Proneural proteins in C. elegans

The lin-32 gene of C. elegans is an ac-sc homolog, sufficient for specification of neuroblast fate (Zhou, 1995). Chicken ac-sc homolog (cash-1) is one element in a multiple parallel pathway involving notochord or floor plate-derived signals for the specification and development of chick sympathetic neurons (Groves, 1995). Xenopus ac-sc homolog (xash-3) when expressed with the promiscuous binding partner xe12, specifically activates the expression of neural genes in naive ectoderm (Ferreiro, 1994). Zebra fish ac-sc homologs zash-1a and zash-1b are expressed in defined regions of the developing central nervous system. Their pattern of expression is modified by the cyclops mutant (Allende, 1994).

The NSM cells of the nematode Caenorhabditis elegans differentiate into serotonergic neurons, while their sisters, the NSM sister cells, undergo programmed cell death during embryogenesis. The programmed death of the NSM sister cells is dependent on the cell-death activator EGL-1, a BH3-only protein required for programmed cell death in C. elegans, and can be prevented by a gain-of-function (gf) mutation in the cell-death specification gene ces-1, which encodes a Snail-like DNA-binding protein. The genes hlh-2 and hlh-3, which encode a Daughterless-like and an Achaete-scute-like bHLH protein, respectively, are required to kill the NSM sister cells. A heterodimer composed of HLH-2 and HLH-3, HLH-2/HLH-3, binds to Snail-binding sites/E-boxes in a cis-regulatory region of the egl-1 locus in vitro that is required for the death of the NSM sister cells in vivo. Hence, it is proposed that HLH-2/HLH-3 is a direct, cell-type specific activator of egl-1 transcription. Furthermore, the Snail-like CES-1 protein can block the death of the NSM sister cells by acting through the same Snail-binding sites/E-boxes in the egl-1 locus. In ces-1(gf) animals, CES-1 might therefore prevent the death of the NSM sister cells by successfully competing with HLH-2/HLH-3 for binding to the egl-1 locus (Thellmann, 2003).

Achaete-Scute basic helix-loop-helix (bHLH) proteins promote neurogenesis during metazoan development. A C. elegans Achaete-Scute homolog, HLH-14, has been characterized in this study. A number of neuroblasts express HLH-14 in the C. elegans embryo, including the PVQ/HSN/PHB neuroblast, a cell that generates the PVQ interneuron, the HSN motoneuron and the PHB sensory neuron. hlh-14 mutants lack all three of these neurons. The fact that HLH-14 promotes all three classes of neuron indicates that C. elegans proneural bHLH factors may act less specifically than their fly and mammalian homologs. Furthermore, neural loss in hlh-14 mutants results from a defect in an asymmetric cell division: the PVQ/HSN/PHB neuroblast inappropriately assumes characteristics of its sister cell, the hyp7/T blast cell. It is argued that bHLH proteins, which control various aspects of metazoan development, can control cell fate choices in C. elegans by regulating asymmetric cell divisions. Finally, a reduction in the function of hlh-2, which encodes the C. elegans E/Daughterless bHLH homolog, results in similar neuron loss as hlh-14 mutants and enhances the effects of partially reducing hlh-14 function. It is proposed that HLH-14 and HLH-2 act together to specify neuroblast lineages and promote neuronal fate (Frank, 2003).

Aside from its conserved sequence, there are a number of similarities between hlh-14 and previously characterized A-S genes. The most obvious similarity is that hlh-14 mutants lack neurons. A-S family members in Drosophila specify external sense organs. In the absence of A-S genes, neuronal cell types needed for the function of these organs are lost. Vertebrate A-S family members generate a wide variety of neuronal precursors, including the progenitors of the cerebral cortex and progenitors in the ventral telencephalon. This study clearly establishes that hlh-14 function is required for normal PVQ, HSN, and PHB neuron development (Frank, 2003).

Yet a close look at the types of neurons specified by hlh-14 reveals an important difference between hlh-14 and other A-S genes. While Drosophila and mammalian A-S genes appear to specify neuroblast lineages dedicated to generating neuronal cells of a particular type or coordinated function, hlh-14 specifies a neuroblast lineage dedicated to generating three disparate types of neuron: an interneuron (PVQ), a motoneuron (HSN) and a sensory neuron (PHB). There is no known coordinated function that these three neurons perform. Why is hlh-14 less specific than fellow A-S family members in this regard? One possibility is that C. elegans must adapt the function of neural bHLH genes such as hlh-14 in order to generate its diverse collection of 302 neurons (of 118 distinct types) out of only 959 somatic cells. Such adaptation may allow hlh-14 to specify lineally related, yet functionally distinct, collections of neurons (Frank, 2003)

A second similarity between hlh-14 and other A-S-like genes is the genetic interaction between hlh-14 and hlh-2, the C. elegans E/DA homolog. In Drosophila, heterodimers between DA and A-S family members are essential for neurogenesis. In C. elegans, the A-S factor HLH-3 can bind E/DA HLH-2 in vitro, and the expression patterns of HLH-3 and HLH-2 overlap in a number of neuronal lineages. Taken together, four facts suggest that HLH-14 and HLH-2 act together in the PVQ/HSN/PHB lineage to regulate neuronal development. (1) A-S proteins, as well as other types of bHLH proteins, are known to interact physically with E/DA family members to form functional heterodimers in a number of organisms. (2) Both HLH-14 and HLH-2 are expressed in the PVQ/HSN/PHB lineage. (3) Loss of function of either gene results in the loss of neurons in this lineage. (4) A weak hlh-2 mutant can enhance the partial neuronal loss defects of hlh-14(RNAi) (Frank, 2003).

Another similarity that HLH-14 shares with certain A-S family members is that it possesses proneural characteristics. Not only is hlh-14 necessary for neuron development, but also it is expressed early in neurogenesis. hlh-14::gfp expression is seen in the PVQ/HSN/PHB neuroblast, the first cell in this lineage solely dedicated to generating neurons (Frank, 2003).

Sexual dimorphism in the nervous system is required for sexual behavior and reproduction in many metazoan species. However, little is known of how sex determination pathways impose sex specificity on nervous system development. In C. elegans, the conserved sexual regulator MAB-3 controls several aspects of male development, including formation of V rays, male-specific sense organs required for mating. MAB-3 promotes expression of the proneural Atonal homolog LIN-32 in V ray precursors by transcriptional repression of ref-1, a member of the Hes family of neurogenic factors. Mutations in ref-1 restore lin-32::gfp expression and normal V ray development to mab-3 mutants, suggesting that ref-1 is the primary target of MAB-3 in the V ray lineage. Proteins related to MAB-3 (DM domain proteins) control sexual differentiation in diverse metazoans. It is therefore suggested that regulation of Hes genes by DM domain proteins may be a general mechanism for specifying sex-specific neurons (Ross, 2005).

MAB-3 promotes development of male-specific sensory neurons by regulating two bHLH factors. In V ray precursors, MAB-3 indirectly promotes expression of the proneural protein LIN-32 by preventing expression of REF-1, a distant homolog of the Hes family of neurogenic proteins. REF-1 is a negative regulator of lin-32; lin-32::gfp expression is dramatically reduced in the mab-3 V ray lineage, but is restored by the introduction of a ref-1 mutation. This REF-1-mediated repression of lin-32 is necessary to prevent V ray formation in mab-3 mutants, as evidenced by the observation that ref-1 mutations restore V ray development in mab-3 mutants. Furthermore, ectopic ref-1 expression is sufficient to cause V ray defects in wild-type males. These results indicate that MAB-3 acts in parallel to Hox proteins to promote activation of lin-32 by preventing expression of ref-1, a gene with antineural activity (Ross, 2005).

