BIOLOGICAL OVERVIEW

It is increasingly apparent that proneural bHLH transcription factors not only confer neural competence for sensory organ precursor (SOP) formation but also endow SOPs with neural subtype information. Amos is required to establish the identity of multiple classes of sense organs. Thus, its complicated, full name of 'Absent MD neurons and olfactory sensilla', suggests that Amos is required for the identity of a class of multidendritic neurons (MD) and for a class of olfactory sensilla. Specifically, Amos is required to established the identity of the so-called solo-MD neurons and to establish the identity of two olfactory sensilla: basiconica and trichodea sensilla. Therefore, this essay must enter the complex world of neurals subtypes and the processes by which their identities are established.

The Drosophila embryonic peripheral nervous system (PNS) consists of three major types of sensory organs: external sensory (ES) organs, chordotonal (CH) organs, and multiple dendritic (MD) neurons. Despite having distinct neuronal activities and sensory structures, the mechanisms of early cell fate specification for these three types of sensory organs are quite similar. The program to develop sensory organs is initiated by the activities of proneural genes. The transcription activation of proneural genes is limited to small patches of ectodermal cells, termed proneural clusters. The expression of proneural genes confers on each cell within the proneural cluster the potential to become a sensory organ precursor (SOP) cell. However, only one, or at most, a few cells within a cluster are selected to become SOP cells. This cell(s) maintains the proneural gene expression by autoactivation. Proneural genes achaete (ac) and scute (sc) of the achaete-scute complex (AS-C) are required for the formation of ES organs, whereas atonal (ato) directs the formation of CH organs. MD neurons are generated in two ways. Most MD neurons are derived from ES or CH organ lineages. The remaining MD neurons, known as the solo-MD neurons, are generated directly from SOP cells, independent of ES and CH lineages. In the case of some solo-MD neurons in the ventral (vmd) and dorsal (dmd) groups, the originating SOP cells divide a few times to generate daughter cells destined exclusively to become MD neurons. A particular solo-MD neuron, the dorsal bipolar (dbp) neuron, is generated from an asymmetric division of a dorsal SOP cell, which gives rise to a dbp neuron and an associated glia cell (Huang, 2000 and references therein).

In mutants lacking both the AS-C and atonal, coding for proteins known to establish SOP cell fate, two to three neurons of the solo-MD type remain in the dorsal region of each abdominal hemisegment. The identity of these remaining neurons are controlled by Amos. Both the AS-C and Atonal physically interact with the protein Daughterless (Da). Since the solo-MD neurons that exist in AS-C;ato double mutants are eliminated in da mutants, this observation implies that the proneural gene for those solo-MD neurons also encodes a transcription factor of the bHLH family (Huang, 2000).

The technique of double-stranded RNA interference (RNAi), a procedure that mimics the effect of mutation, was used to eliminate amos function in embryos. Loss of amos function eliminates MD neurons that remain in AS-C;atonal mutants. These results suggested that amos is required for the formation of dbp and some dmd neurons, the neurons, which remain in AS-C;ato double mutants. When misexpressed, amos induces the formation of all types of sensory neurons. These results provide strong evidenct that intrinsic differences among the proneural genes, specifically genes of the AS-C, atonal and amos play a role in the induction of different types of sensory neurons (Huang, 2000).

The development of olfactory sensilla is not as well characterized as it is for sense organs such as sensory bristles (external sense organs) or chordotonal organs (internal stretch receptors). Nevertheless, the initial developmental steps appear similar to these other sense organs. For each adult olfactory sensillum, a precursor cell is selected from the ectoderm of the early pupal antennal imaginal disc. These cells, generally termed sense organ precursors (SOPs), arise over an extended period of time in the developing pupal antennal imaginal disc. Despite their resemblance to sensory bristles, the cells of an olfactory sensillum do not appear to arise solely by division of the SOP. Instead, the SOP (dubbed the founder cell) apparently recruits surrounding ectodermal cells to become the support cells that form the external sensillum structure (Reddy, 1997). Perhaps surprisingly, the adult olfactory SOPs are not specified by the AS-C (Gupta, 1997), confirming that these sensilla are distinct from external sense organs. Instead, some maxillary palp sensilla and the antennal sensilla coeloconica require atonal (Gupta, 1997). None of these genes, however, are required for the sensilla basiconica and trichodea. Nevertheless, there is good evidence that these sensilla require the activity of an unidentified bHLH proneural gene(s). Gupta (1997) has shown that misexpression of extra machrochaetae in the pupal antennal disc causes a decrease in olfactory sensilla of all types, suggestive of antagonism of a bHLH protein in addition to ato. Interestingly, basiconic and trichoid sensillum formation requires the function of a Runt domain transcription factor encoded by lozenge (lz), with sensilla basiconica being completely absent in strong lz mutants. A recent analysis suggests that lz acts upstream of SOP formation but is not itself a proneural gene (Gupta, 1998). amos expression is highly localized, being mostly restricted to regions in which olfactory precursors arise in both embryo and imaginal discs. Loss-of-function and gain-of-function evidence exists that amos is a proneural gene for a subset of olfactory sensilla, most likely the sensilla basiconica and trichodea. Moreover, amos is partly regulated by lz, and this regulation can account for the effect of lz mutations on olfactory SOP formation (Goulding, 2000).

