneural activity (Hand, 2005).

Several studies have already illustrated the importance of protein-protein interactions and posttranslational modifications in mediating some of the biological functions of bHLH transcription factors. The current results show that the DNA binding-independent function of Ngn2 in the specification of the neuronal migration properties and dendritic morphology of cortical neurons is mediated by phosphorylation of tyrosine 241. This tyrosine residue is part of a larger Ngn2-specific proline-rich domain (YWQPPPP) motif that constitutes a predicted SH2 binding site for non-receptor tyrosine kinase. Future experiments will be aimed at determining which non-receptor tyrosine kinase phosphorylates Ngn2Y241 and how this phosphorylation regulates Ngn2 function (Hand, 2005).

It is hypothesized that the subtype specification activity mediated by phosphorylation of Ngn2Y241 regulates protein-protein interaction, for example, by converting a transcriptional repressor into an activator, thereby inducing the transcription of downstream target genes that regulate neuronal migration and dendritic polarity such as specific Rho-GAPs. This model would explain why Ngn2Y241F is acting as a dominant negative over Ngn2. Current experiments are aimed at identifying phosphorylation-dependent interactors of Ngn2Y241 that might mediate the transcription of specific target genes controlling radial migration and the polarity of dendritic outgrowth (Hand, 2005).

Cell cycle-regulated multi-site phosphorylation of Neurogenin 2 coordinates cell cycling with differentiation during neurogenesis

During development of the central nervous system, the transition from progenitor maintenance to differentiation is directly triggered by a lengthening of the cell cycle that occurs as development progresses. However, the mechanistic basis of this regulation is unknown. The proneural transcription factor Neurogenin 2 (Ngn2) acts as a master regulator of neuronal differentiation. This study demonstrates that Ngn2 is phosphorylated on multiple serine-proline sites in response to rising cyclin-dependent kinase (cdk) levels. This multi-site phosphorylation results in quantitative inhibition of the ability of Ngn2 to induce neurogenesis in vivo and in vitro. Mechanistically, multi-site phosphorylation inhibits binding of Ngn2 to E box DNA, and inhibition of DNA binding depends on the number of phosphorylation sites available, quantitatively controlling promoter occupancy in a rheostat-like manner. Neuronal differentiation driven by a mutant of Ngn2 that cannot be phosphorylated by cdks is no longer inhibited by elevated cdk kinase levels. Additionally, phosphomutant Ngn2-driven neuronal differentiation shows a reduced requirement for the presence of cdk inhibitors. From these results, a model is proposed whereby multi-site cdk-dependent phosphorylation of Ngn2 interprets cdk levels to control neuronal differentiation in response to cell cycle lengthening during development (Ali, 2011).

Neurogenin homologs and gliogenesis

To study the role of basic helix-loop-helix (bHLH) transcription factors in gliogenesis, bHLH transcription factors expressed in glial precursor cells were examined along with the participation of these factors in regulating oligodendrocyte and astrocyte development. As assessed by reverse transcription-polymerase chain reaction (RT-PCR), Neurogenin3 (Ngn3) is transiently expressed in bipotential glial cells fated to become either oligodendrocytes or astrocytes. Mice lacking Ngn3 display a loss of Nkx2.2 expression, a transcription factor required for proper oligodendrogliogenesis. Furthermore, a reduction in the expression of myelin basic protein (MBP), proteolipid protein (PLP), and glial fibrillary acidic protein (GFAP), markers for mature oligodendrocytes and astrocytes, was observed in the Ngn3 null mice. Overexpression of Ngn3 is sufficient to drive expression from the PLP promoter in transient cotransfection assays. Overall, the data suggest that Ngn3 may regulate glial differentiation at a developmental stage prior to the segregation of the oligodendrocyte and astrocyte lineage (J. Lee, 2003).

Oligodendrocytes, the myelinating cell type of the central nervous system, arise from a ventral population of precursors that also produces motoneurons. Although the mechanisms that specify motoneuron development are well described, the mechanisms that generate oligodendrocytes from the same precursor population are largely unknown. By analyzing mutant zebrafish embryos, it has been found that Delta-Notch signaling is required for spinal cord oligodendrocyte specification. Using a transgenic, conditional expression system, it was also learned that constitutive Notch activity promotes formation of excess oligodendrocyte progenitor cells (OPCs). However, excess OPCs are induced only in ventral spinal cord at the time that OPCs normally develop. These data provide evidence that Notch signaling maintains subsets of ventral spinal cord precursors during neuronal birth and, acting with other temporally and spatially restricted factors, specifies them for oligodendrocyte fate (Park, 2003).

Because mouse embryos that are homozygous for null mutations of Delta or Notch genes die at early stages of neural development, there is little information that addresses the requirement of Notch signaling for vertebrate CNS glial specification. This limitation can be circumvented through analysis of mice in which Notch1 is conditionally inactivated in the cerebellum. These mice prematurely express neuronal markers and have reduced number of mutant cerebellar cells that express the glial marker GFAP. In an alternative approach, neurospheres can derived from Delta-like 1 mutant mice. After culturing, mutant neurospheres produce excess neurons and a deficit of oligodendrocytes and astrocytes compared with controls. Additionally, retinas of mice that are homozygous for a mutation of Hes5, which encodes a downstream effector of Notch signaling, have fewer Müller glia than the wild type. These observations are consistent with the idea that Delta-Notch signaling regulates neuronal-glial fate decisions (Park, 2003).

A switch between production of neurons and glial cells has been proposed to be regulated by bHLH proteins. In the ventral spinal cord, motoneuron and oligodendrocyte precursors expressed Olig bHLH proteins, which are structurally similar to proneural Ngns. During the period of motoneuron production, a subset of Olig+ cells expressed Ngns. Later, Ngn expression subsides, coincident with the time at which oligodendrocytes are thought to be specified. These observations, coupled with various functional tests, led to the proposal that Ngn and Olig proteins create a simple bHLH protein code in which Ngn and Olig expression together specify motoneuron development and Olig alone, upon Ngn downregulation, specifies oligodendrocyte development (Park, 2003).

The data provide evidence supporting the importance of a bHLH protein code to motoneuron and oligodendrocyte specification and show that Delta-Notch signaling is required to establish the code. The failure to restrict ngn1 expression to a subset of medial neural plate cells in Notch signaling deficient zebrafish embryos correlates with formation of excess neurons, consistent with observations that Notch signaling inhibits proneural genes expression and neuronal development in vertebrate and invertebrate embryos. Furthermore, dla-/-;dld-/- and mib-/- embryos fail to maintain a proliferative population of olig2+ cells. This is interpreted to mean that, in the absence of Delta-Notch mediated inhibition, uniformly high levels of Ngns cause all olig2+ neural precursors to stop dividing and differentiate as neurons at the expense of oligodendrocytes. Thus, in normal embryos, high levels of Notch activity prevents ngn gene expression in a subset of olig2+ neural precursors, reserving them to produce other cell types, such as oligodendrocytes, at a later time. In this view, Delta-Notch signaling might play a purely permissive role in neural cell fate diversification, by regulating the ability of neural precursors to respond to other instructive signals (Park, 2003).

Combinatorial actions of patterning and HLH transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing spinal cord

During development, the three major neural cell lineages, neurons, oligodendrocytes and astrocytes, differentiate in specific temporal orders at topologically defined positions. How the timing and position of their generation are coordinately regulated remains poorly understood. Evidence is presented that the transcription factors Pax6, Olig2 and Nkx2.2 (Nkx2-2), which define the positional identity of multipotent progenitors early in development, also play crucial roles in controlling the timing of neurogenesis and gliogenesis in the developing ventral spinal cord. Each of these factors has a unique ability to either enhance or inhibit the activities of the proneural helix-loop-helix (HLH) factors Ngn1 (Neurog1), Ngn2 (Neurog2), Ngn3 (Neurog3) and Mash1 (Ascl1), and the inhibitory HLH factors Id1 and Hes1, thereby regulating both the timing of differentiation of multipotent progenitors and their fate. Consistent with this, dynamic changes in their co-expression pattern in vivo are closely correlated to stage- and domain-specific generation of three neural cell lineages. Genetic manipulations of their temporal expression patterns in mice alter the timing of differentiation of neurons and glia. A molecular code model is proposed whereby the combinatorial actions of two classes of transcription factors coordinately regulate the domain-specific temporal sequence of neurogenesis and gliogenesis in the developing spinal cord (Sugimori, 2007).

This study demonstrates the combinatorial actions of two classes of transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing ventral spinal cord. In vitro data have shown that the proneural HLH factors Ngns and Mash1 intrinsically possess the activity to induce neurons and oligodendrocytes, respectively, whereas the inhibitory HLH factors Id1 and Hes1 stimulate astrogenesis. Yet, the timing of differentiation of neurons and glia in vivo is not determined a priori by the expression of these HLH factors. The data have shown that they do so in collaboration with Pax6, Olig2 and Nkx2.2, the primary function of which has been thought to be to specify the positional identity of progenitors (Sugimori, 2007).

These patterning factors participate in controlling both the timing of differentiation and cell fate by two mechanisms. First, they act to maintain progenitors undifferentiated by suppressing otherwise strong neurogenic and astrogenic activities of Ngns and Id1 and/or Hes1. The suppression of the neurogenic activity of Ngn2 by Olig2 is in accordance with the fact that the Olig2+ domain markedly expands while producing a large number of motoneurons. Such an activity, however, is not limited to Olig2, but common among three patterning factors. Second, three patterning factors differentially modulate the activity of Mash1. Mash1 itself promotes differentiation of both neurons and oligodendrocytes. Pax6, however, converts Mash1 to become selectively neurogenic, whereas Olig2 selectively enhances Mash1-dependent oligodendrogenesis. Thus, it is proposed that these two classes of transcription factors comprise a molecular code for the coordinated spatiotemporal control of neuro/gliogenesis. According to this model, the relative expression levels of patterning and HLH factors at the single cell level are crucial to determine the fate of multipotent progenitors. How the timing and expression level of individual factors are precisely controlled remains to be further investigated. How these two classes of transcription factors coordinately regulate genetic programs for differentiation of neurons and glia also needs to be examined in the future studies (Sugimori, 2007).

