Atonal is a bHLH transcription factor, the same structure as genes of the achaete-scute complex. The Atonal bHLH domain has 46% identity to Scute and progressively less identity to Twist, Daughterless and Nautilus (Jarman, 1993).

A mouse bHLH transcription, MATH-1, is related to atonal. MATH-1 is expressed in the dorsal part of the CNS but becomes restricted later to the cerebellum (Akazawa, 1995). A second mammalian atonal homolog, (MATH-2) is expressed in the cortical plate and the mantle layer throughout the CNS but not in the ventricular zone. The cerebrum continues to produce MATH-2 in adults. Both MATH-1 and 2 heterodimerize and collaborate with E47 (Shimizu, 1995).

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).

Identification of Bombyx atonal and functional comparison with the Drosophila atonal proneural factor in the developing fly eye

The proneural genes are fundamental regulators of neuronal development in all metazoans. A critical role of the fly proneural factor Atonal (AtoDm) is to induce photoreceptor neuron formation in Drosophila, whereas its murine homolog, Atonal7Mm (aka Ath5) is essential for the development of the ganglion cells of the vertebrate eye. This study identified the Bombyx mori ato homolog (atoBm). In a pattern strikingly reminiscent of atoDm, the atoBm mRNA is expressed as a stripe in the silkworm eye disc. Its DNA-binding and protein-protein interaction domain is highly homologous to the atoDm bHLH. Targeted expression of atoBm in the endogenous atoDm pattern rescues the eyeless phenotype of the fly ato1 mutant and its ectopic expression induces similar gain-of-function phenotypes as AtoDm. Rescue experiments with chimeric proteins show that the non-bHLH portion of AtoBm (N-region) can effectively substitute for the corresponding region of the fly transcription factor, even though no apparent conservation can be found at the amino acid level. On the contrary, the highly similar bHLH domain of AtoBm cannot similarly substitute for the corresponding region of AtoDm . Thus, the bHLHBm domain requires the AtoBm N-region to function effectively, whereas the bHLHDm domain can operate well with either N-region. These findings suggest a role for the non-bHLH portion of Ato proteins in modulating the function of the bHLH domain in eye neurogenesis and implicate specific aa residues of the bHLH in this process (Yu, 2012).

C. elegans atonal homologs

bHLH transcription factors function in neuronal development in organizms as diverse as worms and vertebrates. In the C. elegans male tail, a neuronal sublineage clonally gives rise to the three cell types (two neurons and a structural cell) of each sensory ray. The bHLH genes lin-32 and hlh-2 are necessary for the specification of multiple cell fates within this sublineage, and for the proper elaboration of differentiated cell characteristics. Mutations in lin-32, a member of the atonal family, can cause failures at each of these steps, resulting in the formation of rays that lack fully-differentiated neurons, neurons that lack cognate rays, and ray cells defective in the number and morphology of their processes. Mutations in hlh-2, the gene encoding the C. elegans E/daughterless ortholog, enhance the ray defects caused by lin-32 mutations. In vitro, LIN-32 can heterodimerize with HLH-2 and bind to an E-box-containing probe. Mutations in these genes interfere with this activity in a manner consistent with the degree of ray defects observed in vivo. It is proposed that LIN-32 and HLH-2 function as a heterodimer to activate different sets of targets, at multiple steps in the ray sublineage. During ray development, lin-32 performs roles of proneural, neuronal precursor, and differentiation genes of other systems (Portman, 2000).

Loss-of-function and ectopic expression analyses have shown that lin-32 function is both necessary and sufficient for hypodermal seam cells to enter the ray sublineage, leading to the idea that lin-32 functions as the proneural gene for the rays. Weak loss-of-function alleles of lin-32 and hlh-2 in combination with specific markers for ray cell fates show that these genes are also required for later steps. In these mutants, partial ray sublineages are observed and also clonal groups in which some, but not all, cells have fully differentiated. These results demonstrate that the development of individual ray cell types can be uncoupled from each other by loss of lin-32 and hlh-2 function, indicating that these genes have separable functions required at different points in ray development. Thus lin-32, in addition to having a proneural-like competence function in the ray neuroblast, specifies later aspects of ray cell determination and differentiation as well (Portman, 2000).

Based upon the genetic evidence presented in this study, it cannot be determined how direct the functions of lin-32 and hlh-2 are on the steps for which they are required. Indeed, it is possible that the LIN-32:HLH-2 heterodimer is acting only at an early step, perhaps in ray precursor cells, to activate a variety of targets, each required for a different subsequent step of ray development. These intermediates might then be segregated as determinants into different branches of the sublineage, allowing them to function in the proper cells at the proper time. Since lin-32 and hlh-2;lin-32 mutations can disrupt these steps separately, it is clear that the multiple functions of these genes are independent of each other to at least some degree, and that failure to activate one target or set of targets can occur without serious effects on another. The alternative hypothesis, that LIN-32:HLH-2 complexes might be activating different targets at different times, seems more likely, and is supported by the observation that the expression of lin-32 reporter genes and HLH-2 protein continues until the final division of the ray sublineage (Portman, 2000).

