InteractiveFly: GeneBrief

cousin of atonal: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

In the expectation that more bHLH genes are required in neurogenesis in Drosophila, new bHLH genes have been sought in PCR experiments using degenerate primers based on homology to atonal. In addition to ato itself, the PCR product contained potential bHLH-encoding sequences from two new closely related genes, which have been termed amos (Goulding, 2000a) and cousin of atonal (cato), the subject of this report. cato is expressed widely in the developing PNS after neural precursor selection but before terminal differentiation. Consistent with this pattern, cato appears to be required for proper sensory neuron morphology (Golding, 2000b).

Numerous vertebrate homologs of Drosophila proneural genes of the achaete-scute complex and of atonal have been cloned, supporting the argument that many aspects of neurogenesis in Drosophila and vertebrates are homologous. Indeed, characteristics consistent with a role in neural precursor determination have been demonstrated for some of these genes, including Xash3 (a Xenopus AS-C homologue), neurogenin1 (a distant ato relative), Math1, and Math5 (ato homologs). Surprising was the fact that expression of many of these homologs are activated later in neural development, after the stage of neural precursor determination. From this observation, it has been proposed that cascades or networks of bHLH factors may function during all stages of vertebrate neural development from commitment of precursors, through to proliferation and migration, and finally to postmitotic terminal differentiation. The implication of this hypothesis is that bHLH proteins control appropriate target genes for each of these stages, including the activation of the next bHLH protein in the cascade (Golding, 2000b and references therein).

Many questions arise pertaining to the different roles of these closely related genes and how these roles are achieved. The Drosophila PNS has provided a particularly amenable model for analyzing proneural genes, but despite this, positive bHLH regulators of later neurogenesis in Drosophila are not well understood. The best known example is asense (ase), an aberrant member of the AS-C that is activated in all neural precursors after their formation. Mutation of ase, however, has no apparent effect in most of these cells, which may be because the phenotype is too subtle or there is redundancy with other factors. In addition to ase, a fly homolog of vertebrate neurogenin has been isolated. This gene, target of Pox-n (tap), is expressed in a handful of differentiating chemosensory neurons, but is incompletely characterized functionally (Ledent, 1998). cato is expressed widely in the developing PNS after neural precursor selection but before terminal differentiation. Consistent with this pattern, cato appears to be required for proper sensory neuron morphology. It is clear that cato is not a proneural gene, despite its close sequence relationship with ato. The available evidence suggests that instead, it is associated later in neurogenesis with correct neuronal morphology. cato therefore potentially represents a gene that functions similar to vertebrate neuronal differentiation regulators, such as neuroD (Golding, 2000b).

There are widespread defects in neuronal morphology in cato-deficient embryos: the dendrites of chordotonal neurons are consistently malformed, appearing longer and often thicker than their wild-type equivalents. Although it is likely that cato mutant sensory neurons would be severely impaired functionally, the morphological defects observed are subtle. This might be the result of redundancy between cato and related gene functions. Of particular interest as a possible cato interactor is ase. because ase encodes a bHLH protein that is expressed in a postproneural pattern similar to that of cato. Although ase is widely expressed in the developing nervous system, most embryonic neurons appear normal in ase mutant embryos, and such individuals are indeed quite viable. Despite this, it was found that additional mutation of ase strongly enhances the neuronal differentiation defects of cato-deficient embryos. In particular, the dendritic defects described above are strongly exacerbated in these embryos. The scolopale is a glial structure that surrounds the PNS neuron. In the most striking cases it appears that the scolopale structure itself is inappropriately stained by neuronal antibodies, suggesting either that the scolopale cell was beginning to exhibit neuronal characteristics or that the dendrite extends around it rather than entering into it. Additionally, some neurons appear to have lost their particular neuronal characteristics and, although stained by 22C10, had either lost or not developed any distinctive neuronal morphology (Golding, 2000b).

In addition to the worsening of neuronal morphology, these double-mutant embryos develop axon-related defects that are not observed in either cato or ase mutant embryos. In particular, many neurons of the dorsal and lateral sensory neuron groups have poorly formed or misrouted axons, and in extreme cases, axons are entirely missing. This suggests that cato and ase are required either for correct axon pathfinding to the CNS or for the general process of axon outgrowth itself. The axon outgrowth defects are similar to those observed in pros mutant embryos. Thus, as in pros mutants, dorsal sensory neurons are more strongly affected -- probably because they are more sensitive in their response to signals provided by the ventral CNS that aid their pathfinding (Golding, 2000b).