Neurogenin 1 (Neurog1) expression in the ventral neural tube is mediated by a distinct enhancer and preferentially marks ventral interneuron lineages

The bHLH transcription factor Neurog1 (Ngn1, Neurod3, neurogenin 1) is involved in neuronal differentiation and cell-type specification in distinct regions of the developing nervous system. In this study transgenic mouse models were developed that use a Bacterial Artificial Chromosome (BAC) containing 208kb flanking the Neurog1 gene to efficiently drive expression of GFP and Cre in all Neurog1 domains. Two characteristics of Neurog1 gene regulation were uncovered. First, a 4kb region previously shown to be sufficient for driving expression of a reporter gene to a subset of the Neurog1 pattern in the developing midbrain, hindbrain, and spinal cord is required uniformly for high levels of expression in all Neurog1 domains, even those not originally identified as being regulated by this region. Second, a 0.8 kb enhancer was identified that is sufficient to drive Neurog1-like expression specifically in the ventral neural tube. Furthermore, Neurog1 progenitor cells in the ventral neural tube are largely fated to interneuron lineages rather than to motoneurons. These studies provide new tools for directing tissue specific expression in the developing neural tube, define Neurog1 lineages in the spinal cord, and further define the complex genomic structure required for obtaining the correct levels and spatial restriction of the neuronal differentiation gene Neurog1 (Quiñones, 2010).

Neurogenin homologs and cell fate determination in the mammalian retina

The expression and function of the basic helix-loop-helix (bHLH) transcription factor NeuroD were studied in the developing neural retina in rodent. neuroD was expressed in areas of undetermined retinal cells as well as developing photoreceptors and amacrine interneurons. Expression is maintained in a subset of mature photoreceptors in the adult retina. Using both loss-of-function and gain-of-function approaches, NeuroD was found to play multiple roles in retinal development. (1) NeuroD is a critical regulator of the neuron versus glial cell fate decision. Retinal explants derived from NeuroD-null mice demonstrate a three- to four-fold increase in Muller glia. Forced expression of neuroD in progenitors in rat using retroviruses hasten cell cycle withdrawal and block gliogenesis in vivo. (2) NeuroD appeared to regulate interneuron development, favouring amacrine over bipolar differentiation. Forced NeuroD expression results in an increase in amacrine interneurons and a decrease in bipolar interneurons. In the complementary experiment, retinae derived from NeuroD-null mice demonstrate a twofold increase in bipolar interneurons and a delay in amacrine differentiation. (3) NeuroD appears to be essential for the survival of a subset of rod photoreceptors. In conclusion, these results implicate NeuroD in a variety of developmental functions including cell fate determination, differentiation and neuron survival (Morrow, 1999).

The basic helix-loop-helix genes Math3 and NeuroD are expressed by differentiating amacrine cells, retinal interneurons. Previous studies have demonstrated that a normal number of amacrine cells is generated in mice lacking either Math3 or NeuroD. In Math3-NeuroD double-mutant retina, amacrine cells are completely missing, while ganglion and Müller glial cells are increased in number. In the double-mutant retina, the cells that would normally differentiate into amacrine cells do not die but adopt the ganglion and glial cell fates. Misexpression studies using the developing retinal explant cultures show that, although Math3 and NeuroD alone promote only rod genesis, they significantly increase the population of amacrine cells when either the homeobox genes Pax6 or Six3 is co-expressed. These results indicate that Math3 and NeuroD are essential, but not sufficient, for amacrine cell genesis, and that co-expression of the basic helix-loop-helix and homeobox genes is required for specification of the correct neuronal subtype (Inoue, 2002).

Thus, in Math3/NeuroD double-mutant retina, amacrine cells are completely missing without significant cell death. The cells that would normally differentiate into amacrine cells remain in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the double-mutant retina. The majority of the lacZ+ cells in the GCL and INL are small in size and show a ganglion cell phenotype, while others display a Müller glia phenotype. In accordance with this observation, ganglion and Müller glial cells are increased in number in the double-mutant retina. Normally, nearly 60% of the GCL cells are displaced amacrine cells and the others are ganglion cells. By contrast, in the double-mutant retina, all of the GCL cells are ganglion cells. Furthermore, there are many ectopic ganglion cells and extra Müller glial cells in the INL of the double-mutant retina. These results indicate that there is a fate switch from amacrine cells to ganglion and Müller glial cells in the absence of Math3 and NeuroD. Since amacrine cell genesis overlaps with ganglion and Müller glial cell genesis, it is likely that the double-mutant cells may adopt the alternatively available cell fates (Inoue, 2002).

The phenotype similar but opposite to the Math3/NeuroD double mutation is observed in the retina lacking the bHLH gene Math5 in mouse and its ortholog in zebrafish, which shows a fate switch from ganglion cells to amacrine cells. Thus, Math5 regulates ganglion versus amacrine cell fate, suggesting that this bHLH gene is involved in the neuronal subtype specification rather than the neuronal versus glial cell fate decision. By contrast, the retina lacking Mash1 and Math3 exhibit a fate switch from bipolar cells to Müller glial cells, indicating that Mash1 and Math3 regulate neuronal versus glial fate determination in the retina. The present data show that Math3-NeuroD double mutation leads to increase of both ganglion cells and Müller glia at the expense of amacrine cells, suggesting that Math3 and NeuroD regulate both neuronal subtype specification and neuronal versus glial fate determination. Although these bHLH genes seem to have distinct activities, it is speculated that the two types of fate switches, neurons to glia and neuronal subtype changes, may simply reflect the different competence of retinal precursors. Because ganglion cell genesis overlaps with amacrine cell genesis but not with Müller glial cell genesis, it is likely that the cells that would differentiate into ganglion cells have a potential to become amacrine cells but not others. Thus, in the absence of Math5, the cells that fail to differentiate into ganglion cells may predominantly become amacrine cells. Likewise, since bipolar and Müller glial cells are the last cell types to be generated, the cells that fail to differentiate into bipolar cells may have the only choice to become Müller glia in the absence of Mash1 and Math3. By contrast, since amacrine cell genesis overlaps with both ganglion and Müller glial cell genesis, the cells that fail to differentiate into amacrine cells may have a potential to become both ganglion and Müller glial cells and thereby adopt these two fates in the absence of Math3 and NeuroD. Thus, the two types of fate switches, neurons to glia and neuronal subtype changes, might mostly reflect the competence of retinal precursors, and it is likely that the cells that are blocked from differentiation to a particular cell type may simply adopt alternatively available cell fates (Inoue, 2002).

RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression but so far they have not been studied extensively in the context of developmental processes. A novel RNA-binding protein, XSEB4R, is strongly expressed in the nervous system. This study is focused on the analysis of Xseb4R in the context of primary neurogenesis and retinogenesis. To study Xseb4R function during eye development, a new protocol was set up allowing in vivo lipofection of antisense morpholino oligonucleotides into the retina. The resulting XSEB4R knockdown causes an impairment of neuronal differentiation, with an increase in the number of glial cells. By contrast, gain-of-function analysis demonstrates that Xseb4R strongly promotes neural differentiation. A similar function is shown during primary neurogenesis. Consistent with this proneural effect, in the open neural plate Xseb4R expression is upregulated by the proneural gene XNgnr1, as well as by the differentiation gene XNeuroD, but is inhibited by the Notch/Delta pathway. Altogether, these results suggest for the first time a proneural effect for a RNA-binding protein involved in the genetic network of retinogenesis (Boy, 2004).

In the developing retina, the production of ganglion cells is dependent on the proneural proteins NGN2 and ATH5, whose activities define stages along the pathway converting progenitors into newborn neurons. Crossregulatory interactions between NGN2, ATH5 and HES1 maintain the uncommitted status of ATH5-expressing cells during progenitor patterning, and later on regulate the transition from competence to cell fate commitment. Prior to exiting the cell cycle, a subset of progenitors is selected from the pool of ATH5-expressing cells to go through a crucial step in the acquisition of a definitive retinal ganglion cell (RGC) fate. The selected cells are those in which the upregulation of NGN2, the downregulation of HES1 and the autostimulation of ATH5 are coordinated with the progression of progenitors through the last cell cycle. This coordinated pattern initiates the transcription of ganglion cell-specific traits and determines the size of the ganglion cell population (Matter-Sadzinski, 2005).

Spatial cell patterning and RGC commitment correlate with the two main phases of ATH5 expression. During the period of patterning, crossregulatory interactions between HES1, NGN2 and ATH5 keep ATH5 expression low, thereby maintaining the uncommitted status of ATH5-expressing cells and enabling the expansion and intermingling of pools of progenitors initially partitioned in distinct domains. Once progenitors are properly distributed throughout the retina, about one-third of ATH5-expressing cells become committed to acquire a definitive RGC fate immediately before exiting the cell cycle. This requires a tight coordination between downregulation of HES1, upregulation of NGN2, cell progression through the last S-phase and the upregulation of ATH5. Cells that upregulate ATH5 expression initiate transcription of early RGC-specific traits, then exit the cell cycle and express Neuro M and other post-mitotic RGC-specific genes. This study highlights how changes in the transcriptional patterns correlate with the progression of progenitors through the last cell cycle and with their commitment to the RGC fate, underlining the role of HES1 as a key prompt of the molecular events leading to RGC genesis (Matter-Sadzinski, 2005).