To identify genes regulating the development of the six touch receptor neurons, the F2 progeny were screened of mutated animals expressing an integrated mec-2::gfp transgene that is expressed mainly in these touch cells. From 2638 mutated haploid genomes, 11 mutations were obtained representing 11 genes that affected the production, migration, or outgrowth of the touch cells. Eight of these mutations were in known genes, and 2 defined new genes (mig-21 and vab-15). The mig-21 mutation is the first known to affect the asymmetry of the migrations of Q neuroblasts, the cells that give rise to two of the six touch cells. vab-15 is a msh-like homeobox gene that appears to be needed for the proper production of touch cell precursors, since vab-15 animals lack the four more posterior touch cells. The remaining touch cells (the ALM cells) are present but mispositioned. lin-32 is a basic helix-loop-helix gene that is most similar to atonal in Drosophila. A lin-32 mutation, u282, produces touch cell defects similar to those produced by vab-15(u781): the AVM, PVM, and PLM cells are missing, and the ALM cells are more anteriorly displaced. To see whether these two genes act together in regulating ALM cell fate, touch cells were examined in a lin-32(u282) vab-15(u781) double mutant by mec-7 immunocytochemistry. The single mutations cause the displacement, but not the loss, of the ALM cells as seen for uIs9 expression and mec-3 expression with u781 and for mec-7 in situ hybridization. In contrast, 49% of the double mutant animals lacked ALM cells and another 38% had only one ALM cell. In addition, when the ALM cells were present, their cell bodies were often anterior to the rear bulb of the pharynx, indicating more severe defects in ALM migration. These additive effects suggest that vab-15 and lin-32 have somewhat redundant roles in activating touch cell fate in ALM cells. Since lin-32 is needed for the generation of the posterior touch cells, by extension, vab-15 may also be needed for their production. In addition to the touch cell abnormalities, vab-15 animals variably exhibit embryonic or larval lethality, cell degenerations, malformation of the posterior body, uncoordinated movement, and defective egg laying (Du, 2001).

Fish and frog Atonal homologs

In zebrafish, neuronal differentiation progresses across the retina in a pattern that is reminiscent of the neurogenic wave that sweeps across the developing eye in Drosophila. Expression of a zebrafish homolog of Drosophila atonal, ath5, sweeps across the eye predicting the wave of neuronal differentiation. By analyzing the regulation of ath5 expression, the mechanisms that regulate initiation and spread of neurogenesis in the retina have been elucidated. ath5 expression is lost in Nodal pathway mutant embryos lacking axial tissues that include the prechordal plate. A likely role for axial tissue is to induce optic stalk cells that subsequently regulate ath5 expression. These results suggest that a series of inductive events, initiated from the prechordal plate and progressing from the optic stalks, regulates the spread of neuronal differentiation across the zebrafish retina (Masai, 2000).

To elucidate how neurogenesis is regulated in the developing vertebrate retina, a zebrafish homolog of Drosophila atonal was isolated by cDNA library screening. The bHLH motif of the zebrafish clone has 87% amino acid identity to Xath5a and 68% to Drosophila Atonal. Phylogenetic tree analysis suggests that the zebrafish gene is an ortholog of Xath5. Unlike the Xenopus gene, in situ hybridization analysis reveals that ath5 is expressed exclusively in the developing retina (Masai, 2000).

ath5 expression is first detected in the ventronasal retina adjacent to the choroid fissure at 25hpf, spreads from nasal to more dorsal and temporal retina over the next few hours and is present throughout most of the neural retina at 36 hr. By 72 hr, when retinal ganglion cell, inner nuclear, and photoreceptor cell layers are distinct, ath5 expression is downregulated in differentiated neurons but remains expressed in retinoblast cells near the ciliary margin. The initiation and fan-shaped spread of ath5 expression are reminiscent of the pattern of appearance of differentiated neurons at slightly later stages. To examine the relationship between ath5 and neuronal differentiation, ath5 expression was compared to that of early neuronal markers. islet-1 and lim3 encode LIM homeodomain proteins that are expressed in retinal ganglion and inner nuclear layer cells. Expression of both genes is initiated in ventronasal retina soon after ath5 and subsequently spreads to dorsal and temporal retina, following the progression of ath5 (Masai, 2000).

Neuronal differentiation is indeed initiated in ventronasal retina. neuroD (Drosophila homolog target of pox-n) encodes a bHLH protein expressed during late phases of neurogenesis. Within the retina, neuroD expression is initiated in the ventronasal region and subsequently spreads dorsally and temporally within the outer retina. Likewise, the first retinal ganglion cells to be labeled with anti-acetylated alpha-tubulin antibody are present in the ventronasal retina as are the earliest FRet43-labeled double cone photoreceptors (Masai, 2000).