Two conserved ref-1 regulatory elements (A and B) have been identified and putative MAB-3 binding sites have been identified within these elements. Wild-type MAB-3, is required to prevent ref-1::gfp expression during V ray development. Based on these observations, the following model is proposed. In wild-type V ray precursors, MAB-3 promotes LIN-32-mediated V ray development by binding within one or both of the conserved elements to prevent activation of ref-1 by an unknown factor, X. In the mab-3 mutant V ray lineage, ref-1 is inappropriately activated by X and disrupts V ray formation by preventing Hox-mediated activation of lin-32. ref-1-inactivating mutations relieve this repression of lin-32, restoring normal V ray formation in mab-3 ref-1 double mutants (Ross, 2005).

ref-1 is initially expressed in the posterior hypodermal seam cells in young males and is downregulated when mab-3 is first expressed. Although the identities of ref-1 activators are unknown, a binding site for one such factor may overlap the MAB-3 binding site in element B; disruption of this MAB-3 site eliminates ref-1 expression in the seam. Thus, MAB-3 may repress ref-1 by physically interfering with binding or function of activators bound to nearby sites. Similarly, overlapping binding sites for DSX and a bZIP transcription factor coordinate regulation of yolk expression in Drosophila. The structure of DM domain proteins may be particularly suited for interaction with transcription factors that bind overlapping DNA sites. DSX binds in the minor groove of DNA, which might allow close apposition with major groove binding transcription factors (Ross, 2005).

It is possible that the weak mab-3-suppressing mutation ref-1(ez6) reduces ref-1 expression by disrupting a second positive regulatory site in element A. However, ref-1 transgenes containing the ez6 lesion are expressed in the seam and rescue the ref-1(ez11) V ray phenotype. The rescuing activity and expression of ref-1 transgenes driven by ez6 mutant regulatory sequences may be a consequence of high copy number of the reporter or may indicate a minor role for this element (Ross, 2005).

All sex-specific development in the C. elegans soma occurs downstream of the zinc finger transcription factor TRA-1, the terminal global regulator in the sex determination cascade. However, the connection between TRA-1 and male-specific effectors that drive V ray development remains obscure. MAB-3 represses ref-1 expression in males to allow specification of V rays by LIN-32. While it might follow that REF-1 normally prevents V ray formation in hermaphrodites, this does not appear to be the case. Although ref-1 is expressed in hermaphrodite seam cells, ref-1 mutant hermaphrodites do not produce ectopic V rays or express ectopic lin-32::gfp. TRA-1 must somehow prevent V ray formation in hermaphrodites. TRA-1 might regulate lin-32 expression directly or might prevent lin-32 activation indirectly by regulating Hox gene activity. EGL-5 expression in the V6 lineage is sex specific and could be regulated by TRA-1. MAB-5 is expressed in the V6 lineage in both sexes, but TRA-1 might modulate MAB-5 activity, for example by controlling factors that modify MAB-5 posttranslationally (Ross, 2005).

Male-specific regulation of ref-1 by MAB-3 also must require additional regulators. Although mab-3 is expressed in hermaphrodites, it only represses ref-1 in males. It is possible that mab-3 requires a male-specific coregulator. Alternatively, MAB-3 may be posttranslationally modified such that it is active only in males (Ross, 2005).

Proteins of the Hes family of neurogenic regulators typically share a characteristic bHLH domain, an Orange domain that may confer functional specificity, and a C-terminal WRPW sequence required for interaction with the corepressor Groucho (Gro). Although the bHLH domains of REF-1 are most similar to those of the Hes family, the overall resemblance of REF-1 to Hes proteins is weak. The six C. elegans REF-1-like proteins are unusual in that they each possess two bHLH domains. Furthermore, the REF-1 bHLH domains are only 28% and 22% identical to the bHLH domain of Hairy and lack a basic domain proline that is conserved in other Hes proteins. By contrast, the bHLH domain of LIN-22, a second C. elegans Hairy homolog, is 51% identical to that of Hairy. Additionally, REF-1 lacks an Orange domain and contains a C-terminal FRPWE, rather than WRPW, sequence (Ross, 2005).

Despite sequence and structural differences, REF-1 bears striking functional homology to other Hes proteins. In flies, Hairy and E(spl) proteins progressively limit domains of neurogenesis in the peripheral nervous system by interfering with the activity of proneural factors like Achaete (Ac), Scute (Sc), and Atonal. During sensory bristle formation, Hairy binds directly to an ac-sc enhancer to restrict spatial expression of ac. E(spl) proteins act later, in response to Notch signaling, to downregulate proneural gene expression in presumptive epidermal cells by interfering with an autostimulatory feedback loop. E(spl) proteins also antagonize proneural proteins by interfering with activation of proneural target genes. In both cases, E(spl)-mediated repression can occur by direct DNA binding or by protein-protein interactions with proneural activators that are bound to their own sites. Repression by either mechanism requires recruitment of Gro. Vertebrate Hes proteins can act as transcriptional repressors and are also thought to prevent activation by sequestering the MASH or MATH proneural proteins in inactive heterodimer complexes (Ross, 2005).

Like Hes proteins, REF-1 prevents neurogenesis by negatively regulating a proneural protein, LIN-32. ref-1-dependent reduction of lin-32::gfp expression in mab-3 mutant males suggests that REF-1 is likely to repress lin-32 transcription. Consistent with this, REF-1 proteins with substitutions in the first basic domain (mu220 and ez11) fail to repress neurogenesis and lin-32::gfp expression, suggesting that DNA binding is required. In addition, the lin-32 promoter contains many potential REF-1 binding sites (E boxes and N boxes). It is unclear whether REF-1 interacts with the Gro homolog UNC-37 to negatively regulate lin-32. ref-1 transgenes lacking the FRPWE domain weakly rescue the ref-1 phenotype, suggesting that this sequence is partially dispensable for ref-1 function. It is possible that another sequence mediates REF-1/UNC-37 interactions or that REF-1 interacts with a different corepressor. The possibility cannot be excluded that REF-1 regulates lin-32 posttranscriptionally, perhaps by forming an unstable heterodimer with LIN-32 or by interfering with a positive feedback mechanism that would normally increase lin-32 expression (Ross, 2005).

Both ref-1 and lin-32 are required for normal development of two neuronal structures derived from seam cells. REF-1 negatively regulates development of V5- and V6-derived sensory rays and production of the postdeirid, a neuroblast normally derived from V5. In contrast, LIN-32 promotes sensory ray and postdeirid formation (Ross, 2005)

The Hes protein LIN-22 prevents neurogenesis in anterior seam cells V1-V4 . In lin-22 mutants, V1-V4 undergo a V5-like lineage to produce a postdeirid and two sensory rays. The ectopic postdeirid depends on lin-32, suggesting that LIN-22 negatively regulates lin-32 in V1-V4. REF-1 and LIN-22 appear to affect postdeirid production regionally, with LIN-22 acting in the anterior and REF-1 in the posterior seam (Ross, 2005).

Ectopic sensory ray production in lin-22 mutants requires Hox, lin-32, and mab-3 activity, suggesting that LIN-22 acts upstream of the network of V ray regulators. ref-1 and lin-22 interact to inhibit V ray formation in V1-V4. ref-1 mutations cause ectopic ray formation in mab-3; lin-22 double mutants, which normally do not produce V1-V4-derived rays. This suggests that, at least in mab-3 mutants, LIN-22 acts upstream of REF-1 in a hierarchy of bHLH proteins controlling V ray neurogenesis (Ross, 2005).