A specific feature of retinogenesis is that it proceeds from the centre to the periphery such that all seven retinal cell types are distributed at the proper ratio throughout the retina. At early stages of development, the retinal neuroepithelium is subdivided into two developmentally distinct territories. Low levels of HES1 transcripts outline a broad region of the posterior retina where ATH5, NGN2 and ASH1 are expressed, whereas a robust accumulation of HES1 transcripts throughout the anterior retina prevents the onset of proneural gene expression. HES1 functions similarly at the onset of neurogenesis in the olfactory placode, where it circumscribes a domain of Mash1 expression. It thus appears that HES1 is acting, much like hairy in Drosophila, as a prepattern gene. Neurogenesis starts within a rather broad central region defined by expression of ATH5, NGN2 and Neuro M. Cells expressing ATH5 at a high level and Neuro M-positive cells are evenly distributed throughout the neurogenic domain, indicating that the first newborn RGCs are produced with similar frequency throughout the central retina. In the posterior retina, cells that initiated expression of proneural genes are initially organized in two separate domains corresponding to two retinal lineages: cells that express NGN2/ATH5 constitute the progenitor pools from which early-born retinal neurons will emerge, whereas ASH1-expressing cells form a pool for late-born neurons. The opposite effects of NGN2 on ATH5 and ASH1 expression combined with the inhibitory activity of ASH1 on ATH5 transcription account for the distribution of ASH1 and ATH5/NGN2 cells in two distinct progenitor domains, the more peripheral expression of ASH1 perhaps reflecting its lower sensitivity towards HES1. The initial patterning of the posterior retina resembles the neuroepithelial partitioning detected in other areas of the developing CNS. However, whereas in other CNS regions the refining of borders is essential for the precise spatial generation of different classes of neurons along the dorsoventral axis, the blurring of borders and intermingling of initially distinct progenitor pools are necessary for a proper spatial distribution of neurons and glia throughout the retina. Although ATH5/NGN2 and ASH1 expressions are mutually exclusive, a small fraction of ATH5-expressing cells co-express ASH1, indicating that they are in a transient state prior to acquiring a definite progenitor status. Because the ATH5, NGN2 and ASH1 genes crossregulate and display different sensitivities towards HES1, it is supposed that various balances between these four factors may mediate alternate fate choices. Such dynamic regulatory interactions are, in part, responsible for the progressive loss of patterning in the posterior retina. The ATH5/NGN2 domain remains restricted to the posterior retina until E4 and expands to keep pace with growth of the whole retina at a rate similar to that reported for the differentiation of RGCs. Despite significant changes in the expression pattern of ATH5, similar proportions of retinal cells express this gene at stages 18 and 29-30, suggesting that ATH5-expressing cells propagate at a rate comparable with that of the other progenitors during the period of patterning (Matter-Sadzinski, 2005).

Even though the population of ATH5-expressing cells is established at E2.5, only a small fraction of these will differentiate into RGCs until E4. Retinogenesis is controlled by components of the Notch pathway, which may employ two strategies to keep the majority of cells in the central retina from differentiating during the patterning period. Cells that express proneural genes may promote the upregulation of HES1 in neighbouring cells, thereby preventing them from expressing proneural genes. The proximity in central retina of individual cells that highly express HES1 or ATH5 is indeed indicative of ongoing lateral inhibition. However, cells strongly expressing Notch effectors are rare in the posterior retina, whereas a high proportion of ATH5-expressing progenitors co-express HES1. Thus, it appears that the low level of HES1 in cells that have already initiated NGN2 and ATH5 expression suffices to prevent the upregulation of these genes. The proliferative state is thereby maintained in most ATH5-expressing cells, as required to ensure the proper ratio of RGC progenitors in the posterior retina and as expected of HES genes, which function to keep neuroepithelial cells undifferentiated, thereby regulating the size and cell architecture of brain structures and retina. In anterior retina, progenitor cell patterning becomes evident by E4 and the expansion of proneural gene expression proceeds, much as in zebrafish, in a wave-like fashion as HES1 expression recedes to the retinal margin. The ASH1 and NGN2 expression domains expand to the periphery at similar rates, whereas the progression of the ATH5 domain is slightly delayed. The full patterning of the retina accomplished around E6 coincides with the upregulation of proneural gene expression throughout the retina and with the peak of RGC production (Matter-Sadzinski, 2005).

To analyse how ATH5 is regulated along the course of RGC specification, a promoter region extending 775 bp upstream of the initiation codon was used. The cloned sequence accurately reproduces the activity and the mode of regulation of the endogenous promoter. It contains essential regulatory elements that are well conserved across distant vertebrate species, but it is unclear whether the different species use similar strategies to regulate ATH5 expression. Whereas a proximal cis-regulatory region of the Xenopus Xath5 gene suffices, much as in the chick retina, to drive retina specific reporter gene expression in a bHLH-dependent manner, the mouse ATH5 promoter appears to be regulated differently. It is tempting to speculate that the different modes regulating ATH5 across species may account for differences in the spatiotemporal progenitor patterning of the retinal neuroepithelium. Differences in the developments of the anterior and posterior retinas may have permitted the evolution of a specialized structure such as the macula (Matter-Sadzinski, 2005).

This study reveals that NGN2 acts at different regulatory levels during RGC specification. In early retina, NGN2 is a principal regulator of ATH5 expression and exerts this function through direct activation of ATH5 transcription and through crossregulatory interactions with HES1. In addition, NGN2 drives ATH5-expressing cells out of S phase. Whereas the capacity of NGN2 to promote cell cycle arrest is part of its panneuronal activities and is in evidence in other compartments of the developing CNS, its capacity to activate ATH5 expression is largely retina specific. The quasi-simultaneous onset of NGN2 and ATH5 expression in the central retina shortly after formation of the eye cup, the capacity of NGN2 to activate ATH5 transcription and to bind the ATH5 promoter at the early stages of development suggest that NGN2 may be directly involved in the activation of ATH5 expression. The finding that the expansion of the NGN2 domain towards the anterior edge of the retina precedes that of ATH5 argues in favour of this interpretation. In the retina of the Ngn2–/– mouse, the much increased expression of ASH1 and the downregulation of ATH5 when compared with the wild type, may result from an increase in the population of ASH1-expressing cells at the expense of the ATH5/NGN2 progenitors, thus underlining the importance of NGN2 in establishing and maintaining a pool of ATH5-expressing cells. Both the NGN2 and ATH5 genes fail to be activated in the retinal precursors of the Pax6–/– mouse and Pax6 has been proposed to regulate NGN2 directly in the mouse retina. There are multiple E-boxes but no consensus Pax6 binding site in the chicken ATH5 promoter, and therefore the idea is favored that Pax6 regulates ATH5 via NGN2. The expression of NGN2 in many regions of the nervous system anlage where ATH5 is not detected and the demonstration that recruitment of NGN2 on the ATH5 promoter is retina specific provide evidence that a retina-specific context accounts for the capacity of NGN2 to activate ATH5 expression. The ability of bHLH factors to regulate the development of distinct neurons has been proposed to depend upon the cellular contexts in which they function. In retina, this context may be determined, among other possibilities, by the balance between NGN2 and HES1: as show, HES1 inhibits the NGN2-mediated activation of ATH5 in a dose-dependent manner. Likewise, the upregulation of NGN2 correlates with the dowregulation of HES1. Moreover, single cell transcriptional analysis reveals that overexpressing NGN2 diminishes the pool of cells that co-express ATH5 and HES1, an indication that NGN2 may contribute to the downregulation of HES1 in early neural progenitors, thereby providing a cellular environment permissive for ATH5 autostimulation (Matter-Sadzinski, 2005).

The upregulation of both NGN2 and ATH5 occurs later in development, around E6, but by then ATH5 has become the main regulator of its own transcription. NGN2 occupies the ATH5 promoter similarly at E3 and at E6, suggesting that it still directly participates in the control of ATH5 transcription. However, its main contribution to ATH5 expression may occur through other, indirect regulatory pathways. As ATH5-expressing progenitors exit the cell cycle, NGN2 promotes the expression first of Neuro M and then of Neuro D, both stimulators of ATH5 promoter activity. These distinct functions of NGN2 in the ontogenesis of RGCs illustrate how, depending on specific combinations of transcription factors and of other cellular components, neurogenic proteins may contribute to neuronal identity (Matter-Sadzinski, 2005).

Rewiring the retinal ganglion cell gene regulatory network: Neurod1 promotes retinal ganglion cell fate in the absence of Math5

Retinal progenitor cells (RPCs) express basic helix-loop-helix (bHLH) factors in a strikingly mosaic spatiotemporal pattern, which is thought to contribute to the establishment of individual retinal cell identity. This study asked whether a tightly regulated pattern is essential for the orderly differentiation of the early retinal cell types and whether different bHLH genes have distinct functions that are adapted for each RPC. To address these issues, one bHLH gene was replaced with another. Math5 is a bHLH gene that is essential for establishing retinal ganglion cell (RGC) fate. The retinas were examined of mice in which Math5 was replaced with Neurod1 or Math3, bHLH genes that are expressed in another RPC and are required to establish amacrine cell fate. In the absence of Math5, Math5Neurod1-KI was able to specify RGCs, activate RGC genes and restore the optic nerve, although not as effectively as Math5. By contrast, Math5Math3-KI was much less effective than Math5Neurod1-KI in replacing Math5. In addition, expression of Neurod1 and Math3 from the Math5Neurod1-KI/Math3-KI allele did not result in enhanced amacrine cell production. These results were unexpected because they indicated that bHLH genes, which are currently thought to have evolved highly specialized functions, are nonetheless able to adjust their functions by interpreting the local positional information that is programmed into the RPC lineages. It is concluded that, although Neurod1 and Math3 have evolved specialized functions for establishing amacrine cell fate, they are nevertheless capable of alternative functions when expressed in foreign environments (Mao, 2008).

Neurogenin and the olfactory sensory neuron lineage

The lineage of olfactory neurons has been relatively well characterized at the cellular level, but the genes that regulate the proliferation and differentiation of their progenitors are currently unknown. Math4C/neurogenin1 and the basic helix-loop-helix gene Mash1 (Drosophila homolog: achaete) are expressed in the olfactory epithelium by different dividing progenitor populations, while another basic helix-loop-helix gene, NeuroD, is expressed at the onset of neuronal differentiation. These expression patterns suggest that each gene marks a distinct stage of olfactory neuron progenitor development in the following sequence: Mash1>Math4C/neurogenin1>NeuroD. Inactivation of Mash1 function leads to a severe reduction in the number of olfactory neurons. Most cells in the olfactory epithelium of Mash1 mutant embryos fail to express Math4C/neurogenin1 or NeuroD. Strikingly, a subset of progenitor cells in a ventrocaudal domain of Mash1 mutant olfactory epithelium still express Math4C/neurogenin1 and NeuroD and differentiate into neurons. Cells in this domain also express Math4A/neurogenin2, another member of the Math4/neurogenin gene family, and not Mash1. These results demonstrate that Mash1 is required at an early stage in the olfactory neuron lineage to initiate a differentiation program involving Math4C/neurogenin1 and NeuroD. Another gene activates a similar program in a separate population of olfactory neuron progenitors (Cau, 1997).

bHLH transcription factors are expressed sequentially during the development of neural lineages, suggesting that they operate in genetic cascades. In the olfactory epithelium (OE), the proneural genes Mash1 and neurogenin1 are expressed at distinct steps in the same olfactory sensory neuron (OSN) lineage. Loss-of-function analysis shows that both genes are required for the generation of OSNs. However, their mutant phenotypes are strikingly different, indicating that they have divergent functions. In Mash1 null mutant mice, olfactory progenitors are not produced and the Notch signaling pathway is not activated, establishing Mash1 as a determination gene for olfactory sensory neurons. In neurogenin1 null mutant mice, olfactory progenitors are generated but they express only a subset of their normal repertoire of regulatory molecules and their differentiation is blocked. Thus neurogenin1 is required for the activation of one of several parallel genetic programs functioning downstream of Mash1 in the differentiation of OSNs. These results illustrate the versatility of neural bHLH genes which adopt either a determination or a differentiation function, depending primarily on the timing of their expression in neural progenitors (Cau, 2002).