The relationship between ath5 expression and proliferation were examined by using an antibody to phosphorylated histone H3 that labels cells in late G2 and M phase. At the stage when ath5 expression is initiated, the nuclei of dividing cells are present throughout the retina near the scleral surface. As ath5 expression spreads, dividing cells become more localized to the margins of the retina, and by 48 hr, most dividing cells are located within the ciliary marginal zone peripheral to, or occasionally within, the domain of ath5 expression. These results indicate that ath5 is neither expressed in the most proliferative cells of the ciliary marginal zone nor within differentiated neurons of the central retina. Instead, in agreement with data in Xenopus on Xath5, it is suggested that ath5 is expressed transiently in retinoblasts and perhaps postmitotic neurons prior to full differentiation (Masai, 2000).

To determine if Ath5 can promote neurogenesis, DNA encoding Ath5 under the control of the CMV promoter was injected. Injected embryos exhibit very strong induction of neuroD, a potential target for Ath5 within the retina. Supporting the possibility that Ath5 may directly regulate neuroD expression, ectopic ath5 often cell autonomously induces neuroD. Confirming that Ath5 could promote formation of neurons, injected embryos also showed strong and precocious expression of huC, an early marker of postmitotic neurons (Masai, 2000).

ath5 expression is initiated in nasal retina and only later spreads to temporal retina, suggesting that signals that induce ath5 expression might initially be localized to the nasal optic cup. To test this possibility, dissected optic cups were divided into nasal and temporal halves and were cultured separately. ath5 expression was observed only in nasal, but not temporal retinal explants. These data suggest that inductive activity is localized to the nasal side of the optic cup at the 18 somite stage. ath5 expression is initiated in cells adjacent to the choroid fissure -- the position at which the optic stalk connects with the retina -- raising the possibility that this tissue may be a source of signals that induce ath5. Optic stalk cells and retinal cells that line the choroid fissure express the paired box transcription factor Pax2.1/Noi and differentiate as reticular astrocytes. Analysis of ath5 and pax2.1 expression revealed that ath5-expressing retinal cells are directly adjacent to pax2.1-expressing optic stalk/choroid fissure cells, suggesting that interactions at the boundary between optic stalk and neural retina may initiate neurogenesis. To test this possibility, the position of the optic stalk/retinal interface was experimentally manipulated. Increasing Shh activity expands the optic stalk into distal regions of the optic cup and represses retinal fates, thereby repositioning the boundary between neural retina and optic stalk. In shh-overexpressing embryos, ath5 expression is always initiated directly adjacent to the most distal pax2.1-expressing cells. These data suggest that the optic stalk or the boundary between neural retina and optic stalk produces signals that induce ath5 expression. To test the possibility that the optic stalk/retinal interface induces ath5 expression, tissue at this boundary was ablated either surgically or using a focused laser beam. When the ablation was performed at 18hpf, ath5 expression was often absent in the retina on the ablated side at 33hpf, whereas normal expression was observed in control eyes. Eyes in which ath5 expression was absent lacked pax2.1-expressing cells directly adjacent to the nasal retina, confirming that the boundary between optic stalk and nasal retina is disrupted by the operation. These data suggest that interaction between the optic stalk and nasal retina is necessary for induction of ath5 expression. Furthermore, the absence of ath5 expression in dorsal and temporal regions of eyes with optic stalk ablations suggests that the spread of ath5 expression to temporal retina is dependent upon initiation of expression in ventronasal retina (Masai, 2000).

To determine if optic stalk tissue can promote ath5 expression, optic stalk cells were transplanted into temporal retina. This was a difficult operation as cells often failed to integrate into the retina. However, in some cases, ectopic ath5 expression is observed adjacent to the transplanted tissue (in three of nine embryos) at a stage when ath5 expression is normally restricted to ventronasal retina, suggesting that optic stalk can, cell nonautonomously, promote ath5 expression in adjacent neural retina (Masai, 2000).

Mutation of the zebrafish lakritz (lak) locus completely eliminates the ganglion cells (RGCs), which are the earliest-born retinal cells. Instead, excess amacrine, bipolar, and Müller glial cells are generated in the mutant. The extra amacrines are found at ectopic locations in the ganglion cell layer. Cone photoreceptors appear unaffected by the mutation. Molecular analysis reveals that lak encodes Ath5, the zebrafish eye-specific ortholog of the Drosophila basic helix-loop-helix transcription factor Atonal. A combined birth-dating and cell marker analysis demonstrates that lak/ath5 is essential for RGC determination during the first wave of neurogenesis in the retina. The results suggest that this wave is skipped in the mutant, leading to an accumulation of progenitors for inner nuclear layer cells (Kay, 2001).

