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).
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).
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 trnascripts 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).
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).
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).
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).
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).
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).
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).
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