The analysis of the Ngn1 mutant phenotype in the OE has revealed the expression in the OE of two groups of regulatory genes distinct in their mode of regulation in basal progenitors. Genes of the first group, which include NeuroD and paired-homeobox gene Phd1, require Ngn1 function for their expression. Genes of the second group, which include the Lim-homeobox gene Lhx2 and the HLH gene Ebf1, are activated in basal progenitors independently of Ngn1 activity. None of these genes are expressed in Mash1 mutant OE, indicating that they belong to distinct regulatory pathways that are activated downstream of Mash1 in basal progenitors. It has been proposed that in neural crest-derived progenitors, Mash1 couples two parallel differentiation programs controlling the expression of neuronal subtype genes (e.g. the homeobox gene Phox2a and genes encoding the neurotransmitter synthesizing enzymes TH and BDH) and pan-neuronal genes (e.g., genes encoding peripherin and NF160). The idea that neuronal differentiation entails the activation of distinct regulatory programs implementing the different aspects of the neuronal phenotype has been amply supported experimentally. The results presented here indicate that Mash1 is likely to similarly couple the various components of the OSN phenotype. Among the regulatory genes expressed in basal progenitors and missing in Mash1 mutant OE, NeuroD and Ebf1 are likely to promote generic neuronal differentiation of OSNs. Both genes are broadly expressed in neurons in the embryonic nervous system and have the capacity to induce ectopic neurons when forcibly expressed in Xenopus embryos. In contrast, other basal OE progenitor genes, e.g., Phd1 and Lhx2, are likely to be involved in the specification of OSN identity. These genes belong to the paired-homeodomain and Lim-homeodomain families of transcription factors, respectively, which have been implicated in specification of various aspects of the neuronal phenotype. It is interesting to note that the Ngn1-dependent and -independent groups of regulators defined in this study do not segregate into 'pan-neuronal' (NeuroD and Ebf1) and 'neuronal subtype' (Phd1 and Lhx2) categories, but are on the contrary distributed in both. This suggests that the regulatory programs supporting OSN differentiation are not specialized in the acquisition of either generic or OSN-specific traits, but may instead control the acquisition of different combinations of both types of traits. The logic behind this complex regulation of the OSN phenotype remains to be elucidated (Cau, 2002).

Mice lacking both Mash1 and Ngn1 present a complete depletion of OSNs at all stages examined, indicating that together, Mash1 and Ngn1 are required for the progression of neurogenesis throughout the OE. Analysis of the olfactory placodes of Mash1;Ngn1 double mutant embryos shows that the two genes have redundant functions in the determination of OSN progenitors at this early stage. In particular, a lack of Dll1 and Hes5 expression reveals that Notch signaling is not activated and that OSN progenitors are likely missing from the olfactory placodes in the absence of both Mash1 and Ngn1 (Cau, 2002).

The redundancy of Mash1 and Ngn1 function in the determination of placodal progenitors at E10.0 is in marked contrast to the distinct and sequential roles of the two genes in the OE at E12.5. Even at E12.5, the Mash1/Ngn1 cascade does not operate in all OSN progenitors, since Ngn1 is expressed and required in a subset of Mash1 mutant OE progenitors at this stage. The fact that Mash1-independent progenitors are found at reproducible locations in different Mash1 mutant embryos argues for the existence of a distinct progenitor population in which a neurogenesis program including Ngn1 expression can be activated without Mash1 function. Whether Ngn1 is required for the determination of Mash1-independent progenitors at E12.5 as is the case for progenitors of olfactory placodes, or for their differentiation as is the case for other E12.5 OE progenitors, is difficult to address given the rarity of these cells. In any case, the epistatic relationship between Mash1 and Ngn1 observed in most OSN progenitors at E12.5 is not a general feature of the neurogenesis program in the OE, but is instead restricted both temporally (to the E12.5 OE and not the E10.0 placode) and spatially (to the rostral OE and less so to the caudal OE) (Cau, 2002).

How can these findings be reconciled with the observation that in regulatory cascades underlying cell lineage development, distinct subfamilies of bHLH factors are usually used as either early expressed determination factors or later expressed differentiation genes? These results support the idea that determination factors also have the necessary properties to participate to neuronal differentiation programs, and that the specific determination or differentiation function of genes like Ngn1 or Mash1 depends primarily on the timing of their expression and on the context of their activity. In contrast, other neural bHLH genes such as NeuroD and related genes, are consistently expressed in late precursors and post-mitotic neurons and have not been implicated in the process of progenitor selection during normal development, although NeuroD shares with Ngns the property to induce neurons and ectopically activate Notch signaling when overexpressed in Xenopus embryos, and NeuroD has been shown to participate to the choice between neuronal and glial fates in the retina. The fact that differentiation genes such as NeuroD are normally not involved in cell determination suggests that they may lack some of the necessary properties. Indeed, the myogenic determination factors MyoD and Myf5 are more efficient than the differentiation factor myogenin at remodelling chromatin and activate transcription at previously silent loci, an activity which is very likely relevant to their determination function. Possibly as a consequence of these divergent activities, myogenin cannot fully substitute for Myf5 when expressed from the Myf5 locus. It will be interesting to test the prediction that neural determination genes (Mash1 and Ngns) can efficiently substitute for differentiation genes (NeuroD and related genes) but not the reverse, in similar gene swapping experiments (Cau, 2002).

NeuroD and Ear Development

BETA2/NeuroD1 is a bHLH transcription factor that is expressed during development in the mammalian pancreas and in many locations in the central and peripheral nervous systems. During inner ear ontogenesis, it is present in both sensory ganglion neurons and sensory epithelia. Although studies have shown that BETA2/NeuroD1 is important in the development of the hippocampal dentate gyrus and the cerebellum, its functions in the peripheral nervous system and in particular in the inner ear are unclear. Mice carrying a BETA2/NeuroD1 null mutation exhibit behavioral abnormalities suggestive of an inner ear defect, including lack of responsiveness to sound, hyperactivity, head tilting, and circling. These defects can be explained by a severe reduction of sensory neurons in the cochlear-vestibular ganglion (CVG). A developmental study of CVG formation in the null mutant demonstrates that BETA2/NeuroD1 does not play a primary role in the proliferation of neuroblast precursors or in their decision to become neuroblasts. Instead, the reduction in CVG neuron number is caused by a combination both of delayed or defective delamination of CVG neuroblast precursors from the otic vesicle epithelium and of enhanced apoptosis both in the otic epithelium and among those neurons that do delaminate to form the CVG. There are also defects in differentiation and patterning of the cochlear duct and sensory epithelium and loss of the dorsal cochlear nucleus. Thus, BETA2/NeuroD1 is the first gene to be shown to regulate neuronal and sensory cell development in both the cochlear and vestibular systems (Liu, 2000b).

A key factor in the genetically programmed development of the nervous system is the death of massive numbers of neurons. Therefore, genetic mechanisms governing cell survival are of fundamental importance to developmental neuroscience. Inner ear sensory neurons are dependent on a basic helix-loop-helix transcription factor called NeuroD for survival during differentiation. Mice lacking NeuroD protein exhibit no auditory evoked potentials, reflecting a profound deafness. DiI fiber staining, immunostaining and cell death assays reveal that the deafness is due to the failure of inner ear sensory neuron survival during development. The affected inner ear sensory neurons fail to express neurotrophin receptors, TrkB and TrkC, suggesting that the ability of NeuroD to support neuronal survival may be directly mediated through regulation of responsiveness to the neurotrophins (Kim, 2001).

The function of the zinc finger transcription factor GATA3 was studied in a newly established, conditionally immortal cell line derived to represent auditory sensory neuroblasts migrating from the mouse otic vesicle at embryonic day E10.5. The cell line, US/VOT-33, expresses GATA3, the bHLH transcription factor NeuroD and the POU-domain transcription factor Brn3a, as do auditory neuroblasts in vivo. When GATA3 was knocked down reversibly with antisense oligonucleotides, NeuroD was reversibly down-regulated. Auditory and vestibular neurons form from neuroblasts that express NeuroD; these neuroblasts migrate from the antero-ventral otic epithelium at E9.5-10.5. On the medial side, neuroblasts and epithelial cells express GATA3 but on the lateral side they do not. At E13.5 most auditory neurons express GATA3 but no longer express NeuroD, whereas vestibular neurons express NeuroD but not GATA3. Neuroblasts expressing NeuroD and GATA3 were located in the ventral, otic epithelium, the adjacent mesenchyme and the developing auditory ganglion. The results suggest that auditory and vestibular neurons arise from different, otic epithelial domains and that they gain their identity prior to migration. In auditory neuroblasts, NeuroD appears to be dependent on the expression of GATA3 (Lawoko-Kerali, 2004).

Cell fate specification during inner ear development is dependent upon regional gene expression within the otic vesicle. One of the earliest cell fate determination steps in this system is the specification of neural precursors, and regulators of this process include the Atonal-related basic helix-loop-helix genes, Ngn1 and NeuroD and the T-box gene, Tbx1. This study demonstrates that Eya1 signaling is critical to the normal expression patterns of Tbx1, Ngn1, and NeuroD in the developing mouse otocyst. A potential mechanism is discussed for the absence of neural precursors in the Eya1-/- inner ears and the primary and secondary mechanisms for the loss of cochleovestibular ganglion cells in the Eya1bor/bor hypomorphic mutant (Friedman, 2005 ).