How does ath5 control RGC fate? In all species examined so far, ath5 expression begins prior to retinal neurogenesis and is rapidly downregulated by neurons exiting the cell cycle. This pattern of expression suggests that ath5 may act at the time of cell cycle exit to promote RGC differentiation. In support of this view, Xath5 overexpression in tadpole retina reduces the total number of retinal neurons by forcing progenitors to differentiate before they have completed the normal number of cell divisions. Accordingly, the lak mutation delays the cell cycle exit of retinal progenitors, preventing the early wave of neurogenesis that normally produces RGCs. These findings are consistent with a model in which ath5 acts solely to drive progenitors out of the cell cycle, leaving specification of cell fate to cell-extrinsic cues. In this model, ath5 would promote RGC genesis by causing cell cycle exit during a time when the retinal environment favors production of RGCs. However, recent gain-of-function experiments in chick suggest that Ath5 can induce transcription of RGC-specific genes. Therefore, it seems likely that ath5 functions both to drive progenitors from the cell cycle at the appropriate time and also to activate an RGC-specific differentiation program (Kay, 2001).

In addition to the absence of RGCs, lak mutants exhibit increased numbers of bipolar, amacrine, and Müller glial cells. The overproduction of inner nuclear layer (INL) cell types at the expense of RGCs suggests that a cell fate switch has occurred. How might the loss of lak/ath5 function lead to this phenotype? One possibility is that INL cell types are made in place of RGCs during the normal period of RGC genesis. This, however, is not what is found; no neurogenesis occurs in lak mutants during the time when RGCs are normally made. The results suggest a more indirect switch from RGC to INL cell fate: loss of neurogenesis during the RGC differentiation wave may increase the number of progenitors available to differentiate during the subsequent INL wave. The first wave of retinal neurogenesis is skipped in lak mutants, whereas the waves of INL and ONL genesis are essentially normal. At 38 hpf, which is around the time that GCL genesis normally tapers off and INL genesis begins, nearly all cells in the lak- retina remain undifferentiated progenitors, as shown by their continued ability to incorporate BrdU. Mutant retinae at this age clearly have more BrdU+ cells than wild-type retinae. Thus, the mutant retina accumulates progenitors during the time when the wild-type retina is making RGCs. This enlarged pool of progenitors should be available to differentiate into bipolar, amacrine, and Müller glial cells once the INL wave begins, explaining why excesses of these cell types are found in the mature mutant retina. By the time of the ONL wave, however, the pool of progenitors is apparently back to wild-type levels, since cone photoreceptors are not overproduced in lak mutants (Kay, 2001).

Structurally, Drosophila atonal (ato) and zebrafish lak/ath5 are highly related, sharing 68% identity in the bHLH domain and 92% identity in the basic portion of the domain. The two genes are functionally related as well. Both genes are expressed at an early stage of eye development, when the first neural precursor cells commit to particular fates, although they are not involved in the early steps of eye formation. Both genes are required for initiation of neurogenesis in the eye. And both genes are specifically required for cell-type specification of the earliest-born eye neurons: R8 photoreceptors in flies and RGCs in fish. Although ato and lak/ath5 appear functionally conserved in the eye, ato has additional functions in the development of the chordotonal proprioceptors in the peripheral nervous system which are not shared by ath5. Instead, a different but highly related vertebrate gene, Math1, determines proprioceptors in the mouse, in addition to having a role in the genesis of cerebellar granule cells and inner ear hair cells. Thus, ath1 and ath5 appear to have split the combined functions of a single, ancestral atonal gene and can therefore both be regarded as (semi-) orthologs of ato. In the fly retina, neuronal differentiation spreads as a wave across the eye imaginal disc behind the morphogenetic furrow (Kay, 2001).

In the developing nervous system, progenitor cells must decide when to withdraw from the cell cycle and commence differentiation. There is considerable debate whether cell-extrinsic or cell-intrinsic factors are most important for triggering this switch. In the vertebrate retina, initiation of neurogenesis has recently been explained by a 'sequential-induction' model -- signals from newly differentiated neurons are thought to trigger neurogenesis in adjacent progenitors, creating a wave of neurogenesis that spreads across the retina in a stereotypical manner. It is shown in this study, however, that the wave of neurogenesis in the zebrafish retina can emerge through the independent action of progenitor cells -- progenitors in different parts of the retina appear pre-specified to initiate neurogenesis at different times. Evidence is provided that midline Sonic hedgehog signals, acting before the onset of neurogenesis, are part of the mechanism that sets the neurogenic timer in these cells. These results highlight the importance of intrinsic factors for triggering neurogenesis, but they also suggest that early signals can modulate these intrinsic factors to influence the timing of neurogenesis many cell cycles later, thereby potentially coordinating axial patterning with control of neuron number and cell fate (Kay, 2005).

This study looked into the cellular and molecular factors that determine the timing of ath5 expression. Retinoblasts do not require signals from the retinal environment in order to correctly time ath5 expression. This study looked into retinal ganglion cell (RGC) derived signals, signals derived from ath5-expressing cells, and finally signals from all retinal cells. In each case at least the relative timing of ath5 expression was normal. These experiments indicate that retinoblasts may possess a cell-intrinsic program that activates neurogenesis. The intrinsic program appears to be at least partially established by the patterning activity of midline-derived Shh. These findings may explain the origins of the wave of RGC differentiation that sweeps across the retina: Because the timing of neurogenesis is pre-specified and staggered according to retinal position, the wave may emerge through the collective timing decisions of individual neuroblasts. In this model, progressive cell-cell signaling is not required to drive the ath5 wave, although such a mechanism may still influence retinoblast differentiation and/or cell fate selection (Kay, 2005).

