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).

GENE STRUCTURE

cDNA clone length - 1467

Bases in 5' UTR - 425

Exons - 1

Bases in 3' UTR - 446

PROTEIN STRUCTURE

Amino Acids - 189

Structural Domains

The bHLH domain of the conceptual Cato protein is 64% identical to Ato and 41% identical to Scute. This compares with 42% identity between Ato and Sc and ~70% identity between members of the AS-C. Among Ato-like bHLH proteins, Cato forms part of the Ato subfamily along with its closest vertebrate homologs and is closer to Ato than to the neuroD or neurogenin subfamilies. Cato is no more closely related to Amos than to Atonal, and there is no sequence conservation outside the bHLH domain. All the residues important for the three-dimensional structure of the bHLH domain are conserved in Cato. Furthermore, all residues required for E-box binding are found in Cato, both in the basic, helix-2 N-terminus and in the loop regions, suggesting that Cato is a positive regulator of transcription. Comparison with the important structural residues of Ato also predicts that Cato probably functions as a heterodimer with Daughterless protein. Differences in the basic region have been shown to confer functional specificity to Ato and Sc. This region is strongly Ato-like in Cato, but there are also a number of differences, which may represent either functional determinants that distinguish Ato and Cato or residues that are not constrained for function. The respective bHLH domains of Ato and Cato are highly conserved in their D. virilis homologs, suggesting functional significance for these differences (Golding, 2000b).

date revised: 20 June 2000

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.