Several lines of evidence support the existence of compartmental boundaries of gene expression within the otocyst governing the divergence of epithelial cell lineages. Examples include the expression of Dlx5 in the dorsal epithelium of the otocyst and its responsibility for development of the semicircular canals and the expression of Otx1 in the ventral otocyst and its essential role in cochlear morphogenesis. Specification of neural progenitors is the earliest identifiable fate determination event in the developing otocyst, beginning around E9. This subpopulation of ventral otic epithelial cells is identifiable by their expression of the Atonal-related basic helix-loop-helix genes, Neurogenin1 (Ngn1) and NeuroD. Ngn1 is necessary for neural progenitor determination and formation of the cochleovestibular ganglion (cvg). Supporting its role in inner ear development, studies in Ngn1 deficient mice show complete absence of the cvg. Ngn1 regulates another gene in this cascade, NeuroD. It is expressed in a spatially and temporally overlapping pattern with Ngn1 and promotes neuroblast delamination into the ventral mesenchyme and growth factor mediated neuronal survival. Tbx1 has recently been shown to act upstream of Ngn1 and NeuroD as a negative regulator of neural fate specification in the otocyst (Friedman, 2005).

NeuroD and neuronal differentiation of adult stem cells

A wide variety of in vivo manipulations influence neurogenesis in the adult hippocampus. It is not known, however, if adult neural stem/progenitor cells (NPCs) can intrinsically sense excitatory neural activity and thereby implement a direct coupling between excitation and neurogenesis. Moreover, the theoretical significance of activity-dependent neurogenesis in hippocampal-type memory processing networks has not been explored. This study demonstrates that excitatory stimuli act directly on adult hippocampal NPCs to favor neuron production. The excitation is sensed via Cav1.2/1.3 (L-type) Ca2+ channels and NMDA receptors on the proliferating precursors. Excitation through this pathway acts to inhibit expression of the glial fate genes Hes1 and Id2 and increase expression of NeuroD, a positive regulator of neuronal differentiation. These activity-sensing properties of the adult NPCs, when applied as an 'excitation-neurogenesis coupling rule' within a Hebbian neural network, predict significant advantages for both the temporary storage and the clearance of memories (Deisseroth, 2004).

Using an array of approaches, the coupling of excitation to neurogenesis in proliferating adult-derived NPCs was studied both in vitro and in vivo. Adult neurogenesis is potently enhanced by excitatory stimuli and involves Cav1.2/1.3 channels and NMDA receptors. These Ca2+ influx pathways are located on the proliferating NPCs, allowing them to directly sense and process excitatory stimuli. No effect of excitation was found on the extent of differentiation in individual cells (measured by extent of MAP2ab expression in the NPC-derived neurons) nor were effects observed on proliferative rate or fraction, survival, or apoptosis. Instead, excitation increased the fraction of NPC progeny that were neurons, both in vitro and in vivo, and total neuron number was increased as well. The Ca2+ signal in NPCs leads to rapid induction of a proneural gene expression pattern involving the bHLH genes HES1, Id2, and NeuroD, and the resulting cells become fully functional neurons defined by neuronal morphology, expression of neuronal structural proteins (MAP2ab and Doublecortin), expression of neuronal TTX-sensitive voltage-gated Na+ channels, and synaptic incorporation into active neural circuits. A monotonically increasing function characterizes excitation-neurogenesis coupling, and incorporation of this relationship into a layered Hebbian neural network suggests surprising advantages for both the clearance of old memories and the storage of new memories. Taken together, these results provide a new experimental and theoretical framework for further investigation of adult excitation-neurogenesis coupling (Deisseroth, 2004).

In the hippocampal formation, neural stem cells exist either within the adjacent ventricular zone or within the subgranular zone proper at the margin between the granule cell layer and the hilus, where proliferative activity is most robust. These cells do not express neuronal markers but proliferate and produce dividing progeny that incrementally commit to differentiated fates (such as the neuronal lineage) over successive cell divisions. Native NPC populations in vivo are therefore heterogenous with regard to lineage potential, and markers are not available that distinguish between the multipotent stem cell and the subtly committed yet proliferative progenitor cell. Excitation may therefore act on either or both types of proliferating precursor, in vitro and in vivo. The functional consequences of coupling excitation to insertion of new neurons for the neural network, however, is independent of which precursor cell types respond to excitation (Deisseroth, 2004).

The enhancement of hippocampal neurogenesis by behavioral stimuli such as environmental enrichment and running may, at least in part, be implemented at the molecular level by excitation-neurogenesis coupling. Notably, running and environmental enrichment increase adult neurogenesis in the hippocampus but not in the subventricular zone. Of course, not every neurogenic region in the brain need follow the excitation-neurogenesis coupling rule outlined here. An activity rule appropriate for the unique information processing or storage function of that brain region might be expected to operate. In this context, it is interesting to note that, while subventricular zone/olfactory bulb precursor neurogenesis is not enhanced by behavioral activity, proliferation and survival in this system can be influenced by olfactory sensory stimuli. This suggests that a different form of activity rule, appropriate for that local circuit, may govern olfactory bulb neurogenesis (Deisseroth, 2004).

Neurogenin and cortex development

The mechanisms by which neural stem cells give rise to neurons, astrocytes, or oligodendrocytes are beginning to be elucidated. However, it is not known how the specification of one cell lineage results in the suppression of alternative fates. In addition to inducing neurogenesis, the bHLH transcription factor neurogenin (Ngn1) inhibits the differentiation of neural stem cells into astrocytes. While Ngn1 promotes neurogenesis by functioning as a transcriptional activator, Ngn1 inhibits astrocyte differentiation by sequestering the CBP-Smad1 transcription complex away from astrocyte differentiation genes, and by inhibiting the activation of STAT transcription factors that are necessary for gliogenesis. Thus, two distinct mechanisms are involved in the activation and suppression of gene expression during cell-fate specification by neurogenin (Sun, 2001).

The mammalian cerebral cortex originates from a single layer of proliferating neuroepithelial cells. These progenitor cells line the ventricular cavities and sequentially give rise to the three major cell types of the brain: neurons, astrocytes, and oligodendrocytes. Neurogenesis precedes gliogenesis throughout the nervous system, and retroviral labeling techniques have further shown that a single progenitor can give rise to both neurons and astrocytes. It thus appears that a common cortical progenitor cell gives rise first to a variety of layer-specific neurons and then switches to producing astrocytes, and ultimately oligodendrocytes. The molecular mechanisms that orchestrate these sequential events during development are unclear (Sun, 2001 and references therein).

In culture, cortical progenitor cells isolated at different embryonic stages behave in a manner that mimics the normal process of development. Progenitors from rat embryonic day 14 (E14) cortex (at the peak of neurogenesis) primarily give rise to neurons and to dividing precursor cells. In E14 cultures, astrocytes are only generated after several days in vitro. By contrast, E17 progenitors give rise to astrocytes immediately. Multipotent neural stem cells can be isolated and expanded from primary cortical cultures after serial passaging in the presence of mitogenic growth factors. Although neural stem cell cultures are more homogenous than primary cortical cultures, both types of cultures have been used successfully to identify extracellular factors that specifically promote differentiation of stem cells into neurons, astrocytes, or oligodendrocytes (Sun, 2001 and references therein).

Neuronal differentiation is promoted by both platelet-derived growth factor (PDGF) and by neurotrophin-3 (NT3). The cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are potent inducers of astrocyte production, and thyroid hormone induces oligodendrocyte differentiation. LIF and CNTF exert their effects primarily via the JaK/STAT signaling pathway. LIF and CNTF bind to related receptors, which activate a receptor-associated tyrosine kinase, the Janus kinase (JaK1). Activated JaK1 phosphorylates two cytoplasmic proteins, the signal transducers and activators of transcription 1 and 3 (STAT1 and STAT3). This leads to STAT dimerization and translocation to the nucleus where the STATs activate cell type and stimulus-specific programs of gene expression (Sun, 2001 and references therein).

Other factors, such as bone morphogenetic protein (BMP), can enhance both neuronal and astrocyte differentiation, depending on the age of the stimulated cortical progenitors. BMP-induced astrocyte differentiation appears to be mediated by the downstream Smad signaling proteins. BMPs bind a multimeric receptor, which in turn results in the direct phosphorylation of Smad1. This permits Smad1 to dimerize with Smad4 and to translocate to the nucleus, where these factors cooperate with STATs to activate glial-specific programs of gene expression (Sun, 2001 and references therein).

The cooperation between Smads and STATs on glial promoters such as the glial fibrillary acidic protein (GFAP) promoter appears to be facilitated by a family of coactivator proteins termed p300/CBP. CBP (CREB binding protein) and p300 are ubiquitously expressed and are involved in the transcriptional coactivation of many different transcription factors. STATs and Smads bind to different domains of CBP/p300, and the STAT/p300/Smad complex, acting at the STAT binding element in the astrocyte-specific GFAP promoter, is particularly effective at inducing astrocyte differentiation in neural stem cells (Sun, 2001 and references therein).

The signaling mechanisms by which neuronal or oligodendrocytic cell fates are specified are less well understood. However, several basic helix-loop-helix (bHLH) transcription factors have been implicated as mediators of neuronal or oligodendrocyte differentiation in the developing CNS. These bHLH factors include oligo1 and oligo2 for oligodendrocyte specification, and neurogenin1 and 2 (Ngn1 and Ngn2), Mash1, and Math1 for neuronal differentiation. In the developing mammalian cerebral cortex, two closely related bHLH factors, Ngn1 and Ngn2, are expressed exclusively in the cortical ventricular zone, where neuroepithelial precursor cells reside, and only during the limited period of time when neurogenesis is taking place. Both Ngn1 and Ngn2 dimerize with ubiquitous bHLH proteins, such as E12 or E47. These heterodimers then bind via their positively charged basic domains to DNA sequences that contain the E box consensus motif, CANNTG. E box binding has been found to be critical for bHLH proteins to activate tissue-specific gene expression that promotes neuronal differentiation. The role for neurogenic bHLH proteins in the developing CNS has been substantiated by several recent knockout studies (Sun, 2001 and references therein).

Despite recent progress, there is much that is still not known about how different cell fates are determined in the developing cortex. A critical issue is how alternative cell fates are suppressed. Inducers of neuronal fate must somehow suppress glial differentiation, and likewise, glial differentiation can only proceed if the neuronal fate is blocked. In this report, it is demonstrated that Ngn1 effectively inhibits astrocyte differentiation. Two mechanisms have been identified by which Ngn1 represses glial-specific gene transcription, (1) by sequestering the CBP/p300/Smad1 complex away from glial promoters, and (2) by suppressing the JaK/STAT signaling pathway. Since neurogenin levels are high during cortical neurogenesis and low during gliogenesis, neurogenin's ability to suppress glial differentiation may explain why astrocytes fail to develop during the period of neurogenesis even in the presence of glial-inducing cues (Sun, 2001).