During Drosophila peripheral neurogenesis, Hairy acts early to establish a prepattern of cells competent to become neurons. E(spl) proteins subsequently define the subgroup of these cells that will form sensory organs. The inappropriate neurogenesis in V1-V4 in lin-22 mutants suggests that LIN-22, like Hairy, acts globally to define which seam cells are competent to produce neuronal lineages. The experiments suggest that REF-1, like E(spl), may then act downstream within these lineages to refine which cells will become neurons (Ross, 2005).

One mechanism by which DM domain proteins regulate sexual dimorphism is the sex-specific modulation of developmental programs. For example, DSXF inhibits Wingless and FGF pathway activity and DSXM sex specifically inhibits Dpp signaling. Although direct targets are not known, this inhibition is likely to occur by transcriptional regulation of key pathway components. The DM domain protein MAB-3 represses the Hes family bHLH protein REF-1 in males to modulate sex-specific nervous system development (Ross, 2005).

Hes proteins regulate both the extent of neurogenesis and the specification of neuronal subtypes. In Drosophila, E(spl) mutations lead to ectopic neurogenesis, while overexpression prevents neurogenesis. In the mouse brain, Hes proteins control timing of cell differentiation to regulate brain size, shape, and cell arrangement, possibly via interactions with cell-cycle regulators. Thus, it is clear that sex-specific regulation of Hes activity in the developing nervous system could achieve sexual dimorphism in organ shape, size, cell fate, or timing of differentiation. MAB-3/REF-1 interactions provide an example of such regulation (Ross, 2005).

mab-3 mutant males produce epidermal cells at the expense of neuronal cells, a phenotype like that caused by Hes overexpression in other organisms. In addition, V ray precursor cell divisions of mab-3 mutants are often delayed relative to wild-type. This delay may reflect an interaction between ref-1 and cell-cycle regulators, similar to that seen for mouse Hes proteins. While no interactions between bHLH proteins and DSX have been described, these seem likely based on functional homology between MAB-3 and DSX. Male sex combs are a likely candidate for this mode of regulation, since bristle formation in flies is regulated by Hes proteins. DSX also regulates sexual dimorphism in abdominal neuroblasts, which undergo more cell divisions in males than in females. It is possible that this sex-specific proliferation is controlled by DSX/bHLH interactions (Ross, 2005).

This work establishes that sex-specific regulation of REF-1 and LIN-32 by MAB-3 can regulate development of male-specific neurons in C. elegans. Future studies will reveal whether sex-specific regulation of bHLH proteins by DM domain transcription factors is a conserved mechanism for generating sexual dimorphism in the nervous system (Ross, 2005).

Basic helix-loop-helix (bHLH) proteins of the Achaete/Scute (Ac/Sc) family are required for neurogenesis in both Drosophila and vertebrates. These transcription factors are commonly referred to as 'proneural' factors, as they promote neural fate in many contexts. Although Ac/Sc proteins have been studied in Hydra, jellyfish, many insects, and several vertebrates, the role of these proteins in C. elegans neurogenesis is relatively uncharacterized. The C. elegans genome consists of three Ac/Sc genes, previously identified as hlh-3, hlh-6, and hlh-14. This study characterized the role of hlh-3 in nervous system development. Although hlh-3 appears to be expressed in all neural precursors, hlh-3 null mutants have a mostly functional nervous system. However, these mutants are egg-laying defective, resulting from a block in differentiation of the HSN motor neurons. Detectable HSNs have misdirected axon projection, which appears to result from a lack of netrin signaling within the HSNs. Thus, these findings suggest a novel link between Ac/Sc bHLH proteins and the expression of genes required for proper interpretation of axon guidance cues. Lastly, based on sequence identity, expression pattern, and a role in neural differentiation, hlh-3 is most likely an ortholog of Drosophila asense (Doonan, 2008).

Linking asymmetric cell division to the terminal differentiation program of postmitotic neurons in C. elegans

How asymmetric divisions are connected to the terminal differentiation program of neuronal subtypes is poorly understood. In C. elegans, two homeodomain transcription factors, TTX-3 (a LHX2/9 ortholog) and CEH-10 (a CHX10 ortholog), directly activate a large battery of terminal differentiation genes in the cholinergic interneuron AIY. This study establishes a transcriptional cascade linking asymmetric division to this differentiation program. A transient lineage-specific input formed by the Zic factor REF-2 and the bHLH factor HLH-2 directly activates ttx-3 expression in the AIY mother. During the terminal division of the AIY mother, an asymmetric Wnt/β-catenin pathway cooperates with TTX-3 to directly restrict ceh-10 expression to only one of the two daughter cells. TTX-3 and CEH-10 automaintain their expression, thereby locking in the differentiation state. This study establishes how transient lineage and asymmetric division inputs are integrated and suggests that the Wnt/β-catenin pathway is widely used to control the identity of neuronal lineages (Bertrand, 2009).

Several examples have by now well illustrated that the differentiation of individual neuron types is governed by terminal selector genes that encode transcription factors which directly activate large batteries of terminal differentiation genes. However, how these terminal selector genes are regulated by earlier specification processes, in particular asymmetric divisions, remains poorly understood. This study has uncovered a direct regulatory cascade that links the asymmetric division machinery to the activation of the terminal selector genes ttx-3 and ceh-10 during embryogenesis in C. elegans. These results will first be discussed in the context of the broad concept of progressive regulatory states before analyzing two other general implications of these studies, namely, a common theme of Zic gene function in neural precursors and a potentially broadly conserved role of Wnt signaling in neuronal specification (Bertrand, 2009).

The Zic transcription factor REF-2 is transiently expressed in the SMDD/AIY mother, where it directly activates the expression of the ttx-3 LIM homeobox gene in cooperation with the bHLH transcription factor HLH-2. Following division of the mother cell, TTX-3 is inherited in both SMDD and AIY and activates ceh-10 expression in AIY, but not in SMDD. The difference in ttx-3 activity between AIY and SMDD is due to the Wnt/β-catenin asymmetry pathway. The transcriptional mediators of this pathway, the TCF transcription factor POP-1 and its coactivator the β-catenin SYS-1, are asymmetrically localized after division of the SMDD/AIY mother. In AIY, the POP-1 nuclear concentration is low and SYS-1 concentration is high. This may allow most of the POP-1 proteins to be associated with the coactivator SYS-1 and to activate the transcription of ceh-10 via the predicted POP-1 binding sites present in its promoter. In SMDD, where the POP-1 nuclear concentration is high and SYS-1 concentration is low, most of the POP-1 proteins may not be associated with SYS-1 and therefore repress ceh-10 transcription. Finally, once coexpressed in postmitotic AIY, TTX-3 and CEH-10 directly activate a large battery of terminal differentiation genes responsible for AIY differentiation and specific function. TTX-3 and CEH-10 also maintain their own expression so that the system is locked during larval and adult stages (Bertrand, 2009).

It has been proposed that during development a cell progresses through a succession of 'regulatory states' each characterized by a combination of specific gene regulatory factors. In the case of the AIY terminal division, two regulatory states are observed. The first one (state 1) is characterized by the transient expression of REF-2 and HLH-2 in the SMDD/AIY mother. The second (state 2p) corresponds to the terminal differentiation state defined by the expression of the terminal complex TTX-3/CEH-10 and the battery of terminal differentiation genes. The transition between those two states is driven by a binary decision system based on the Wnt/β-catenin asymmetry pathway (Bertrand, 2009).