Central retinoblasts are already competent to express ath5 independent of signals from ventronasal retina, by 22 somites (~20 hpf at 28.5°C), and temporal retinoblasts are independent of retinal signals by 20 somites (~ 19 hpf at 28.5°C). When the retina is removed at 18 somites and the nasal and temporal halves are cultured separately, the temporal explant is delayed or fails to express ath5. This result may indicate that an important intra-retinal signaling event occurs between the 18- and 20-somite stages (a time window of about 1 hour). Alternatively, explanting the retina may remove a source of extra-retinal signals, such as Shh, that are required for timely differentiation of temporal retinoblasts. Regardless of the precise cellular mechanism through which the timing of ath5 expression is set, the results are significant for showing that the time window during which signals act to influence neurogenic activation is substantially earlier than previously suspected (Kay, 2005).

If retinoblasts have an intrinsic tendency to activate neurogenesis at a particular time, there must be some mechanism that establishes the neurogenic timing for each cell. What is this mechanism, and when does it act? Retinal location is found to be a key variable, implying that an asymmetric spatial signal might set the neurogenic timer in order to impart location-specific timing information. A good candidate for such a signal is Shh, derived from the ventral midline of the diencephalon. Midline Hh signals have been reported to be required for ath5 expression. Shh acts between 13 and 25 hpf not as an absolute prerequisite for ath5 expression, but rather to ensure timely expression of ath5 during the wave, hours later. Before 25 hpf, shh and its relative, tiggy-winkle hedgehog (twhh), are expressed in the diencephalic ventral midline but not in the retina. In fact, shh expression is not detectable in the retina before the ath5 wave has already reached the temporal retina. The combined results suggest that the midline source of Hh signals, in addition to patterning the DV axis of the eye, also has a role in patterning the timing of retinal neurogenesis (Kay, 2005).

Hair cells of the inner ear develop from an equivalence group marked by expression of the proneural gene Atoh1. In mouse, Atoh1 is necessary for hair cell differentiation, but its role in specifying the equivalence group (proneural function) has been questioned and little is known about its upstream activators. These issues have been addressed in zebrafish. Two zebrafish homologs, atoh1a and atoh1b, are together necessary for hair cell development. These genes crossregulate each other but are differentially required during distinct developmental periods, first in the preotic placode and later in the otic vesicle. Interactions with the Notch pathway confirm that atoh1 genes have early proneural function. Fgf3 and Fgf8 are upstream activators of atoh1 genes during both phases, and foxi1, pax8 and dlx genes regulate atoh1b in the preplacode. A model is presented in which zebrafish atoh1 genes operate in a complex network leading to hair cell development (Millimaki, 2007).

There have been differing opinions as to whether vertebrate Atoh1 genes act as classic proneural genes or only as terminal differentiation factors. Specific comparisons between zebrafish atoh1 genes and Drosophila ato reveal striking parallels. More generally, various authors have used four criteria to define proneural function that can be applied to zebrafish atoh1 genes. (1) Proneural genes are expressed before sensory fate specification. atoh1b is induced broadly in the preotic placode at 10.5 hpf, whereas specification of tether cells (stabilization of atoh1 expression) does not occur until 14 hpf. (2) Proneural genes are subject to lateral inhibition (and the related process of domain restriction) via Notch-mediated repression. Zebrafish atoh1 genes, once induced, are readily repressed by Notch activity. Moreover, both atoh1 genes facilitate their own repression by autonomously activating delta expression. (3) Proneural function is necessary for producing the equivalence group for the entire sensory structure. atoh1a;atoh1b morphants produce only a simple epithelium lacking hair cells; and while support cell markers are not known in zebrafish, it is important to note that the epithelium continues to express atoh1a. Since loss of atoh1 expression marks the first step in support cell specification, these cannot be support cells. (4) Proneural function is sufficient to induce ectopic sensory development. Misexpression of atoh1a induces ectopic hair cells, although only in limited regions near the otic vesicle or endogenous sensory epithelia, as has been shown for Atoh1 in mammals. Competence to respond appropriately to Atoh1 may require a unique combination of additional factors. The zone of competence could be influenced by pax2-5-8 genes, which are co-regulated with atoh1 genes by Fgf signaling. Other signaling pathways have also been implicated in this process. Misexpressing components of the Notch or Wnt pathways in chick can also induce ectopic sensory patches, but only in restricted regions near endogenous sensory patches. Combinatorial signaling and restricted zones of competence also influence the functions of proneural genes in Drosophila. Thus, while many additional details need to be resolved, zebrafish atoh1 genes meet all four criteria used to define proneural function (Millimaki, 2007).