Besides inhibiting gliogenesis, Ngn1 and Ngn2 actively promote neurogenesis. In the cerebral cortex, Ngn1 and Ngn2 mRNAs are detected exclusively in the ventricular zone, where the precursor cells reside, and only during the period of cortical neurogenesis. To examine whether Ngn1 can influence cell fate commitment, a retroviral vector was generated to introduce exogenous Ngn1 into dividing cortical precursor cells cultured from rat E14 cortices. When cortical precursor cells were infected with the Ngn1, exogenous Ngn1 expression leads to a significant increase in the number of precursors that become neurons, as compared with the control. Neurons were identified by cell morphology, and by the expression of neuronal-specific markers such as the beta-tubulin/TuJ1 antigen and the microtubule-associated protein MAP2. In addition, the expression of Ngn1 leads to downregulation of the expression of the neuroepithelial precursor marker Nestin. This finding suggests that ectopic Ngn1 effectively induces the process of neuronal differentiation, rather than simply inducing the expression of selected neuronal-specific markers in progenitor cells (Sun, 2001).

The induction of neurogenesis by Ngn1 could be due to enhanced neuronal survival or increased proliferation of committed neuroblasts rather than neuronal differentiation per se. However, the exogenous Ngn1-expressing cells do not show increased cell survival or proliferation rates as compared with nonexpressing cells, ruling out either of these alternative explanations. The possibility that Ngn1 instructively promotes neuronal cell fate determination, as opposed to merely promoting neuronal differentiation, implies that Ngn1 induces neurogenesis at the expense of gliogenesis. During cortical development, neurogenesis precedes glial differentiation. One possibility is that glial-inducing cues are only expressed after neuronal differentiation is complete. Alternatively, Ngn1 might actively inhibit gliogenesis even in the presence of glial-inducing cues, ensuring that the process of neurogenesis is completed before gliogenesis can begin (Sun, 2001).

To explore whether Ngn1 can still induce neurogenesis in the presence of glial-inducing factors, the effect of Ngn1 on neuronal and glial differentiation was examined in cells treated with LIF/CNTF. Ectopically expressed Ngn1 is able to inhibit cytokine-induced astrocyte differentiation in both cortical precursor and neural stem cell cultures. Astrocytes were identified by their morphology and by the expression of GFAP. In E14 cortical cultures treated for four days with CNTF, neurogenin expression reduces the number of GFAP-expressing astrocytes by 80%). In addition, Ngn1-expressing cells display a neuronal-like morphology (small round cell bodies with one or two simple processes) that is quite distinct from the stellate GFAP-positive astrocytes. This implies that Ngn1 is not only regulating GFAP expression but may also affect other aspects of astrocyte differentiation (Sun, 2001).

The change in morphology is also seen when Ngn1 is expressed in neural stem cells in the presence of CNTF, which would normally cause the differentiation of virtually all of the stem cells into astrocytes. Some of the Ngn1-expressing cells express the neuronal markers TuJ1 and MAP2. The remaining Ngn1-expressing cells are TuJ1 negative, but nonetheless fail to display astrocytic morphology or GFAP expression (Sun, 2001).

Many transcription factors require CBP/p300 in order to activate transcription, and there is evidence that the levels of CBP/p300 are limiting, i.e., that there is competition among the various families of transcription factors for CBP/p300 binding. For example, nuclear steroid receptors indirectly inhibit AP-1-dependent transcription by sequestering CBP/p300 away from AP-1 and onto sites where the nuclear receptors are bound. Similarly, the anti-adenoviral actions of interferon are attributed to interferon's ability to activate STATs, which then sequester CBP/p300 away from the adenoviral transcription factor E1A. During early cortical development, endogenous Ngn1 associates with both CBP and Smad1, and the presence of neurogenin blocks STAT binding to CBP. Xenopus neurogenin has been shown to recruits CBP/p300 to the NeuroD promoter to activate transcription and induce neurogenesis. The characterization of the domains of CBP that interact with neurogenin reveal that both an N- and a C-terminal domain are involved. Interestingly, the neurogenin binding domains of CBP overlap with the STAT binding sites on CBP (but not with the Smad binding sites). This is consistent with the finding that neurogenin competes with STAT proteins for binding to CBP. By sequestering CBP, neurogenin may not only inhibit STAT-mediated transcription, but may also inhibit the function of other CBP-dependent transcription factors. Ngn1 also inhibits AP-1-dependent transcription. This may be relevant to Ngn's ability to inhibit astrocyte differentiation since the analysis of the GFAP promoter identifies multiple sites, including an AP-1 site, that contribute to neurogenin's inhibition of the GFAP promoter. Taken together, these findings suggest that CBP/p300 may orchestrate broad programs of gene expression that are relevant to cell fate determination. The effect of CBP/p300 on cell fate may then be determined by the relative binding affinity and abundance of different transcription factors that either compete or cooperate with one another for binding to CBP/p300 (Sun, 2001 and references therein).

In addition to sequestering the CBP-Smad1 complex, neurogenin also inhibits the activation of astrocyte-specific genes by blocking STAT activation. The mechanism by which Ngn1 reduces the level of phospho-STAT1 and -STAT3 is unknown. The Ngn1 deficient in binding DNS can also inhibit STAT phosphorylation, though not to the extent seen with wild-type Ngn1. This suggests that Ngn1 inhibits STAT phosphorylation only in part by a mechanism that is independent of Ngn1 binding to DNA (Sun, 2001).

The dorsal and ventral domains of the telencephalon are delineated by a unique boundary structure that restricts the migration of dorsal and ventral cells to a different extent. While many cells invade the dorsal cortex from the ventral ganglionic eminence (GE), hardly any cortical cells cross the boundary into the GE. Several molecules have been implicated in the regulation of ventral to dorsal cell migration, but so far nothing is known about the molecular mechanisms restricting cortical cell migration in vivo. In the absence of the transcription factor neurogenin 2, cells from the cortex migrate into the GE in vitro and in vivo as detected in transgenic mice containing a lacZ gene in the neurogenin 2 locus. In contrast, the migration of cells from the GE is not affected. Molecular and cellular analysis of the cortico-striatal boundary reveal that neurogenin 2 regulates the fasciculation of the cortico-striatal boundary which may explain the non cell-autonomous nature of the migration defect as detected by in vitro transplantation. Taken together, these results show that distinct cues located in the cortico-striatal boundary restrict cells in the dorsal and ventral telencephalon (Chapouton, 2001).

Mature neocortical layers all derive from the cortical plate (CP), a transient zone in the dorsal telencephalon into which young neurons are continuously delivered. To understand cytogenetic and histogenetic events that trigger the emergence of the CP, a slice culture technique has been used. Most divisions at the ventricular surface generate paired cycling daughters (precursor/precursor divisions, or P/P divisions) and the majority of the P/P divisions are asymmetric in daughter cell behavior; they frequently send one daughter cell to a non-surface position, the subventricular zone (SVZ), within a single cell-cycle length while keeping the other mitotic daughter for division at the surface. The non-surface-dividing cells are mostly positive for Hu, an RNA-binding protein that has been used as a neuronal marker in the developing cerebral wall, and their daughters are also Hu+, suggesting their commitment to the neuronal lineage and supply of early neurons at a position much closer to their destiny than from the ventricular surface. The release of a cycling daughter cell to SVZ was achieved by collapse of the ventricular process of the cell, followed by its non-surface division. Neurogenin2 (Ngn2) was immunohistochemically detected in a certain cycling population during G1 phase and was further restricted during G2-M phases to the SVZ-directed population. Its retroviral introduction converted surface divisions to non-surface divisions. The asymmetric P/P division may therefore contribute to efficient neuron/progenitor segregation required for CP initiation through cell cycle-dependent and lineage-restricted expression of Ngn2 (Miyata, 2002).

The role of the proneural bHLH genes Neurogenin2 (Ngn2) and Mash1 in the selection of neuronal and glial fates by neural stem cells has been addressed. Mice mutant for both genes present severe defects in development of the cerebral cortex, including a reduction of neurogenesis and a premature and excessive generation of astrocytic precursors. An analysis of wild-type and mutant cortical progenitors in culture shows that a large fraction of Ngn2;Mash1 double-mutant progenitors fail to adopt a neuronal fate, instead remaining pluripotent or entering an astrocytic differentiation pathway. Together, these results demonstrate that proneural genes are involved in lineage restriction of cortical progenitors, promoting the acquisition of the neuronal fate and inhibiting the astrocytic fate (Nieto, 2001).

The analysis of bHLH gene function in progenitor cultures led to three important observations: (1) by studying separately the properties of Ngn2+ and Ngn2- progenitors, it has been demonstrated that Ngn2 expression is restricted to committed neuronal and astrocytic progenitors and is absent from pluripotent progenitors; (2) both neuronal and astrocytic lineages of the cortex are heterogenous for Ngn2 expression and Mash1 has an essential function in the Ngn2- subpopulation of cortical progenitors; (3) in vitro studies show that Ngn2 and Mash1 mutations result primarily in defects in fate commitment of cortical progenitors, which remain pluripotent or adopt a glial fate rather than becoming restricted to neuronal differentiation. Together, the data obtained in vivo and in culture support the conclusion that Ngn2 and Mash1 act in different populations of cortical progenitors to promote the neuronal fate and inhibit the astrocytic fate (Nieto, 2001).

It was hypothesized that Ngn2 might be involved in the morphological changes that cause a cycling daughter cell to lose its ventricular attachment and migrate to the SVZ or IZ for NS division. To test this possibility, a retrovirus containing ngn2 and GFP genes, were injected, along with injecting a control GFP virus, into lateral ventricles of mouse embryos. Considering the delayed onset of expression of introduced genes, injections were made at E12 and examinations to identify the position of GFP and pH3 double positive cells were mostly performed 48 hours later. In the control treated group (7 embryos from 3 independent experiments), the majority of GFP+ dividing cells (45 cells per embryo on average) were found at the ventricular surface, providing a percentage non surface-division of 26±3% (mean±s.e.m.). In contrast, embryos injected with Ngn2 virus showed a reversed surface:non-surface ratio. The proportion of GFP+ dividing cells (10 cells per embryo on average) that were found at the non-surface position (73±3%) was significantly higher than that seen in control treated samples. The expression of Ngn2 in GFP+ NS dividing cells was confirmed. A similar predominance of non-surface divisions was detected at 24 hours and 36 hours after injection of Ngn2 virus at E12. These results, together with the spatiotemporal pattern of Ngn2 expression, strongly suggest that Ngn2 is involved in the choice of mitosis position during E13-E14 (Miyata, 2002).