Experimental misexpression of proneural genes in developing ectoderm results in ectopic sense organ formation owing to excessive SOP commitment. For instance, the scutellum (rear of thorax) normally bears four external sense organs (bristles) and no chordotonal organs. UAS-scute flies develop extra external sense organs when expression is driven by Gal4^109-68, a driver line specific for proneural cluster cells. Conversely, ato is functionally distinct in that its misexpression results in ectopic chordotonal organs within the scutellum, although numbers of external sense organs are also formed, which is postulated to be the result of dissociation of ato's SOP-determining and chordotonal identity-determining properties in this assay. Interestingly, when ase is misexpressed, phenotypes identical to those of sc are observed. Thus, although ase is not normally expressed during SOP commitment, it is capable of proneural function that is indistinguishable from ac and sc (Golding, 2000b).

It was asked whether cato would similarly be functionally indistinguishable from ato in this ectopic sense organ assay. Initial observations of Gal4^109-68/UAS-cato flies indicate that cato can indeed behave as an ato-like proneural gene in directing the formation of a mixture of ectopic external sense organs and chordotonal organs. Like ato, at low misexpression levels cato induces extra external sense organs, but at higher levels external sense organs fail to form. Concomitantly, ectopic chordotonal organs are now present within the scutellum. A more detailed analysis, however, showed several qualitative and quantitative differences between the UAS-cato and the UAS-ato phenotypes. One noteworthy difference is that only under conditions of very strong cato misexpression are ectopic chordotonal organs generated, and even then far fewer chordotonal organs are formed than for UAS-ato. Also, these sensilla tend to be isolated or in small, poorly formed clusters of chordotonal organs. Such clusters are quite different in appearance from the densely packed arrays produced in Gal4^109-68/UAS-ato flies. These results suggest that although cato can behave inappropriately as a chordotonal proneural gene, it cannot efficiently bypass the requirement for ato in chordotonal SOP formation, and thus the proteins are not functionally equivalent (Golding, 2000b).

A further difference with ato is that the few chordotonal organs formed by UAS-cato tend to be malformed. Indeed it is often difficult to identify ectopic chordotonal organs on morphology alone, their scolopale structures being poorly differentiated. Additionally, the scolopale cells often stain inappropriately with the neuronal marker 22C10. The differentiation of external sense organs is also affected, again most notably with the malformation of support cells (socket and hair). It seems likely that disrupting the normal pattern of cato expression (by misexpression) affects sense organ cellular differentiation. This effect is also seen after misexpression in the embryo using a hairy-Gal4 driver line. With this driver, misexpression of ato results in expansion of lateral chordotonal organ clusters (usually 6-10 neurons instead of 5) but the neurons are of normal morphology. In contrast, misexpression of cato results in less expansion of the chordotonal clusters (usually 5-7 neurons); instead, there are organizational and dendritic abnormalities in the chordotonal neurones that are reminiscent of those seen upon cato loss (Golding, 2000b).

Among the vertebrate members of the ato-like subfamily, Xath5 is also expressed during neural differentiation like cato (Kanekar, 1997). Successive expression of different bHLH genes has been observed in a number of areas of vertebrate neurogenesis, such as the olfactory placode, the retina, and the neural tube. In a similar fashion, cato expression defines a cascade of bHLH gene activation in Drosophila neurogenesis. Thus, in chordotonal development, the order of bHLH gene activation is ato followed by cato and then followed by ase. In external sense organs, this appears to become scute/achaete followed by ase and then followed by cato. The overlap of ato and cato expression during chordotonal SOP development conforms to the notion of cascades of interlinked bHLH transcription factors in neurogenesis. It is not clear, however, whether ato does regulate cato. Examination of the cato genomic sequence for potential Ato/Da heterodimer binding sites (E boxes) reveals a number of good sites. However, in at least one chordotonal SOP (the P cell), factors other than Ato must be capable of activating cato, since its expression persists in ato mutant embryos. Interestingly, it has been a paradox that the P cell is still able to form a chordotonal organ in ato mutant embryos. It is likely that sc can replace ato function in forming the P cell, but the paradox is that among the proneural genes only ato has been shown to provide chordotonal identity information. The presence of cato in this precursor may resolve this problem, since cato may be able to endow this cell with chordotonal identity in the absence of ato (Golding, 2000b).