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).

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).

ATH-3, a novel basic helix-loop-helix (bHLH) gene from Xenopus and mouse was isolated and found to be homologous to the Drosophila proneural gene atonal. Homology of the mouse protein to Drosophila Atonal is 54% in the bHLH domain. ATH-3 is expressed in the developing nervous sytem, with high levels of expression in the brain, retina and cranial ganglions. Injection of ATH-3 RNA into Xenopus embryos dramatically expands the neural tube and induces ectopic neural tissues in the epidermis but inhibits non-neural development. This ATH-3-induced neural hyperplasia does not require cell division, indicating that surrounding cells that are normally non-neural types adopt a neural fate. In a Xenopus animal cap assay, ATH-3 is able to convert ectodermal cells into neurons expressing anterior markers without inducing mesoderm. Interestingly, a single amino acid change from Ser to Asp in the basic region, which mimics phosphorylation of Ser, severely impairs the anterior marker-inducing ability without affecting general neurogenic activities. These results provide evidence that ATH-3 can directly convert non-neural or undetermined cells into a neural fate, and suggest that the Ser residue in the basic region may be critical for the regulation of ATH-3 activity by phosphorylation. ATH-3 proves not to be regulated by Pax-6, suggesting that the two genes may constitute different genetic pathways for retinal development (Takebayashi, 1997).

Math5, a mouse basic helix-loop-helix (bHLH) gene that is closely related to Drosophila atonal and Xenopus Xath5, is largely restricted to the developing eye. Math5 retinal expression precedes differentiation of the first neurons and persists within progenitor cells until after birth. To position Math5 in a hierarchy of retinal development, Math5 and Hes1 expression were compared in wild-type and Pax6-deficient (Sey) embryos. Math5 expression is downregulated in Sey/+ eyes and abolished in Sey/Sey eye rudiments, whereas the bHLH gene Hes1 is upregulated in a similar dose-dependent manner. These results link Pax6 to the process of retinal neurogenesis and provide the first molecular correlate for the dosage-sensitivity of the Pax6 phenotype. During retinogenesis, Math5 is expressed significantly before NeuroD, Ngn2 or Mash1. To test whether these bHLH genes influence the fates of distinct classes of retinal neurons, Math5 and Mash1 were ectopically expressed in Xenopus retinal progenitors. Unexpectedly, lipofection of either mouse gene into the frog retina causes an increase in differentiated bipolar cells. Directed expression of Math5, but not Xath5, in Xenopus blastomeres produces an expanded retinal phenotype. It is proposed that Math5 acts as a proneural gene, but has properties different from its most closely related vertebrate family member, Xath5 (Brown, 1998).

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 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).

The vertebrate Six3 gene (See Drosophila 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).

Recent studies on vertebrate eye development have focused on the molecular mechanisms of specification of different retinal cell types during development. Only a limited number of genes involved in this process has been identified. In Drosophila, BarH genes are necessary for the correct specification of R1/R6 eye photoreceptors. Vertebrate Bar homologs have been identified and are expressed in vertebrate retinal ganglion cells during differentiation; however, their retinal function has not yet been addressed. The roles have been examined of the Xenopus Bar homolog Xbh1 in retinal ganglion cell development and its interaction with the proneural genes Xath5 and Xath3, whose ability to promote ganglion cell fate has been demonstrated. XHB1 plays a crucial role in retinal cell determination, acting as a switch towards ganglion cell fate. Detailed expression analysis, animal cap assays and in vivo lipofection assays, indicate that Xbh1 acts as a late transcriptional repressor downstream of the atonal genes Xath3 and Xath5. However, the action of Xbh1 on ganglion cell development is different and more specific than that of the Xath genes, and accounts for only a part of their activities during retinogenesis (Poggi, 2004).

To elucidate in detail how Xbh1 expression is related to retinal neurogenesis in time and space, its expression was compared with that of Xath5. Xath5 expression starts in the retina at around stage 24, preceding the reported onset of retinal differentiation. Expression is initially present throughout most of the neural retina, but displays a dorsal to ventral gradient that is consistent with neurogenesis commencing slightly earlier in the dorsal retina than in the ventral retina. When RGC, inner nuclear, and photoreceptor cell layers become distinct, Xath5 expression is downregulated in differentiated neurons, but remains in the ciliary marginal zone (CMZ), where retinoblasts are generated throughout life. Xbh1 expression in the retina is first detected in the dorsal inner optic cup around stage 26-27, shortly after the onset of Xath5 expression, and subsequently spreads from dorsal to ventral until it covers the entire retina, thus following the wave of retinal differentiation. At stage 38, when the three main retinal cell layers become distinct, Xbh1 is detected in the ganglion cell layer, in some scattered cells in the inner part of the inner nuclear layer (INL), and also in the most central part of the CMZ. At stage 42, Xbh1 expression is almost completely restricted to cells of the central differentiated ganglion cell layer, and to the central CMZ; a few cells in the INL also showed expression. At stage 42, double in situ hybridization shows expression of Xbh1 and Xath5 in the central most part of the CMZ. High magnification of these sections shows that, in spite of some superposition, Xbh1 does not extend as far peripherally as Xath5. Xbh1 expression in the CMZ is also more central than that of XNotch1, predominantly restricted to proliferating cells. Interestingly, a few cells of the ventral-most central retina still express both Xath5 and Xbh1. These cells may be in a similar commitment state as those co-expressing the two genes in the CMZ, and may reflect the delay in differentiation in the ventral retina with respect to the dorsal retina. After stage 42, when differentiation occurs almost exclusively in the CMZ, Xbh1 is also progressively downregulated in the ganglion cell layer, but persists in the CMZ. Thus, both in the retina and in the CMZ, Xbh1 expression strictly follows, in time and space, the dynamics of Xath5 expression (Poggi, 2004).