The telencephalon shows vast morphological variations among different vertebrate groups. The transcription factor neurogenin1 (ngn1) controls neurogenesis in the mouse pallium and is also expressed in the dorsal telencephalon of the evolutionary distant zebrafish. The upstream regions of the zebrafish and mammalian ngn1 loci harbour several stretches of conserved sequences. The upstream region of zebrafish ngn1 is capable of faithfully recapitulating endogenous expression in the zebrafish and mouse telencephalon. A single conserved regulatory region is essential for dorsal telencephalic expression in the zebrafish, and for expression in the dorsal pallium of the mouse. However, a second conserved region that is inactive in the fish telencephalon is necessary for expression in the lateral pallium of mouse embryos. This regulatory region, which drives expression in the zebrafish diencephalon and hindbrain, is dependent on Pax6 activity and binds recombinant Pax6 in vitro. Thus, the regulatory elements of ngn1 appear to be conserved among vertebrates, with certain differences being incorporated in the utilisation of these enhancers, for the acquisition of more advanced features in amniotes. These data provide evidence for the co-option of regulatory regions as a mechanism of evolutionary diversification of expression patterns, and suggest that an alteration in Pax6 expression was crucial in neocortex evolution (Blader, 2004).

To delineate the regulatory regions responsible for brain expression of ngn1 in older zebrafish embryos, transgenic lines carrying wild-type and deletion variants of ngn1 transgenes were analysed. Two regulatory regions were mapped that are required for transgene expression in the brain of post-somitogenesis-stage embryos. The first region, residing at position –6702 to –6490 bp upstream of the ATG start site, which harbours the LSE (lateral stripe element), drives expression in the dorsal telencephalon. A second regulatory region referred to as LATE was mapped to position –1775 to –1368. The LATE region, like the LSE, is highly conserved in mouse and human homologues of ngn1. Comparative functional studies were carried out in mouse embryos to investigate the activity of these conserved regulatory elements. Focus was placed on the dorsal telencephalon of the mouse, since this is undoubtedly the most derived brain region to have arisen during vertebrate evolution. The LSE drives expression in the dorsal telencephalon in both mouse and zebrafish embryos, indicating a conserved function with respect to telencephalic expression. Curiously, the LATE region of the zebrafish ngn1 gene drives expression in the lateral telencephalon of the mouse embryo but not in the zebrafish telencephalon. The area of activity of LATE overlaps with that of the paired-homeodomain transcription factor Pax6, suggesting a role of Pax6 in regulating the activity of LATE. Pax6 was shown to bind to a conserved Pax6-binding site in the LATE region. Moreover, the lack of pax6 activity in zebrafish by simultaneous knockdown of both pax6.1 and pax6.2 leads to a small eye phenotype and strongly reduces endogenous ngn1 and transgene expression. These results are consistent with a direct regulatory role of Pax6 on the activity of LATE. Based on the highly modular structure of vertebrate regulatory regions, which are usually composed of multiple short and degenerate binding sites for transcription factors, it is commonly assumed that elaboration of novel patterns of gene expression is accomplished by changes in the regulatory sequence. These data suggest that a pre-existing enhancer was co-opted, and that the evolution of the pax6 expression pattern led to the recruitment of LATE into the newly developed territories of the mouse telencephalon (Blader, 2004).

Neural precursor cells (NPCs) have the ability to self-renew and to give rise to neuronal and glial lineages. The fate decision of NPCs between proliferation and differentiation determines the number of differentiated cells and the size of each region of the brain. However, the signals that regulate the timing of neuronal differentiation remain unclear. Wnt signaling is shown to inhibit the self-renewal capacity of mouse cortical NPCs, and instructively promotes their neuronal differentiation. Overexpression of Wnt7a or of a stabilized form of ß-catenin in mouse cortical NPC cultures induces neuronal differentiation even in the presence of Fgf2, a self-renewal-promoting factor in this system. Moreover, blockade of Wnt signaling leads to inhibition of neuronal differentiation of cortical NPCs in vitro and in the developing mouse neocortex. Furthermore, the ß-catenin/TCF complex appears to directly regulate the promoter of neurogenin 1, a gene implicated in cortical neuronal differentiation. Importantly, stabilized ß-catenin does not induce neuronal differentiation of cortical NPCs at earlier developmental stages, consistent with previous reports indicating self-renewal-promoting functions of Wnts in early NPCs. These findings may reveal broader and stage-specific physiological roles of Wnt signaling during neural development (Hirabayashi, 2004).

The mechanism by which Wnt signaling regulates neurogenesis was investigated. Since the results implicated the ß-catenin/TCF complex in neuronal differentiation, a proneural gene was sought that might be under the control of these transcription factors. One such candidate is the bHLH transcription factor Ngn1, because this gene is expressed during early neurogenesis in the neocortex, and its expression, together with that of the Ngn2 gene, is essential for development of the neocortex. A consensus sequence for TCF binding was found located at nucleotide (nt) positions -1167 to -1160 relative to the transcription start site of the mouse Ngn1 gene. This region within the promoter has been shown to be responsible for expression of the gene in the dorsal neocortex during neurogenesis. To determine whether this TCF binding element is functional, the activities of the Ngn1 gene promoter (nt -2670 to +74) containing either an intact or mutated version of this DNA sequence were compared. Cultured NPCs were transfected with a luciferase reporter construct under the control of the wild-type or mutant Ngn1 gene promoter. The transcriptional activity of the mutant promoter was markedly reduced compared with that of the wild-type (Hirabayashi, 2004).

Next, a ChIP assay was used to examine whether endogenous ß-catenin was associated with the Ngn1 gene promoter in cultured NPCs. Lysates of cultured NPCs were subjected to shearing of genomic chromatin followed by immunoprecipitation with antibodies to ß-catenin. Polymerase chain reaction (PCR) analysis with primers targeted to the TCF binding element of the Ngn1 gene promoter revealed the presence of this element in the immunoprecipitates. The level of Ngn1 mRNA was determined by reverse transcription (RT)-PCR in cultured NPCs. Expression of S33Y ß-catenin markedly increased the level of Ngn1 mRNA but not that of the control glyceraldehydes-3-phosphate dehydrogenase (Gapdh) mRNA, suggesting that transcription of the Ngn1 gene is indeed under the control of the canonical Wnt pathway. Together, these results suggest that the ß-catenin/TCF complex directly regulates transcription of the Ngn1 gene during neuronal differentiation of cortical NPCs (Hirabayashi, 2004).

The results clearly indicate that stabilized ß-catenin instructs neuronal differentiation of cortical NPCs prepared from mouse E11.5 neocortex and cultured for 3 days. However, it has been shown that ectopic expression of stabilized ß-catenin by the nestin enhancer results in the expansion of NPC cell number and suppression of cell cycle exit. This difference might be due to the timing at which stabilized ß-catenin was expressed, since the nestin enhancer is known to become active at around E8.5. To test this idea, the effects were compared of ß-catenin on NPCs prepared from different stages of mouse neocortex development. Expression of stabilized ß-catenin increased the population of TuJ1+ cells in NPCs prepared from E13.5 neocortex, but reduced somewhat the population of TuJ1+ cells among neuroepithelial cells acutely prepared from E10.5 neocortex. This suggests that the response of NPCs to the canonical Wnt pathway depends on the stage of neural development (Hirabayashi, 2004).

Recent experiments have suggested that Wnt signaling has the capacity to promote self-renewal in various tissue stem cells including neural stem cells and hematopoietic stem cells. In the central nervous system, cells located in the midbrain or hippocampus are deleted in mice deficient in Wnt1 or Wnt3a, respectively. Mice lacking both Wnt1 and Wnt3a also manifest a reduction in the size of the caudal midbrain, rostral hindbrain, cranial and spinal ganglia, and dorsal neural tube. Furthermore, ectopic expression of Wnt1 or stabilized ß-catenin was shown to lead to a net increase in the size of the precursor pool in the chick spinal cord, in part through transcriptional regulation of cyclinD, and infection of cortical explants with Wnt7aHA-expressing retrovirus induced expansion of neuronal precursors which accompanied expression of the Egf receptor. Consistently, transgenic mice expressing stabilized ß-catenin in NPCs under the control of the nestin enhancer or Brn4 promoter also exhibited overgrowth of the brain and spinal cord, reflecting an expansion of the precursor population without alteration of the primary patterning of cell identities. By contrast, in the present study, activation of the canonical Wnt pathway reduced the size of the precursor pool and promoted neuronal differentiation in the developing neocortex. It is speculated that this difference might be attributable to differences in the developmental stage of the NPCs. Indeed, activation of the canonical Wnt pathway promoted neuronal differentiation of NPCs derived from E13.5 embryos, but not those acutely dissected from E10.5 embryos. It is possible that the chromatin region encompassing regulatory elements of genes crucial for neuronal differentiation (such as Ngn1) undergoes a change during development from a closed to an open state (Hirabayashi, 2004).

Neurogenin (Ngn) 1 and Ngn2 encode basic-helix-loop-helix transcription factors expressed in the developing neocortex. Like other proneural genes, Ngns participate in the specification of neural fates and neuronal identities, but downstream effectors remain poorly defined. This study set out to identify Ngn2 effectors in the cortex using a subtractive hybridization screen; several regionally expressed genes were identified that are misregulated in Ngn2 and Ngn1;Ngn2 mutants. Included were genes down-regulated in germinal zone progenitors (e.g., Nlgn1, Unc5H4, and Dcc) and in postmitotic neurons in the cortical plate (e.g., Bhlhb5 and NFIB) and subplate (e.g., Mef2c, srGAP3, and protocadherin 9). Further analysis revealed that Ngn2 mutant subplate neurons are misspecified and that thalamocortical afferents (TCAs) that normally target this layer instead inappropriately project towards the germinal zone. Strikingly, EphA5 and Sema3c, which encode repulsive guidance cues, were down-regulated in the Ngn2 and Ngn1;Ngn2 mutant germinal zones, providing a possible molecular basis for axonal targeting defects. Thus, several new components of the differentiation cascade(s) activated downstream of Ngn1 and Ngn2 were identified and novel insights were provided into a new developmental process controlled by these proneural genes. Further analysis of the genes isolated in this screen should provide a fertile basis for understanding the molecular mechanisms underlying corticogenesis (Mattar, 2004).