The function and regulation of the bHLH gene, cato, in Drosophila neurogenesis

bHLH transcription factors play many roles in neural development. cousin of atonal (cato) encodes one such factor that is expressed widely in the developing sensory nervous system of Drosophila. However, nothing definitive was known of its function owing to the lack of specific mutations. This study characterised the expression pattern of cato in detail using newly raised antibodies and GFP reporter gene constructs. Expression is predominantly in sensory lineages that depend on the atonal and amos proneural genes. In lineages that depend on the scute proneural gene, cato is expressed later and seems to be particularly associated with the type II neurons. Consistent with this, evidence was found that cato is a direct target gene of Atonal and Amos, but not of Scute. Two specific mutations of cato were generated. Mutant embryos show several defects in chordotonal sensory lineages, most notably the duplication of the sensory neuron, which appears to be caused by an extra cell division. In addition, cato is required to form the single chordotonal organ that persists in atonal mutant embryos. It is concluded that although widely expressed in the developing PNS, cato is expressed and regulated very differently in different sensory lineages. Mutant phenotypes correlate with cato's major expression in the chordotonal sensory lineage. In these cells, it is proposed that cato plays roles in sense organ precursor maintenance and/or identity, and in controlling the number of cell divisions in the neuronal branch of the lineage arising from these precursors (zur Lage, 2010).

Although cato is a PNS-specific gene, its expression and function appear to be different in distinct lineages of the PNS. Its expression begins in Ch precursors just after their formation, but appears much later in ES lineages. Correlating with this pattern, it was found that cato is directly regulated by ato in Ch SOPs but it is not a direct target of Sc in ES SOPs. This expression pattern, and its underlying regulation, appears to be characteristic of a number of genes, including the transcription factor Rfx and a number of its targets. The pattern is referred to as 'Ch-enriched' and it is suggested that such genes mediate part of Ato's subtype specificity in neurogenesis. Interestingly, in different sensory lineages, it seems that cato is regulated by Amos and Ato through the same E box binding site (zur Lage, 2010).

The functions characterised for cato relate to its major site of expression: the Ch organs. The most obvious defect in cato mutant embryos involves supernumerary cell divisions in the neuronal branch of Ch lineages. This is reminiscent of the known roles of the other non-proneural bHLH proteins, dpn and ase. Thus, in the larval optic lobe, dpn expression maintains proliferation, whilst ase promotes cell cycle exit and neuronal differentiation. The function of cato and ase in limiting cell division resembles the well-known function of vertebrate proneural-like bHLH factors in promoting the cell cycle exit of neuronal progenitors as a prelude to differentiation. This is opposed by HES factors (homologous to dpn), which maintain proliferation (zur Lage, 2010).

In the case of the larval optic lobe, ase functions in part via the CDK inhibitor, dap. dap itself is generally required for cells to terminate cell division appropriately and cells generally undergo one extra division in dap mutants. dap expression is highly dynamic in embryos, and it appears that a pulse of dap expression helps to ensure the timely shut down of cyclin function for appropriate cell cycle exit. This study shows that dap is similarly required for Ch neurons. Moreover, the PNS phenotype of dap mutant embryos is strikingly similar to that of cato. This suggests that cato regulates dap in Ch neurons. Genetic analysis suggests this might be so, but no clear change was seen in dap expression in cato mutant embryos. However, the complex and highly dynamic expression of dap may make small lineage-specific changes in expression difficult to detect. The idea that cato might regulate dap is consistent with previous observations that dap is under the control of multiple developmental regulators rather than of cell cycle regulators themselves, and also that dap is regulated by Ato in the developing eye. dap is one of several cell cycle regulators (cyclin E and string that have complex modular cis-regulatory regions. It is notable that cato appears to regulate only the division of the neuron and not support cells. It is speculated that this division may require independent regulation from those of the support cells, because the number of neurons within a Ch organ varies in different locations, presumably as a result of extra neuronal cell divisions. For instance, some Ch organs in the adult femur have two neurons, whilst Ch organs in the antenna have three neurons (zur Lage, 2010).