The fact that Xath5 expression precedes and later partially overlaps Xbh1 expression in the CMZ, suggests a possible regulatory interaction. The animal cap assay was used to investigate whether Xbh1 can be transcriptionally regulated by Xath5. One-cell-stage embryos were injected into the animal pole with 1 ng of Xath5 RNA. Animal caps were cut at blastula stage, harvested at stage 28, and processed for RT-PCR assays to detect possible activation of Xbh1, and of the ganglion cell markers Xbrn3.0 and Xbrn3d, the earliest markers of RGCs, known as Xath5 downstream genes. Xath5 is able to activate Xbh1, Xbrn3d and Xbrn3.0 transcription in injected animal caps, whereas none of these genes was transcribed in control caps. It was also found that Xath3 is able to activate Xbh1, as well as Xbrn3.0 and Xbrn3d. Interestingly, injection of 500 pg of XneuroD mRNA, although able to trigger Xbrn3d in animal caps, is not able to activate Xbh1 expression. This suggests that Xbh1 transcription may be specifically controlled by atonal-like factors, but not by any bHLH factor (Poggi, 2004).

To test whether Xbh1 could activate Xbrn3.0 and Xbrn3d, one ng of RNA encoding Xbh1 was injected into 1-cell-stage embryos and assayed for the expression of Xbrn3 genes in stage 28 animal caps. Xbh1 was found to trigger both Xbrn3.0 and Xbrn3d transcription in animal caps. Whether Xbh1 was able to activate Xath5 and/or Xath3 was also tested in animal caps. Xbh1 does not activate Xath5, but does activate Xath3 transcription (Poggi, 2004).

In a wide range of vertebrate species, the bHLH transcription factor Ath5 is tightly associated with both the initiation of neurogenesis in the retina and the genesis of retinal ganglion cells. At least two modes of regulating the expression of Ath5 during retinal development are described. A proximal cis-regulatory region of the Xenopus Ath5 gene (Xath5) is highly conserved across vertebrate species and is sufficient to drive retinal-specific reporter gene expression in transgenic Xenopus embryos. Xath5 proximal transgene expression depends upon two highly conserved bHLH factor binding sites (E-boxes) as well as bHLH factor activity in vivo. However, bHLH activity is not required for expression of a longer Xath5 transgene, suggesting that additional mechanisms contribute to Xath5 expression in vivo. Consistent with this, a more distal fragment that does not include the conserved proximal region is sufficient to promote transgene expression in the developing retina. In mouse, a longer fragment of the cis-regulatory region of either the mouse or Xenopus Ath5 gene is necessary for transgene expression, and expression of a mouse Math5 (Atoh7) transgene is not dependent upon autoregulation. Thus, despite extensive conservation in the proximal region, the importance of these elements may be species dependent (Hutcheson, 2005).

This study has identified bHLH-dependent and -independent modes of Ath5 gene regulation in Xenopus, raising the issue of how they contribute to endogenous Xath5 expression. It is possible that the distal and proximal cis-regulatory sequences serve overlapping or redundant functions. Alternatively, the distal and proximal regions may regulate different phases of Xath5 expression. For example, during Drosophila eye development atonal gene expression is initiated in a bHLH-independent fashion by factors such as Hedgehog, then expression becomes dependent upon Atonal itself. It is therefore possible that Xath5 gene regulation is similar, with initiation of gene expression being bHLH-independent and maintenance of expression requiring Xath5 and/or other bHLH factors such as X-Ngnr-1. In chick, bHLH factors are clearly involved in regulation of Cath5, but it remains to be determined whether there is bHLH-independent regulation as well. In mouse, no evidence was found for bHLH-dependent Atoh7 gene regulation, demonstrating that Math5 expression is bHLH independent. Thus, it has been shown that although some mechanisms of Ath5 gene regulation are conserved, there are intriguing species-specific differences that remain to be explored (Hutcheson, 2005).