These findings provide explicit support for a theoretical model initially proposed by Priess and coworkers (Lin, 1998). In this model a transcription factor 'B' expressed in both daughter cells following the division cooperates with a high POP-1 level in the anterior cell to specify state 2a and cooperates with a low POP-1 level in the posterior cell to specify state 2p. In the case of AIY, this lineage-specific factor 'B' corresponds to the transcription factor TTX-3 (Bertrand, 2009).

Before discussing general principles of Wnt/β-catenin signaling in neuronal specification, ref-2, one specific member of the regulatory network studied here, will be discussed. ref-2 is expressed in several neuronal precursors in the embryo; in contrast, there is no detectable expression of ref-2 in postmitotic neurons at larval and adult stages. Similarly, in Hydra and vertebrates, Zic transcription factors are also expressed in several neural progenitors, while expression in adult postmitotic neurons is only rarely seen. This indicates that Zic transcription factors may have a conserved function in neural precursor development. While in vertebrates Zic transcription factors have been shown to play a role in promoting the proliferation of the progenitors, it is conceivable that they also function as transient initiators of the terminal differentiation program of specific neurons, as observed in the case of AIY. For example, an intriguing parallel can be drawn between the development of the AIY interneurons and the cholinergic projection neurons/interneurons of the vertebrate basal forebrain. These vertebrate cholinergic neurons have an important function in memory formation, as is the case for the cholinergic interneuron AIY. In vertebrates, these postmitotic neurons and their progenitors express the TTX-3-related LIM-homeodomain transcription factor Lhx7/8, which is required for their differentiation. It has been recently reported that the Zic transcription factors Zic1 and Zic3 are also expressed in these progenitors and that inactivation of both genes reduces the number of cholinergic neurons. While these Zic factors seem to regulate primarily the proliferation of the precursors, it would be interesting to test whether, in analogy to ttx-3 initiation by REF-2, they also initiate the expression of Lhx7/8 and endow the progenitors with the ability to generate cholinergic neurons (Bertrand, 2009).

A particular Wnt pathway, the Wnt/β-catenin asymmetry pathway, is involved in many asymmetric blastomere divisions in the early embryo as well as some asymmetric divisions during larval development in C. elegans. Analysis of temperature-sensitive mutants of the upstream kinase gene lit-1(t1512) has shown that this pathway is involved in six successive asymmetric division rounds in the early embryo. However, this pathway has not been shown so far to be implicated in the terminal division of embryonic neuroblasts. This study has observed that the three terminal neuroblast divisions analyzed (giving rise to AIY, AIN, and ASER, respectively) are affected by disrupting this Wnt pathway. Moreover, lit-1(t1512); mom-4(ne1539) embryos shifted at restrictive temperature just before most embryonic neuroblasts undergo their last division give rise to larvae showing strongly uncoordinated movements, suggesting additional defects in motor neuron lineages. These observations predict that the Wnt/β-catenin asymmetry pathway is widely used in terminal neuroblast division in the C. elegans embryo (Bertrand, 2009).

While it was shown that the transcriptional mediators of this pathway, POP-1/TCF and SYS-1/β-catenin, are asymmetrically localized after the terminal division of embryonic neuroblasts, how the asymmetry in this pathway is initially established remains obscure. Both POP-1 and SYS-1 are regulated by this pathway at a posttranslational level (Mizumoto, 2007). In the early embryo POP-1 asymmetry in the AB lineage requires an initial MOM-2/Wnt signal coming from the P1 lineage that may be transmitted among AB blastomeres by a relay mechanism, but POP-1 asymmetry becomes later independent of MOM-2/Wnt. MOM-5/Frizzled is enriched in the posterior pole of early AB derivatives, and in analogy to the planar cell polarity in Drosophila, a Wnt-independent asymmetric Frizzled localization could be responsible for generating asymmetric cell divisions. Additional studies on Wnt requirement and Frizzled localization are required to assess their mode of function in the context of the terminal division of embryonic neuroblasts (Bertrand, 2009).

Neurons are also generated via asymmetric divisions in Drosophila and vertebrates. Recent results suggest a possible role for β-catenin in the asymmetric division of neural progenitors in the mouse brain. For example, it has been proposed that β-catenin may regulate the asymmetric division generating intermediate progenitors from radial glial cells during corticogenesis. A Wnt/β-catenin system, similar to the one shown in this study to operate in terminal neuroblast divisions in C. elegans, may therefore be used in binary cell fate decisions during the development of the nervous system in other organisms (Bertrand, 2009).

Achaete-Scute complex of Drosophilids derived from simple ur-complexes preserved in mosquito and honeybee

In Drosophila melanogaster the Enhancer of split-Complex [E(spl)-C] consists of seven highly related genes encoding basic helix-loop-helix (bHLH) repressors, intermingled with four genes that belong to the Bearded (Brd) family. Both gene classes are targets of the Notch signalling pathway. The Achaete-Scute-Complex [AS-C] comprises four genes encoding bHLH activators. Focussing on Diptera and the Hymenoptera Apis mellifera, the question arose how these complexes evolved with regard to gene number in the evolution of insects. In Drosophilids, both gene complexes are highly conserved, spanning roughly 40 million years of evolution. However, in species more diverged, like Anopheles or Apis , dramatic differences are found. Here, the E(spl)-C consists of one bHLH () and one Brd family member (malpha) in a head to head arrangement. Interestingly in Apis but not in Anopheles, there are two more E(spl) bHLH like genes within 250 kb, which may reflect duplication events in the honeybee that occurred independently of those in Diptera. The AS-C may have arisen from a single sc/l'sc like gene which is well conserved in Apis and Anopheles and a second ase like gene that is highly diverged, however, located within 50 kb. Thus, E(spl)-C and AS-C presumably evolved by gene duplication to the current complex composition in Drosophilids in order to govern the accurate expression patterns typical for these highly evolved insects. The ancestral ur-complexes, however, consisted most likely of just two genes: (1) E(spl)-C contains one bHLH member of type and one Brd family member of malpha type, and (2) AS-C contains one sc/l'sc and a highly diverged ase like gene (Schlatter, 2005).

The Achaete-Scute complex (AS-C) is well conserved in D. virilis: all four genes, achaete (ac), lethal of scute (l'sc), scute (sc) and asense (ase) are found in the same order and orientation on the X-chromosome. As in D. melanogaster, the genes are without introns. All proteins share the typical bHLH motif of the AS-C proteins and this domain reveals the lowest evolutionary rate. However, compared with the bHLH proteins of the E(spl)-C the bHLH proteins of the AS-C evolve faster. The complex can be separated into two clusters that are distinguished by their rates of conservation. L'sc and Sc are well conserved with an identity between D. melanogaster and D. virilis of more than 75% ; in contrast, Ac and Ase are conserved with an identity of less than 69%. Note that the highest divergence that was found between these two species in the E(spl)-C was for M8 with still almost 81% identity (Schlatter, 2005).

Of the four AS-C gene members in D. melanogaster, ase stands out because it is much larger than the other three. In D. virilis, the size increase is even more striking: D.v.Ase is predicted to comprise 619 residues, whereas D.m.Ase is only 486 residues in length. This extension of more than 20% additional residues is caused by multiple insertions of repetitive sequences that code for poly-glutamine (Q), poly-alanine (A) and poly-asparagine (N) stretches. Like in D. melanogaster the unrelated gene pepsinogen-like (pcl) is located between l'sc and ase (Schlatter, 2005).