It is increasingly apparent that proneural bHLH factors not only confer neural competence for SOP formation but also endow SOPs with neural subtype information. While expression and loss-of-function analyses show that amos, ato, and the AS-C are required for different subtypes of sense organs, a direct test of the functional specificity of proneural proteins comes from comparison of their capabilities in misexpression assays. Misexpression of all proneural genes results in ectopic SOP formation, but the subtype of the sense organ depends on both the gene misexpressed and the developmental context of misexpression. In the context of antennal development, misexpression of amos or ato, but not an AS-C gene, drives ectopic olfactory sensillum formation. This suggests that the shared features of the amos and ato bHLH domains provide functional information important for olfactory sensillum determination. One of the specific functions ascribed to ato in chordotonal SOP formation is the inhibition of cut, a subtype selector gene for external sense organs. Olfactory sensilla also do not require cut, and so inhibition of cut expression might be an important conserved function of ato and amos in olfactory SOP formation. Another shared function may be the activation of cell recruitment, since amos- and ato-dependent sense organs (olfactory sensilla, chordotonal organs, and ommatidia) all require some form of local cell recruitment in their development. The recruitment of cells to an ommatidium and the recruitment of SOPs into chordotonal clusters both require Egfr signaling, perhaps under the direct control of ato. It is not yet known whether Egfr is involved in the process of cell recruitment by olfactory SOPs, but it is clearly possible that ato and amos directly control the recruitment process. Outside the antenna, misexpressed ato drives ectopic chordotonal organ formation. This functional specificity has been found to reside in Ato's bHLH domain and largely in its basic region. Ato's basic region is completely conserved in Amos, apart from an R to K conservative substitution. Not surprisingly, therefore, amos can also mimic ato in directing chordotonal organ formation when misexpressed outside the antenna. Nevertheless, despite amos's ability to mimic ato and their common requirement in olfactory sensillum formation, these genes are not functionally interchangeable. Differences in their ability to determine olfactory sensillum subtypes are initially suggested by their differing loss-of-function phenotypes, despite their partly overlapping expression domains. Also, Gupta (1997) reported that ato misexpression in the antenna specifically results in increased sensillum coeloconica numbers, while amos increases all sensilla types in the experiments described here and can rescue the loss of sensilla basiconica in lz mutants. At the least, this shows that amos can supply some functional information that ato apparently cannot (i.e., basiconic/trichoid fate). Most significantly, only amos misexpression can drive ectopic sensilla of olfactory-like morphology outside the antenna. Thus, unlike ato, amos function in olfactory sensillum formation appears to be partially able to overcome the constraints of developmental context. Presumably, one or more target genes must be differentially regulated by amos and ato (Goulding, 2000 and references therein).

The proneural gene amos promotes multiple dendritic neuron formation in the Drosophila peripheral nervous system

Misexpression has also been used to examine the abilities of various proneuronal genes to determine SOP cell fate in the embryonic PNS. To determine whether different types of proneural genes confer SOP cells with distinct neuronal specificities, a test was made of the abilities of three proneural genes (sc, ato, and amos) to induce the formation of ES, CH, and MD neurons. For MD neuron formation, the lacZ expression was tested in E7-2-36 embryos carrying proneural genes misexpressed by sca-GAL4. Misexpression of amos leads to a significant increase of MD neurons through the whole segment. In the ventral region, the number of MD neurons (vmd5 + vpda) increases significantly. Misexpression of sc or ato slightly induces the formation of MD neurons, but not as significantly as misexpression of amos. To compare the induction abilities of these three proneural genes in CH neuron formation, embryos were stained with MAb22C10, and the lateral CH neurons were scored. In embryos with ato or amos misexpressed by sca-GAL4, a slight increase in the number of CH neurons in the lateral region was observed. Few ectopic CH neurons were induced in embryos that misexpressed sc. A test was performed to see if amos could rescue ato mutants in CH organ formation. In ato¹ mutants, misexpression of amos driven by hairy-GAL4 restores CH neuron formation in odd, but not even, segments. The extent of CH neurons rescued by amos is comparable to that of neurons rescued by ato, suggesting that amos can replace ato functionally (Huang, 2000).