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

The dorsal hindbrain includes distinct classes of neurons for processing various sensory stimuli, but the developmental aspects of these neurons remain largely unknown. Two distinct classes of neurons have been identified in the dorsal hindbrain of developing zebrafish: (1) neurons that express the inhibitory neuronal marker Gad1/2, and (2) neurons that express the zn-5 antigen and Lhx2/9 and require the basic helix-loop-helix transcription factor Atoh1a for development. Neurons were traced to their axon terminals by expressing green fluorescent protein using the Gal4VP16-UAS (UAS, upstream activating sequences) system in combination with the promoter/enhancer regions of gad2 for the Gad1/2(+) neurons and zic1 for the zn-5(+)Lhx2/9(+) neurons. The Gad1/2(+) neurons projected to the contralateral hindbrain, while the zn-5(+)Lhx2/9(+) neurons projected to the contralateral midbrain torus semicircularis, suggesting a role in auditory and lateral line sensory processing. Comparison of these projections with those from the cochlear nuclei to the inferior colliculus in mammals suggests similarities across vertebrate species (Sassa, 2007).

Chick Atonal homologs

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).

During retinogenesis, the Xenopus basic helix-loop-helix transcription factor Xath5 has been shown to promote a ganglion cell fate. In the developing mouse and chicken retinas, gene targeting and overexpression studies have demonstrated critical roles for the Brn3 POU domain transcription factor genes in the promotion of ganglion cell differentiation. However, the genetic relationship between Ath5 (an atonal homolog) and Brn3 genes is unknown. To understand the genetic regulatory network(s) that controls retinal ganglion cell development, the relationship between Ath5 and Brn3 genes was analyzed by using a gain-of-function approach in the chicken embryo. It was found that during retinogenesis, the chicken Ath5 gene (Cath5) is expressed in retinal progenitors and in differentiating ganglion cells but is absent in terminally differentiated ganglion cells. Forced expression of both Cath5 and the mouse Ath5 gene (Math5) in retinal progenitors activates the expression of cBrn3c following central-to-peripheral and temporal-to-nasal gradients. As a result, similar to the Xath5 protein, both Cath5 and Math5 proteins have the ability to promote the development of ganglion cells. Moreover, forced expression of all three Brn3 genes also can stimulate the expression of cBrn3c. Ath5 and Brn3 proteins are capable of transactivating a Brn3b promoter. Thus, these data suggest that the expression of cBrn3c in the chicken and Brn3b in the mouse is initially activated by Ath5 factors in newly generated ganglion cells and later maintained by a feedback loop of Brn3 factors in the differentiated ganglion cells (Liu, 2001).

The data suggest that Ath5 and Brn3 factors constitute a transcriptional cascade regulating the specification, differentiation, and survival of retinal ganglion cells. During chicken retinogenesis, Cath5 may function in competent progenitors to promote commitment of ganglion cell fates and activate cBrn3c expression. The activation of cBrn3c expression in postmitotic ganglion cell precursors then promotes ganglion cell differentiation and survival. In differentiated ganglion cells, cBrn3c may activate the expression of cBrn3a and cBrn3b, and its expression may be subject to positive autoregulation and feedback control by cBrn3a and cBrn3b. During mouse retinogenesis, an analogous transcriptional cascade controls ganglion cell development. Math5 is responsible for the initiation of expression of Brn3b, which in turn activates the expression of Brn3a and Brn3c. The expression of Brn3b in differentiated ganglion cells is then maintained by a combination of autoactivation and feedback regulation by Brn3a and Brn3c. Although it remains to be determined what regulates the expression of Cath5 and Math5, multiple midline signals including Nodal signaling have been implicated in the control of ath5 expression in the developing zebrafish retina (Liu, 2001).

Math1 is a basic helix-loop-helix transcription factor expressed in progenitor cells that give rise to dorsal commissural interneurons in the spinal cord, granule cells of the cerebellum, and sensory cells in the inner ear and skin. Transcriptional regulation of this gene is tightly controlled both temporally and spatially during nervous system development. The signals that mediate this regulation are likely integrated at the Math1 enhancer, which is highly conserved among vertebrate species. The zinc-finger transcription factor Zic1 has been identified as a regulator of Math1 expression. Zic1 binds a novel conserved site within the Math1 enhancer, and represses both the expression of endogenous Cath1 (chicken homolog of Math1) and the activity of a Math1 enhancer driven lacZ reporter when expressed in chick neural tubes. Repression by Zic1 blocks the autoregulatory activity of Math1 itself. Although previous reports have shown that Zic1 and Math1 are both induced by BMP signaling, these genes appear to have opposing functions, since Math1 acts to promote neuronal differentiation in the chick neural tube and excess Zic1 appears to block differentiation. Zic1-mediated repression of Cath1 transcription may modulate the temporal switch between the progenitor state and differentiating dorsal cell types during neural tube development (Ebert, 2003).

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).

Mammalian Atonal homologs

Atonal Evolutionary homologs part 2/2

atonal: Biological Overview | Regulation | Targets of Activity and Protein Interactions | Developmental Biology | Effects of Mutation | References

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