Genes related to achaete or scute have been identified in a large number of species, from hydra to mouse, and so these are also to be expected in the different insects. The AS-C was most intensely studied in various species of Schizophora flies, apart from Drosophila. The number of genes varies between one and four, however, is not strictly correlated with the position in the phylogenetic tree. For example, AS-C of Calliphora vicina contains three genes, whereas other dipteran flies like Drosophila contain four. Two genes are found in the branchiopod crustacean Triops longicaudatus. In Dipteran flies the expression patterns of the proneural genes are largely varied. This is regulated by positional information through the Iroquois Complex and pannier and in addition by a transcriptional feed-back loop involving AS-C proteins. Eventually, neural precursors are selected by the repressive activity of E(spl) bHLH proteins. In this way, location and number of the large bristles on the notum is precisely controlled. The mosquito is covered with rows of large sensory bristle, where number and position varies between individuals. This is in accordance with the fact that there is only one scute-like gene, A.g.ash that is expressed all over the presumptive notum in a modular pattern. Recently it was shown that the Anopheles A.g.ash gene can mimic the endogenous Drosophila genes and that overexpression leads to many ectopic bristles (Schlatter, 2005).

Although the bristle pattern on the notum of different Drosophilids varies slightly, bristle number and position is highly stereotyped. Therefore, it is not surprising to find the AS-C highly conserved within Drosophilids. Yet, the rate of change came unexpectedly and is quite remarkable outside of the bHLH domain. Compared to E(spl) bHLH proteins, those encoded by AS-C have a rather low degree of similarity, most notably Ac. In fact, the big flesh fly Calliphora vicina, which like Drosophila belongs to the Schizophora, is totally lacking the ac gene and is covered with bristles. In agreement, no ac was found in Anopheles or Apis, arguing for rapid evolution. The best conservation rate is found in Sc and L'sc suggesting high evolutionary pressure and maybe common ancestry. Not only the bHLH domain, but also two small stretches outside (aa 203; SPTPS in D. melanogaster L'sc) and also the C-terminus are of high similarity, the latter found identical in Calliphora. Presumably these protein domains are of functional importance. Indeed, the C-terminus acts as a transcriptional activation domain and is also used to recruit E(spl) bHLH proteins. Although the alignments of the respective genes of honeybee and mosquito to sc and l'sc are very similar, the tendency is toward a closer relationship to l'sc. However, it is proposed that this gene pair arose by duplication in the course of Drosophilid evolution, such that a common ancestor may be present in the other two species (Schlatter, 2005).

The rate of conservation is very limited for the Ase homologs. Decent conservation is found within the bHLH domain, and moreover, a further well-conserved box is present (NGxQYxRIPGTNTxQxL; x are differences between A. gambiae and D. melanogaster). This sequence is likewise detected in the Ase protein of C. vicina, which shares many more similarities with D.m.Ase. In Apis, there is no such conservation outside of the bHLH domain, which itself is highly diverged. The overall degree of conservation is so poor that further statements about the relationship are difficult. It is argued that this gene represents A.m.ase by its close proximity to A.m.ash, although other interpretations are similarly possible. An analysis of its expression pattern in honeybee may help to solve these questions (Schlatter, 2005).

In conclusion this study found that both E(spl)-C and AS-C expanded rather recently because they are only present in their current complex structures in Drosophilids. In Apis and in Anopheles, very similar arrangements are found indicative of an ancient ur-complex. The E(spl)-C seems to have evolved from two genes, one HES-like and one Brd-like that are arranged in a head to head orientation. Both types of genes are responsive to Notch signalling in Drosophila. The data suggest that the most ancient genes are E(spl) bHLH and E(spl) malpha from which the other E(spl)-C genes derived by duplication and subsequent change. Moreover, an E(spl) ur-complex is likewise detected in Tribolium castaneum that belongs to the order Coleoptera. In Drosophila the complex also gained unrelated genes like m1 and gro. The latter is highly conserved, however, located at different genomic positions. Whereas in Anopheles the ur-complex seems to exist in its original form, two additional -like bHLH genes are found in the Apis genome that possess introns. These introns are at similar positions as the introns of two other HES-like genes, dpn and h which themselves are highly conserved in the three insect species, arguing for a common evolutionary history. Presumably, the introns are evolutionarily ancient because they are also found in the C. elegans E(spl)/h like gene lin-22. The AS-C seems to originate from a single sc/l'sc like bHLH gene and a second largely diverged bHLH gene that shares similarity with Drosophila ase. The high degree of variation in the latter makes it difficult to conclusively decide on the original arrangement of this gene complex (Schlatter, 2005).

Expression pattern of a butterfly achaete-scute homolog reveals the homology of butterfly wing scales and insect sensory bristles

Lepidopteran wing scales are the individual units of wing color patterns and represent a key innovation during Lepidopteran evolution. On the basis of developmental and morphological evidence, it has been proposed that the sensory bristles of the insect peripheral nervous system and the wing scales of Lepidoptera are homologous structures. In order to determine if the developmental pathways leading to Drosophila sensory bristle and butterfly scale formation use similar genetic circuitry, a homolog of the Drosophila achaete-scute (AS-C) genes--which encode transcription factors that promote neural precursor formation--were cloned from the butterfly Precis coenia and its expression pattern was examined during development. During embryonic and larval development, the expression pattern of the AS-C homolog, ASH1, forecasts neural precursor formation. ASH1 is expressed both in embryonic proneural clusters--within which an individual cell retains ASH1 expression, enlarges, segregates, and becomes a neural precursor. ASH1 is also expressed in larval wing discs in putative sensory mother cells. However, ASH1 is expressed in pupal wings in evenly spaced rows of enlarged cells that segregate from the underlying epidermis, but, rather than give rise to neural structures, each cell contributes to an individual scale. If scales and bristles are in fact homologous structures, then the non-innervation of scales is consistent with the observed programmed cell death of the basal daughter cell of the putative scale precursor, the cell which in Drosophila is fated to produce the neuron and glia. It is concluded that ASH1 appears to perform multiple functions throughout butterfly development, apparently promoting the initial events of selection and formation of both neural and scale precursor cells. The similarity in the cellular and molecular processes of scale and neural precursor formation suggests that the spatial regulation of an AS-C gene has been modified during Lepidopteran evolution to promote scale cell formation (Galant, 1998).

Proneural proteins in other invertebrates

The study of achaete-scute (ac/sc) genes is a paradigm to understand the evolution and development of the arthropod nervous system. The ac/sc genes have been identified in the coleopteran insect species Tribolium castaneum. Two Tribolium ac/sc genes have been identified -- 1) a proneural achaete-scute homolog (Tc-ASH) and 2) asense (Tc-ase), a neural precursor gene that reside in a gene complex. These genes reside 55 kb apart from each other and thus define the Tribolium ac/sc complex. Focusing on the embryonic central nervous system it is found that Tc ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies it has been shown that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. These studies, however, do not support a role for Tc-ASH in specifying the individual fate of neural precursors, suggesting that the ability of ac and sc to separately regulate this process may represent a recent evolutionary specialization within the Diptera. Furthermore, it is found that Tc-ase is expressed in all neural precursors, suggesting an important and conserved role for asense genes in insect nervous system development. This analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development (Wheeler, 2003).