Ectopic ES bristles in wing blades can be induced by misexpression of proneural genes right after puparium formation. To drive gene expression during this period, flies carrying hs-GAL4 and UAS-ato or UAS-amos transgenes were heat treated for 10 min. The hs-GAL4/UAS-sc flies were heat treated for 5 min to avoid lethality. The ectopic bristles were generated most efficiently in the flies misexpressing sc, although they were heat treated for only half of the time period. The ectopic bristle number induced by amos was higher than the number induced by ato. In summary, all three proneural genes are capable of promoting sensory bristle formation, with sc being the strongest. ato and amos, but not sc, can induce CH neuron formation. Finally, only amos strongly induces MD neuron formation. These data suggest that proneural genes of different types confer SOP cells with differential potentials to develop into various types of sensory neurons (Huang, 2000).

Each member of the AS-C, when misexpressed, exclusively produces ES organs, while ato produces both CH and ES organs. Experiments that involve swapping the various domains of Ato and Sc suggest that the ability to generate CH organ formation resides in the basic domain of the Ato bHLH protein. The basic domain of Amos is identical to that of Ato except for a conserved change of Lys to Arg at the boundary of the basic domain. This conservation explains how amos replaces ato in CH organ formation. However, when misexpressed, only amos promotes a significant number of MD neurons, implying that the almost identical DNA-binding domains of Amos and Ato are not sufficient to account for MD neuron formation. Taken together, these data suggest that regions residing outside the DNA-binding domain of Amos contribute to the formation of MD neurons and olfactory sensilla other than the coeloconic type. Identification of the regions for neuronal subtype determination and how they interact with neuronal selector genes will be interesting topics for future study (Huang, 2000 and references therein).

amos promotes olfactory sensillum formation and suppresses bristle formation

amos is a new candidate Drosophila proneural gene related to atonal. Having isolated the first specific amos loss-of-function mutations, it has been shown definitively that amos is required to specify the precursors of two classes of olfactory sensilla. Unlike other known proneural mutations, a novel characteristic of amos loss of function is the appearance of ectopic sensory bristles in addition to loss of olfactory sensilla, owing to the inappropriate function of scute. This supports a model of inhibitory interactions between proneural genes, whereby ato-like genes (amos and ato) must suppress sensory bristle fate as well as promote alternative sense organ subtypes (zur Lage, 2003).

Three mutant alleles of amos were generated in an EMS screen. amos¹ is predicted to result in a protein truncation that removes the second half of bHLH helix 2 and the C-terminal region thereafter. amos² is a missense mutation that changes a Ser to an Asn in helix 1 of the bHLH domain. This position is not part of the bHLH core consensus sequence and is not predicted to directly affect DNA binding or dimerization. Moreover, Asn is found in this position in the ato bHLH domain, and so the effect of this mutation would be predicted to be mild. amos³ contains a 230 bp deletion within the ORF, which also causes a frame-shift that brings a spurious downstream stop codon in frame. This allele gives a predicted peptide of 74 amino acids, of which only the first 30 are shared with amos. It therefore lacks the entire bHLH domain and is likely to be a null (zur Lage, 2003).

amos¹ mutant embryos lack two dorsal sensory neurons per segment, including the dorsal bipolar dendritic neuron. Nevertheless, all amos alleles are adult viable as homozygotes and hemizygotes. The antennae of mutant adult flies were mounted and examined by light microscopy in order to quantify the number and type of olfactory sensilla. Compared with wild-type, amos mutant antennae carried dramatically reduced numbers of sensilla and, as a consequence, the third segment is significantly smaller. In particular, sensilla basiconica and trichodea were completely absent in the probable genetic nulls, amos¹ and amos³, whereas sensilla coeloconica appear unaffected. These phenotypes support the assertion that amos is the proneural gene for sensilla basiconica and trichodea, whereas ato is the proneural gene for sensilla coeloconica. However, mutant antennae exhibit a further unexpected phenotype. In wild type, the third segment bears only olfactory sensilla; in amos mutant flies, this segment bears a number of ectopic external sensory bristles and other abnormally structured sensilla. These bristles do not have bracts (unlike bristles on the leg), and so this phenotype does not represent a transformation of antenna to leg. These phenotypes are highly unusual; a characteristic of all other loss-of-function proneural gene mutations is that they cause the loss of sense organ subsets without the concomitant appearance of new or abnormal sensory structures. Therefore, the amos null phenotype is unique for a Drosophila proneural gene (zur Lage, 2003).