The other functions detected for cato appear to be unrelated to the neuronal duplication function and show at least some redundancy with other bHLH regulators (ato and ase). In both these cases it is suggested that cato plays a partially redundant role in maintaining SOP fate. In the absence of ase and cato, some Ch SOPs fail to form scolopidia. A similar situation applies to C1 in the absence of ato and cato. The apparent redundancy between ato and cato suggests that C1 SOP can form via alternative routes involving ato and cato. However, cato is expressed too late to be a proneural gene, and so another factor must supply the proneural function in the absence of ato. It seems likely that this factor is sc, which is expressed in C1 despite being the ES proneural gene. Embryos with a mutation of the achaete-scute complex often show one missing scolopidium in the lch5 cluster, while AS-C/ato mutant embryos have no Ch cells at all. Such interchangeability of proneural functions between ato and sc is surprising since sc does not generally have the capacity to direct Ch subtype specification, as shown in misexpression experiments. In contrast, ato's subtype specificity function is reflected in its ability to convert ES SOPs to a Ch fate. It is suggested that expression of cato in a sc-dependent C1 cell may provide sufficient subtype determination information when ato is absent. It is not clear why such a complicated exception should have arisen. One possibility is that C1 forms a unique neuronal type among Ch organs. Certainly there are a number of genes that are only expressed in, or are only absent from, this one neuron. For instance, MAb49C4 detects an antigen that is expressed in all lch5 neurons except lch5a. Moreover, C1 appears to be functionally unique in that it acts to induce surrounding cells to differentiate as oenocytes via EGFR signalling. This function of C1 appears to be 'rescued' by cato function, since the C1 cells present in ato mutants are able to recruit oenocytes (zur Lage, 2010).

Expression of Cato in ES lineages appears to be mainly as a prelude to late expression in the md/da neurons that derive from both ES and Ch lineages. As yet, no function has been discerned for this late expression, but it is speculated that cato mutant larvae may exhibit a physiological defect in da neurons, which are thought to be required for nociception and thermoreception (zur Lage, 2010).

Characterisation of the first mutations for cato has revealed roles in maintenance and cell division in Ch lineages. These roles are relatively subtle considering that cato is expressed widely in the developing PNS. Moreover, cato orthologues can be readily recognised among Drosophila species and other Diptera, suggesting strong conservation. It is possible that further functions remain to be uncovered, perhaps in da neuron physiology or in the complex cephalic sense organs (zur Lage, 2010).

REGULATION

Transcriptional Regulation

It seems that loss or gain of cato function causes neuronal defects. Like cato, embryos mutant for pros show neuronal defects, although the basis of these defects is not known. It was asked whether cato expression depends on pros function. Strikingly, loss of pros function results in the ectopic appearance of cato transcription within the ganglion mother cells (GMCs) and neurons of the developing CNS. This correlates with the wild-type expression of Pros in GMCs, indicating that Pros is normally a transcriptional repressor of cato in the CNS. There are indications that cato derepression begins in neuroblasts, even though pros is not thought to function until GMC formation: although Pros protein is present in the neuroblasts, it becomes localized only in the nucleus in the GMCs. Interestingly, although pros is apparently recessive, a weak derepression of cato is also observed in heterozygote pros/+ embryos. In this case cato expression is observed most clearly as nuclear sites of nascent transcription (Golding, 2000b).

DEVELOPMENTAL BIOLOGY

Embryonic

In situ hybridization to mRNA in whole-mount embryos reveals that cato is expressed dynamically during neurogenesis and is confined to the developing PNS. The manner of expression is very different from that of proneural genes: it is initiated in SOPs after their formation. Expression continues in the division products of the precursors (after proneural genes are normally switched off) and the pattern broadly resembles that of the PNS enhancer trap line A37. Expression is switched off as cells begin to undergo terminal differentiation. cato expression appears to be activated earliest in chordotonal SOPs. Thus the initial pattern of cato expression in stage 10/11 strongly marks the appearance of chordotonal SOPs, and in this respect the pattern distinctly resembles the later expression of ato as it becomes refined from proneural clusters to the SOPs. Double-labelling experiments using antibodies to Ato in conjunction with a Cato RNA probe confirm that the onset of cato expression correlates with the refinement of Ato expression to isolated SOPs. Later, after ato is switched off, cato remains expressed in the division products of the SOPs (Golding, 2000b).