Homologs of ac/sc genes have been described in a number of insect and non-insect species. These data support and augment the model in which the last common ancestor of arthropods contained a single prototypical ac/sc gene that carried out both proneural and asense functions. In support of this model, the sole Hydra ac/sc gene, CnASH, does not group with either the proneural or asense genes in phylogenetic analysis and contains motifs indicative of both the proneural and asense genes. In addition, phylogenetic analysis of the two ac/sc genes found in a spider, the chelicerate Cupiennius salei, indicates these genes are more closely related to each other than any other ac/sc genes. These data raise the possibility that a single ancestral ac/sc gene underwent independent duplication events in chelicerates and insects. Given this possibility, it is interesting that one of the Cupiennius ac/sc genes, Cs-ASH1, exhibits a proneural-like expression pattern and appears to carry out a proneural-like function and the other, Cs-ASH2, exhibits an asense-like expression pattern and appears to carry out an asense-like function. These data suggest that independent duplications of an ancestral ac/sc gene have independently given rise to proneural-like and asense-like functions in the chelicerate and insect groups. Alternatively, phylogenetic analysis may inappropriately partition chelicerate ac/sc genes from insect ac/sc genes because of evolutionary selection for species-specific amino acid changes in chelicerate as compared to insect proteins (Wheeler, 2003).

Within the insects, it has become clear that serial duplications of a single proneural ac/sc gene gave rise to multiple proneural ac/sc genes in the more derived groups. For example, Tribolium and the basal dipteran Anopheles each contain a single proneural ac/sc gene. However, Ceratitis, a more derived dipteran, contains two proneural ac/sc genes. Thus, a duplication of the ancestral proneural ac/sc gene occurred within the dipteran lineage after the divergence of Ceratitis and Anopheles. The presence of three proneural ac/sc genes in Drosophila, a highly derived genus of dipterans, identifies a second duplication event. The simplest explanation for these data is that the second duplication occurred after the divergence of Drosophila and Ceratitis. However, comparative sequence analysis suggests this duplication may have preceded the divergence of Drosophila and Ceratitis and that Ceratitis has either lost an ac/sc homolog or it has yet to be identified (Wheeler, 2003).

In contrast to the plasticity in proneural ac/sc genes within insects, asense genes appear to be well conserved. A single asense gene exists in Tribolium and Anopheles as well as in the derived dipteran species Ceratitis and Drosophila. In addition, Cupiennius contains a single non-orthologous ac/sc gene with asense-like properties (Cs-ASH2). Thus, the potential that the asense function evolved independently in insects and chelicerates suggests an important role for the asense function in arthropod neural development (Wheeler, 2003).

The existence of ac/sc genes in complexes in Drosophila, Anopheles and Tribolium suggests that this genomic arrangement has been conserved in most if not all holometabolous insects. Shared cis-regulatory regions probably explain why proneural ac/sc genes remain linked in insects and perhaps other species. However, this does not explain why asense is retained in the ac/sc complex as the regulation of asense expression is distinct from that of the proneural ac/sc genes. This phenomenon may be explained by the presence of proneural ac/sc gene cis-regulatory regions surrounding the asense gene. In this model, chromosomal rearrangements that separate asense from the ac/sc complex would probably disrupt proneural ac/sc gene expression and neural precursor formation, thus leading to decreased viability. Consistent with this idea, cis-regulatory regions that drive proneural ac/sc gene expression in the Drosophila PNS appear to flank the ase gene. Thus, the modular cis-regulatory regions that control proneural ac/sc gene expression may also be responsible for the evolutionary conservation of the ac/sc complex. Alternatively, other as yet unidentified genomic forces may preserve the linkage between asense and proneural ac/sc genes (Wheeler, 2003).

These findings raise a number of interesting points. (1) They highlight the potential for evolutionary plasticity of ac/sc genes. Significant changes in ac/sc gene number and expression have occurred over relatively short evolutionary distances and have been correlated with modifications to neural pattern and/or gene function. For example, alterations to ac/sc gene expression in Diptera appear to account for the different patterns of sensory organs found on dipteran species. In addition, data on the role of proneural genes in MP2 fate specification suggest that the increase in ac/sc gene number in Drosophila appears to have facilitated the evolution of new developmental roles for ac and sc in this lineage. (2) The possibility that independent duplication events in chelicerates and insects each gave rise to proneural-like and asense-like genes, indicates that dividing these genetic functions between two genes may be developmentally advantageous. (3) The hypothesis that the last common ancestor of all arthropods contained a single ancestral ac/sc gene suggests it may be possible to identify direct descendants of the prototypical ac/sc gene in extant basal members of each arthropod group. The recent emphasis on the development of genomic resources in non-model organisms should greatly aid progress along this line of inquiry. Thus, continued analysis of ac/sc gene expression, organization and function in arthropods should provide additional insight into the genetic basis of the development and evolution of nervous system pattern (Wheeler, 2003).

The work presented in this paper together with studies on ac/sc gene function in Drosophila provide strong evidence that serial duplications of proneural ac/sc genes in the dipteran lineage led to the diversification of proneural ac/sc gene function in Drosophila. In Drosophila, ac and sc carry out functions distinct from l'sc in specifying the individual fate of the MP2 precursor. Tc-ASH can function in Drosophila as a proneural gene but like Drosophila l'sc fails to specify efficiently the MP2 fate in the CNS. Together these results suggest the ability of ac and sc to specify MP2 fate in Drosophila arose after the divergence of Drosophila and Tribolium. These data provide an example whereby a subset of duplicated genes has evolved a new genetic function while the entire set of duplicate genes has retained the ancestral function (Wheeler, 2003).

In addition to functional changes, the generation of multiple proneural ac/sc genes in the insects was paralleled by modifications to the expression profiles of these genes. In Anopheles (a basal dipteran), and Tribolium a single proneural ac/sc gene is expressed in all CNS proneural clusters. In more derived Diptera the presence of multiple ac/sc genes allows for more complex proneural ac/sc gene expression patterns. For example, Ceratitis contains two proneural ac/sc genes, l'sc and sc; l'sc is expressed in all CNS proneural clusters while sc is expressed in a subset of these clusters. In Drosophila, ac and sc are expressed in the identical pattern of proneural clusters and their expression is largely complementary to that of l'sc. The sum of proneural ac/sc expression in each species then marks all CNS proneural clusters despite differences in the expression pattern of individual proneural ac/sc genes. Thus, in Drosophila, the complete expression pattern of proneural ac/sc genes is divided between the largely complementary expression profiles of ac and sc relative to l'sc. The division of labor between proneural ac/sc genes in Drosophila has resulted in mutually exclusive expression patterns for ac and sc relative to l'sc in proneural clusters like MP2. This spatial separation of proneural gene expression probably facilitated the potential for ac and sc to acquire developmental functions distinct from l'sc (Wheeler, 2003).

Together this work and that of others on arthropod ac/sc genes highlights the utility of studying ac/sc genes in elucidating the genetic basis of the development and evolution of arthropod nervous system pattern. These studies illustrate the dynamic nature of ac/sc gene number, expression and function over a relatively short evolutionary time. Based on this, future work on ac/sc genes in additional arthropod species should continue to provide insight into the molecular basis of the evolution of arthropod nervous system development (Wheeler, 2003).