During cortical development, both activity-dependent and genetically determined mechanisms are required to establish proper neuronal connectivity. While activity-dependent transcription may link the two processes, specific transcription factors that mediate such a process have not been identified. The basic helix-loop-helix (bHLH) transcription factor Neurogenic Differentiation 2 (NeuroD2) was identified in a screen for calcium-regulated transcription factors and it was shown to be required for the proper development of thalamocortical connections. In neuroD2 null mice, thalamocortical axon terminals fail to segregate in the somatosensory cortex, and the postsynaptic barrel organization is disrupted. Additionally, synaptic transmission is defective at thalamocortical synapses in neuroD2 null mice. Total excitatory synaptic currents are reduced in layer IV in the knockouts, and the relative contribution of AMPA and NMDA receptor-mediated currents to evoked responses is decreased. These observations indicate that NeuroD2 plays a critical role in regulating synaptic maturation and the patterning of thalamocortical connections (Ince-Dunn, 2006).

Motility is a universal property of newly generated neurons. How cell migration is coordinately regulated with other aspects of neuron production is not well understood. This study shows that the proneural protein neurogenin 2 (Neurog2), which controls neurogenesis in the embryonic cerebral cortex, directly induces the expression of the small GTP-binding protein Rnd2 in newly generated mouse cortical neurons before they initiate migration. Rnd2 silencing leads to a defect in radial migration of cortical neurons similar to that observed when the Neurog2 gene is deleted. Remarkably, restoring Rnd2 expression in Neurog2-mutant neurons is sufficient to rescue their ability to migrate. These results identify Rnd2 as a novel essential regulator of neuronal migration in the cerebral cortex and demonstrate that Rnd2 is a major effector of Neurog2 function in the promotion of migration. Thus, a proneural protein controls the complex cellular behaviour of cell migration through a remarkably direct pathway involving the transcriptional activation of a small GTP-binding protein (Heng, 2008).

Neural stem cell self-renewal, neurogenesis, and cell fate determination are processes that control the generation of specific classes of neurons at the correct place and time. The transcription factor Pax6 is essential for neural stem cell proliferation, multipotency, and neurogenesis in many regions of the central nervous system, including the cerebral cortex. Pax6 was used as an entry point to define the cellular networks controlling neural stem cell self-renewal and neurogenesis in stem cells of the developing mouse cerebral cortex. The genomic binding locations were identified of Pax6 in neocortical stem cells during normal development, and the functional significance of genes were ascertained that were found to be regulated by Pax6. Pax6 was found to positively and directly regulate cohorts of genes that promote neural stem cell self-renewal, basal progenitor cell genesis, and neurogenesis. Notably, a core network regulating neocortical stem cell decision-making was identified in which Pax6 interacts with three other regulators of neurogenesis, Neurog2, Ascl1, and Hes1. Analyses of the biological function of Pax6 in neural stem cells through phenotypic analyses of Pax6 gain- and loss-of-function mutant cortices demonstrated that the Pax6-regulated networks operating in neural stem cells are highly dosage sensitive. Increasing Pax6 levels drives the system towards neurogenesis and basal progenitor cell genesis by increasing expression of a cohort of basal progenitor cell determinants, including the key transcription factor Eomes/Tbr2, and thus towards neurogenesis at the expense of self-renewal. Removing Pax6 reduces cortical stem cell self-renewal by decreasing expression of key cell cycle regulators, resulting in excess early neurogenesis. It was found that the relative levels of Pax6, Hes1, and Neurog2 are key determinants of a dynamic network that controls whether neural stem cells self-renew, generate cortical neurons, or generate basal progenitor cells, a mechanism that has marked parallels with the transcriptional control of embryonic stem cell self-renewal (Sansom, 2009).

Polycomb limits the neurogenic competence of neural precursor cells to promote astrogenic fate transition

During neocortical development, neural precursor cells (NPCs, or neural stem cells) produce neurons first and astrocytes later. Although the timing of the fate switch from neurogenic to astrogenic is critical for determining the number of neurons, the mechanisms are not fully understood. This study shows that the polycomb group complex (PcG) restricts neurogenic competence of NPCs and promotes the transition of NPC fate from neurogenic to astrogenic. Inactivation of PcG by knockout of the Ring1B or Ezh2 gene or Eed knockdown prolonged the neurogenic phase of NPCs and delayed the onset of the astrogenic phase. Moreover, PcG was found to repress the promoter of the proneural gene neurogenin1 in a developmental-stage-dependent manner. These results demonstrate a role of PcG: the temporal regulation of NPC fate (Hirabayashi, 2009).

During neocortical development, the neurogenic phase normally persists for a limited time period (about 11 cell cycles on average in the mouse neocortex, and this restricted period may be a major parameter in determining the final number of neurons produced during development. PcG proteins contribute to the termination of the neurogenic phase, which normally takes place between E18.5 and E19.0 in the neocortex. Indeed, birthdating analysis showed that cells labeled by BrdU at E19.0 still contributed to upper-layer neurons at P6.5 in Ring1B- or Ezh2-deficient mice but not in control mice. Interestingly, the excess neurons produced at around the end of neurogenic phase appear to be eliminated (probably by cell death) later during postnatal development in both wild-type and Ring1B-deficient mice, suggesting that these late-born excess neurons fail to integrate into the appropriate neuronal networks and therefore cannot be supported by activity/target-dependent survival signals. In other words, the correct timing of the end of neurogenesis might help avoid production of excess (unnecessary, undesirable) neurons (Hirabayashi, 2009).

The roles of PcG in ES cells strikingly differ from those in NPCs. Components of the PcG are known to localize and repress a variety of target genes and play an essential role in the maintenance of pluripotency of ES cells by suppressing differentiation into multiple lineages. A previous report has shown that different arrays of genes are labeled with H3K27me3 in ES cells and ES-derived neuronal progenitors, suggesting that PcG targets are different between these cell types. Indeed, Ring1B deletion in late-stage neocortical NPCs preferentially increases the expression of genes associated with neuronal differentiation/development over those associated with other lineages based on microarray analyses, whereas developmental genes in multiple lineages are derepressed by Ring1B deletion in ES cells (Hirabayashi, 2009).

The fate restriction of ES cells during differentiation is accompanied by diminished occupancy of H3K27me3 at specific 'bivalent' gene promoters involved in the corresponding differentiation process, in contrast to the increased H3K27me3 at ngn loci during fate restriction of NPCs. Moreover, deletion of Ring1B or Suz12 in ES cells results in the loss of neurogenic capacity, whereas deletion of Ring1B in the late NPCs extended neurogenic capacity. These observations further support the difference of PcG functions between these cell types. Thus, this study has unveiled an in vivo role of PcG, namely, temporal (stage-dependent) fate conversion of multipotent progenitors during development (Hirabayashi, 2009).

This study found that PcG is responsible for ngn1 suppression in late-stage NPCs. Since misexpression of ngn1 extends neurogenesis in late-stage NPCs, it is clear that suppression of ngn1 is a prerequisite for the neuronal-to-glial transition of NPC fate. Therefore, the suppression of ngn1 by PcG may partly account for the PcG restriction of neurogenic potential and transition to gliogenesis in the neocortex. However, it is unclear whether PcG also regulates other genes with similar functions. Ngn2 might be such a target, given that the level of H3K27me3 increases at the ngn2 locus in the late stage of neocortical NPCs. However, Ring1B deletion by itself did not cause much increase in ngn2 expression, suggesting that additional mechanisms might account for suppression of ngn2 at late stages of neocortical development (Hirabayashi, 2009).

Besides ngn1, no other proneural genes were found that were greatly upregulated by Ring1B deletion in neocortical NPCs. For instance, there was no elevation in neurogenic genes expressed in the neocortex such as Pax6, Math1, and Mash1. Among the basic helix-loop-helix or homeodomain-containing transcription factors expressed in brain, Dlx2 was significantly derepressed by Ring1B deletion. Although Dlx2 can contribute to neurogenesis in the ventral telencephalon in some contexts, it is not thought that this gene is responsible for the PcG suppression of neurogenesis in the neocortex, since Dlx2 is associated with differentiation of GABAergic interneurons rather than the glutamatergic neurons observed in the Ring1B-deficient mice. Nonetheless, a recent report has shown that the chromatin remodeling factor Mll1 suppresses the accumulation of H3K27me3 at the dlx2 locus and thus confers neurogenic potential in the adult neural stem cells. This implies a very interesting possibility that PcG participates in a common mechanism that suppresses neurogenic potential in both dorsal and ventral telencephalon in the late stages of development, and a small NPC population that escapes from this mechanism by Mll1 is selected to become adult neural stem cells that continue to produce neurons for lifetime (Hirabayashi, 2009).

Although knockdown of Bmi1, a component of PRC1, resulted in NPC loss and brain size reduction in a previous study, these phenotypes were not observed in the Ring1B-deficient mouse, implicating that Bmi1 and Ring1B may form distinct complexes that exert different functions. These functions of Bmi1 may not be related to PRC2, since it was found that brain size reduction was not seen in mice deficient for Ezh2 in the central nervous system, although H3K27me3 modification was barely seen in NPCs from these mice. Functional differences between Bmi1 and PRC2 have also been suggested in the hematopoietic system and tumors. For example, Bmi1 deletion reduced the numbers of myeloid and preB cells, whereas Eed deletion increased these cell types (Hirabayashi, 2009).

The levels of H3K27me3 gradually increase over time at the ngn1 promoter, and it is plausible that, at a certain threshold, their chromatin state becomes inactivated by PRC1, resulting in the suppression of ngn expression and the transition of NPC fate. It is proposed that the developmental-stage-dependent accumulation of H3K27me3 at specific gene loci functions as a timer to drive cell fate switching. Exactly how this accumulation occurs is not clear at present but might involve either a global increase in PcG activity or local recruitment of PcG to the ngn1 locus in late stages of neocortical NPCs. In either case, further analysis of this accumulation may shed light on the mechanism that underlies the developmental regulation of differentiation potential (Hirabayashi, 2009).


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Search PubMed for articles about Drosophila target of Poxn

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date revised: 23 August 2014

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