In PNS cells other than the chordotonal SOPs, cato expression appears delayed relative to SOP formation. This is most clearly seen when the first external sense organ SOP (the A cell) and chordotonal SOP (the P cell) are formed in each abdominal segment during embryonic stage 10. cato is initially activated only in the P cell, appearing later in the A cell during stage 11. Although subsequent SOP formation is dynamic and individual SOPs are difficult to follow, this delay seems to hold true for other external sense organ cells too. Thus, colabelling experiments show that cato expression in external sense organ precursors appears later than either Cut (a specific marker of these SOPs) or Asense (a marker of all neural precursors). Interestingly, the difference in timing of expression between cato and Ase appears reversed in chordotonal SOP formation. Thus, unlike cato, Ase expression appears relatively late in most chordotonal SOPs with the exception of the P cell and shows little overlap with Ato (Golding, 2000b).

Expression of cato appears to continue in the progeny of many or all SOPs during late stage 11 and early stage 12 and at this point resembles the pattern of ase. At this stage cato expression is too complex to discern the identity of individual cells. Nevertheless, during stage 12/13, Cato mRNA appears to become restricted to one of the daughter cells of each sense organ before being finally switched off. Double-labelling experiments with Cut and Pros antibodies suggest that this cell is the neuron. By the end of stage 13, cato expression is largely extinguished, and there is little apparent overlap with markers of neuronal differentiation, such as Elav (Golding, 2000b).

The temporal difference in cato activation between different SOP subtypes suggests that there may be separable functions for cato. It also suggests that different regulatory processes are at play. In particular, the overlap in Ato and cato expression in chordotonal SOPs suggests that cato may be directly activated by Ato. However, no evidence has been found for this. Although most chordotonal expression of cato is indeed absent in ato mutant embryos, this could merely reflect the failure of chordotonal SOP formation that occurs in this mutant. Indeed, cato expression is still detectable in the P cell chordotonal precursor, whose formation is not completely dependent on ato function. Therefore, at least in the P cell, factors other than Ato are capable of activating cato’s expression (Golding, 2000b).

Larval

Expression in the larval imaginal discs mirrors the embryonic expression by being PNS specific and confined to developing SOPs and their progeny. In the wing disc, for instance, the pattern of cato expression broadly resembles that of the SOP enhancer trap marker, A101. Like ase, it persists through at least one round of cell division in the case of the external sense organ precursors. As in the embryo, Cato RNA appears strongly in chordotonal SOPs soon after their formation in the wing, antenna, and leg discs, whereas it is strongly delayed in external sense organ cells. Significantly, cato is not expressed in developing photoreceptors in the eye disc. In the leg disc, cato expression is more transient in the precursors of the femoral chordotonal organ than elsewhere; its expression here is switched off abruptly at the time when Ato itself is abruptly downregulated (Golding, 2000b).

REFERENCES

Search PubMed for articles about Drosophila cato

Goulding, S. E., zur Lage, P. and Jarman, A. P. (2000a). amos, a proneural gene for Drosophila olfactory sense organs that is regulated by lozenge. Neuron 25: 69-78. PubMed Citation: 10707973

Goulding, S. E., White, N. M. and Jarman, A. P. (2000b). cato encodes a basic helix-loop-helix transcription factor implicated in the correct differentiation of Drosophila sense organs. Dev. Biol. 221: 120-131. PubMed Citation: 10772796

Kanekar, S., Perron, M., Dorsky, R., Harris, W. A., Jan, L. Y., Jan, Y. N. and Vetter, M. L. (1997). Xath5 participates in a network of bHLH genes in the developing Xenopus retina. Neuron 19: 981-994. PubMed Citation: 9390513

Ledent, V., Gaillard, F., Gautier, P., Ghysen, A. and Dambly-Chaudiere, C. (1998). Expression and function of tap in the gustatory and olfactory organs of Drosophila. Int. J. Dev. Biol. 42(2): 163-170. PubMed Citation: 9551861

zur Lage, P. I. and Jarman, A. P. (2010). The function and regulation of the bHLH gene, cato, in Drosophila neurogenesis. BMC Dev. Biol. 10: 34. PubMed Citation: 20346138

date revised: 15 April 2011

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