The morphological and functional evolution of appendages has played a critical role in animal evolution, but the developmental genetic mechanisms underlying appendage diversity are not understood. Given that homologous appendage development is controlled by the same Hox gene in different organisms, and that Hox genes are transcription factors, diversity may evolve from changes in the regulation of Hox target genes. Two impediments to understanding the role of Hox genes in morphological evolution have been the limited number of organisms in which Hox gene function can be studied and the paucity of known Hox-regulated target genes. An analysis was carried out of Hindsight, a butterfly homeotic mutant in which portions of the ventral hindwing pattern are transformed to ventral forewing identity, and the regulation of target genes by the Ultrabithorax (Ubx) gene product was compared in Lepidopteran and Dipteran hindwings. Ubx gene expression is lost from patches of cells in developing Hindsight hindwings, which correlates with changes in wing pigmentation, color pattern elements, and scale morphology. This mutant was used to study how regulation of target genes by Ubx protein differs between species. Drosophila Serum response factor (blistered), Achaete-Scute Complex, and wingless are repressed in Drosophila halteres. Portions of the expression pattern of Lepidopteran homologs of these genes are not repressed in butterfly hindwings. Unlike the expression patterns of the homologous genes in halteres, butterfly wg is not repressed along the posterior margin in the hindwing, nor is butterfly SRF repressed in intervein regions, and the AS-C homologs are not repressed in cells flanking the dorsal-ventral boundary. These differences in the regulation of wg, SRF and AS-C between Drosophila halteres and butterfly hindwings suggest that these genes became repressed by Ubx when an ancestral hindwing evolved into a haltere in the dipteran lineage, with a concomitant reduction of appendage size, loss of margin bristles, and changes in shape. Two additional exampes of Ubx-regulated differences in gene expression between fly and butterfly flight appendages were found. (1) wg is expressed in two stripes in butterfly forewings that roughly correspond to the future location of the proximal band elements. This protein of the wg pattern is absent from butterfly hindwings and has not counterpart in flies and represents a novel feature regulated by Ubx in butterflies. (2) Dll is expressed along the margin of both butterfly wings and the Drosophila forewing, but this expression is modified in halteres and may be regulated by Ubx. Changes in Hox-regulated target gene sets are, in general, likely to underlie the morphological divergence of homologous structures between animals (Weatherbee, 1999).

Molecular data suggest that myriapods are a basal arthropod group and may even be the sister group of chelicerates. To find morphological indications for this relationship neurogenesis has been analyzed in the myriapod Glomeris marginata (Diplopoda). Groups of neural precursors, rather than single cells as in insects, invaginate from the ventral neuroectoderm in a manner similar to that in the spider: invaginating cell groups arise sequentially and at stereotyped positions in the ventral neuroectoderm of Glomeris, and all cells of the neurogenic region seem to enter the neural pathway. As in the spider, 30-32 invaginating cell groups are arranged in a regular pattern of seven rows consisting of four to five invaginaton sites each. However, analysis of serial transverse sections reveals that up to 11 cells contribute to an individual invagination site, while in the spider only five to nine cells were counted. Furthermore, in contrast to the spider, the ventral neuroectoderm has a multi-layered structure: the apical region covered by a single invagination site seems to be larger and the spacing between the individual invagination sites is narrower than in the spider. The reason for these morphological features is that the invaginating cell groups are located closer together and because of limited space come to lie over and above each other. The invaginating cells of a group do not all occupy a basal position as in the spider, but they also form stacks of cells. Since more cells contribute to an invagination site and the cell processes of the invaginating cells are not as constricted as in the spider, the apical region occupied by an individual invagination site is larger than in the spider (Dove, 2003).

Homologs for achaete-scute, Delta and Notch have been identified in Glomeris. The genes are expressed in a pattern similar to that found in spider homologues and show more sequence similarity to the chelicerates than to the insects (Dove, 2003).

The data support the hypothesis that myriapods are closer to chelicerates than to insects. The spider and the millipede share several features that cannot be found in equivalent form in the insects: in both the spider and the millipede, about 30 groups of neural precursors invaginate from the neuroectoderm in a strikingly similar pattern. Furthermore, in contrast to the insects, there is no decision between epidermal and neural fate in the ventral neuroectoderm of both species analysed. It is concluded that the myriapod pattern of neural precursor formation is compatible with the possibility of a chelicerate-myriapod sister group relationship (Dove, 2003).

Fish Achaete Homologs

Two zebrafish bHLH genes, neurogenin-related gene I (ngn1) and neuroD (nrd), have been isolated. ngn1 expression is initiated at the end of gastrulation in the neural plate and defines broad domains of cells that probably possess an ability to develop as neurons. This finding suggests that ngn1 may play a role during determination of cell fate in neuroblasts. ngn1 and pax-b are expressed in a mutually exclusive manner. nrd expression follows that of ngn1 in restricted populations of cells selected from ngn1-positive clusters of cells. The earliest nrd-positive cells in the brain and the trunk are a subset of the primary neurons. ngn1 is not expressed in the eye. In this study nrd transcription was activated at 25 hours postfertilization in the ventral retina. Expression of islet-1 occurs in nrd-positive cells after expression of nrd, and the expression of the two genes partially overlaps in time. These observations suggest that during eye development nrd expression may follow expression of some other neurodetermination gene(s). This supports the idea that expression of nrd is a necessary step leading toward overt neuronal differentiation (Korzh, 1998).

deltaD is one of the four zebrafish Delta homologs presently known. Experimental evidence indicates that deltaD participates in a number of important processes during embryogenesis, including early neurogenesis and somitogenesis, whereby the protein it encodes acts as a ligand for members of the Notch receptor family. In accordance with its functional role, deltaD is transcribed in several domains of mesodermal and ectodermal origin during embryogenesis. The organization of the regulatory region of the deltaD gene has been analysed using fusions to the reporter gene gfp and germline transgenesis. Cis-regulatory sequences are dispersed over a stretch of 12.5 kb of genomic DNA, and are organized in a similar manner to those in the regulatory region of the Delta-like 1 gene of mouse. Germline transformation (using a minigene comprising 10.5 kb of this genomic DNA attached to the 3' end of a full-length cDNA clone) rescues the phenotype of embryos homozygous for the amorphic deltaD mutation after eightAR33. Several genomic regions that drive transcription in mesodermal and neuroectodermal domains have been identified. Transcription in all the neural expression domains, with one exception, is controlled by two relatively small genomic regions, which are regulated by the proneural proteins neurogenin 1 and zash1a/b acting as transcriptional activators that bind to so-called E-boxes. Transcriptional control of deltaD by proneural proteins therefore represents a molecular target for the regulatory feedback loop mediated by the Notch pathway in lateral inhibition (Hans, 2002).

Transgenic analysis of the deltaD locus has revealed six distinct cis-regulatory regions, five upstream and one downstream of the transcription start site, that direct gene expression in neuroectodermal and mesodermal subdomains of the embryo. The upstream region of the mouse Dll1 locus shows a similar organization. It is proposed that both promoters are organized in five modules, of which at least three are phylogenetically conserved. Two of these modules correspond to regions HI and HII, identified on the basis of their high sequence similarity; in addition, both regions are located in the same relative positions and in the same orientation in both species. However, there is a difference in the pattern of expression driven by these elements in zebrafish and mouse. In stably transformed mouse embryos, HI coupled to the minimal Dll1 promoter is able to direct reporter gene expression primarily to the ventral tube and some derivatives of the neural crest, such as dorsal root and spinal ganglia. By contrast, transformants bearing HII fused to the minimal Dll1 promoter direct expression in the marginal zone of the dorsal region of the neural tube. In zebrafish, the expression patterns of HI and HII do not exhibit a restriction to dorsal or ventral regions of the neural tube. It seems probable that this apparent difference is due to the different expression of neurogenin 1 and Mash1 (Ascl1 -- Mouse Genome Informatics), and neurogenin 1 and zash1a/b, in mouse and zebrafish, respectively. Indeed, in the mouse embryo the expression patterns of neurogenin 1 and Mash1 show a similar restriction in the neural tube, and it appears that there is a complete overlap in the expression patterns of HI and HII with neurogenin 1 and Mash1. Thus, the regulatory network appears to be conserved in zebrafish and mouse, although the expression domains of the corresponding proneural genes have changed during evolution (Hans, 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).

Unlike mammals, teleost fish mount a robust regenerative response to retinal injury that culminates in restoration of visual function. This regenerative response relies on dedifferentiation of Müller glia into a cycling population of progenitor cells. However, the mechanism underlying this dedifferentiation is unknown. This paper reports that genes encoding pluripotency factors are induced following retinal injury. Interestingly, the proneural transcription factor, Ascl1a, and the pluripotency factor, Lin-28, are induced in Müller glia within 6 h following retinal injury and are necessary for Müller glia dedifferentiation. Ascl1a is necessary for lin-28 expression, and Lin-28 suppresses let-7 microRNA (miRNA) expression. Furthermore, let-7 represses expression of regeneration-associated genes such as, ascl1a, hspd1, lin-28, oct4, pax6b and c-myc. hspd1, oct4 and c-myc(a) exhibit basal expression in the uninjured retina and let-7 may inhibit this expression to prevent premature Müller glia dedifferentiation. The opposing actions of Lin-28 and let-7 miRNAs on Müller glia differentiation and dedifferentiation are similar to that of embryonic stem cells and suggest novel targets for stimulating Müller glia dedifferentiation and retinal regeneration in mammals (Ramachandran, 2010).

Xenopus Achaete Homologs

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. 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).

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).

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).

In a differential screen for downstream genes of the neural inducers, two extremely early neural genes induced by Chordin and suppressed by BMP-4 have been identified: Zic-related-1 (Zic-r1), a zinc finger factor related to the Drosophila pair-rule gene odd-paired, and Sox-2, a Sry-related HMG factor. Expression of the two genes is first detected widely in the prospective neuroectoderm at the beginning of gastrulation, following the onset of Chordin expression and preceding that of Neurogenin (Xngnr-1). Zic-r1 mRNA injection activates the proneural gene Xngnr-1, and initiates neural and neuronal differentiation in isolated animal caps and in vivo. In contrast, Sox-2 alone is not sufficient to cause neural differentiation, but can work synergistically with FGF signaling to initiate neural induction. Thus, Zic-r1 acts in the pathway bridging the neural inducer with the downstream proneural genes, while Sox-2 makes the ectoderm responsive to extracellular signals, demonstrating that the early phase of neural induction involves simultaneous activation of multiple functions (Mizuseki, 1998).

The Drosophila homeoproteins Araucan and Caupolican are members of a combination of factors (prepattern) that control the highly localized expression of the proneural genes achaete and scute. Two Xenopus homologs of ara and caup (Xiro1 and Xiro2) have been identified. Like their Drosophila counterparts, they control the expression of proneural genes and, probably as a consequence, the size of the neural plate. In Xenopus, ectopic expression of these genes expands the neural plate, similar to the effect of overexpressing XASH-3 and ATH-3. Xiro expression precedes expression of the proneural genes, and partially overlaps the domains of expression of XASH-3 and ATH-3 and those of X-ngnr-1, another proneural gene. When overexpressed, X-ngnr-1 causes the differentiation of ectopic neurons. Xiro1 and Xiro2 are themselves controlled by noggin and retinoic acid. Like ara and caup, they are overexpressed in Xenopus embryos as a result of the expression of Drosophila cubitus interruptus gene, suggesting that neurogenesis is induced by the hedgehog family of proteins. These and other findings suggest the conservation of at least part of the genetic cascade that regulates proneural genes, and the existence in vertebrates of a prepattern of factors important to control the differentiation of the neural plate (Gomez-Skarmeta, 1998).

In both Xenopus and the mouse, two highly related genes, Xiro3 and Irx3 respectively, have been identified that encode a Drosophila Iroquois-related homeobox transcription factor. Xiro3 in Xenopus and Irx3 in the mouse are expressed early in the prospective neural plate in a subset of neural precursor cells. In Xenopus, injection of Xiro3 mRNA expands the neural tube and induces ectopic neural tissue in the epidermis, based on the ectopic expression of early neural markers, such as Xsox3. In contrast, the differentiation of the early forming primary neurons, as revealed by the expression of the neuronal marker N-tubulin, is prevented by Xiro3 expression. Activation of Xiro3 expression itself requires the combination of a neural inducing (noggin) and a posteriorizing signal (basic fibroblast growth factor). Overexpression of Xiro3 outside the areas where XASH3 is normally express does not result in ectopic expression of XASH3. However, when Xiro3 is directed into the neural plate, where XASH3 is normally expressed, the domain of XASH3 is slightly expanded. This result suggests that additional factors might cooperate with Xiro3 to induce proneural gene expression. These factors might be downstream of Xiro3 but are not activated until the levels of Xiro3 decline. Ectopic expression of X-Delta-1 in Xiro3-injected embyos is compatible with the idea that Xiro3 induces proneural gene expression, which subsequently induces the expression of X-Delta-1. These results suggest that Xiro3 activation constitutes one of the earliest steps in the development of the neural plate and that it functions in the specification of a neural precursor state (Bellefroid, 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) 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).

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).

Chicken Achaete Homologs

In vertebrate embryos, the precursor cells of the central nervous system (CNS) are induced by signaling from the organizer region. A novel vertebrate achaete-scute homolog, cash4, is expressed in the presumptive posterior nervous system in response to such signaling. CASH4 basic region clearly resembles that of the Drosophila ASC proteins and is distinct from that of other classes of bHLH proteins. However, in common with vertebrate ASC proteins isolated previously, the loop region of the CASH4 bHLH domain is shorter than equivalent regions of the fly ASC proteins. cash4 is first expressed in epiblast cells flanking the late-phase organizer (Hensen's node). cash4 is induced by Hensen's node as evidenced by the ability of Hensen's node cells to induce cash4 when grafted into the presumptive anterior neural plate, a region that does not normally generate cells that express cash4. Hensen's node retains its ability to induce cash4 during regression to the caudal end of the embryo. These node-derived signals can be mimicked in vivo by the activity of fibroblast growth factor (see Drosophila Branchless), suggesting that a member of the FGF family of signaling molecules is involved in the activation of cash4 expression in the presumptive posterior CNS. cash4 can substitute for the achaete/scute genes in the fly and it also has proneural activity in vertebrate embryos. Flies expressing cash4 in the scutellar primordium develop numerous ectopic bristles. cash4 expression in Xenopus blastomeres leads to enlargement of the neural plate and an increase in neural precursor cells as measured by the expansion of the domain of NCAM expression. cash4 is also expressed in prospective blood islands in extra-embryonic tissue. cash4 expression in the blood islands resembles that of gata2 (Drosophila homolog: Serpent), expressed very early in the blood stem cells. cash4 expression precedes that of gata2 ,suggesting a very early role for cash4 in the development of the primitive blood stem cells. As far as neural expression is concerned, cash4 functions as a proneural gene downstream of node-derived signals (including FGF) to promote the formation of the neural precursors that will give rise to the posterior CNS in the chick embryo (Henrique, 1997).

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).

Mammalian Achaete Homologs

Continued Achaete Evolutionary homologs part 2/3 | part 3/3

achaete: Biological Overview | Transcriptional regulation | Targets of activity | Protein Interactions and Post-transcriptional Regulation | Developmental Biology | Effects of Mutation | References

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