Given its subtle molecular basis, the putative hypomorph amos² has a surprisingly strong phenotype: it has no sensilla trichodea, and sensilla basiconica are reduced very substantially. There are also fewer ectopic bristles than in the null alleles, but there are many sensilla of unusual morphology. In the case of this allele, these seem to represent intermediates between sensilla basiconica/trichodea and external sense organs (zur Lage, 2003).

Late pupal antennae were stained with a sensory neuron marker, MAb22C10, to visualize olfactory receptor neurons (ORNs). Olfactory sensilla are innervated by multiple sensory neurons, which can be seen as groups in the wild-type antenna. amos mutant antennae have many fewer neuronal groups, corresponding in number to the sensilla coeloconica and the bristles. There are instances of sensilla innervated by a single neuron, which appear to correspond to the ectopic bristles. In wild-type flies, ORN axons form three olfactory nerves leading to the antennal lobe of the brain. In amos mutant antennae, all three antennal nerves are still present, although consisting of fewer axons (comprising the axons of ato-dependent ORNs). Although thinner, the fascicles appear normal in structure and location. Thus, in contrast to ato, mutations of amos do not cause defects in routing or fasciculation of the olfactory nerves. This supports the conclusion that the ato-dependent sensory lineage provides the information for fasciculation of these nerves (zur Lage, 2003).

Olfactory precursors arise in the pupal antennal imaginal disc over an extended period of time. Given their high density, the appearance of olfactory precursors is complex and incompletely characterized. The evolution of this pattern was characterized by studying Senseless (Sens; a.k.a. Lyra) expression, which is a faithful indicator of proneural-derived sensory precursors and is probably a direct target of proneural proteins. Precursor formation occurs in three waves. Initially, Sens expression begins a few hours before puparium formation (BPF) in an outer semicircle of cells. A second wave begins at 0-4 hours after puparium formation (APF) to give a very characteristic pattern, including three semicircles of precursors. After this, a third wave appears over an extended period of time, with increasing numbers of cells appearing intercalated between the early precursors until no spatial pattern features can be observed (zur Lage, 2003).

Using a polyclonal antibody raised against the entire Amos protein, it was determined that amos expression begins at puparium formation in three distinct semicircles and then continues for the next 16 hours, with the semicircles becoming indistinct by around 8 hours APF. The characteristic early waves of SOPs arise between the Amos domains of expression and do not show overlap with Amos expression. However, the third wave of SOPs appears to arise from the Amos expression domains. These late SOPs co-express Amos, and their nuclei lie beneath the Amos expression domains, consistent with these cells being olfactory SOPs. Unusually, overlying amos proneural cluster expression is evidently not affected by lateral inhibition upon the appearance of the SOPs. ato is expressed much earlier than amos. All wave 1 and 2 SOPs appear to express Ato or to have arisen from Ato-expressing cells. Consistent with this, the entire early SOP pattern is missing in antennal discs from ato¹ mutant pupae. SOPs only begin to appear between 4 and 8 hours APF, corresponding to the third wave of precursors. These coincide very precisely with Amos expression, which itself appears unaffected (zur Lage, 2003).

In summary, there are three waves of olfactory precursor formation. The first and second waves are well defined, giving rise to the sensilla coeloconica of the sacculus and the antennal surface, respectively. These precursors express and require ato. The third wave of precursors is much more extensive and has little obvious pattern, giving rise to the more numerous sensilla basiconica and trichodea. Amos is expressed in a pattern entirely consistent with it being the proneural gene for the late wave precursors. Expression of Amos is complementary with that of Ato and is independent of Ato function. Thus, Ato- and Amos-expressing SOPs show a degree of spatial and temporal separation (zur Lage, 2003).

amos appears to be expressed in proneural domains and then in SOPs. For sc and ato, these two phases of expression are driven by separate enhancers, and SOP-specific enhancers have been identified. A 3.6 kb fragment upstream from amos was found to support GFP reporter gene expression in the pupal third antennal segment. Comparison with Amos and Sens expression shows that GFP coincides with the Amos but not Ato SOPs. This fragment therefore contains an amos SOP enhancer. Perduring GFP expression driven by the enhancer can be observed in large numbers of sensilla on the maturing pupal antenna. From their morphology, it is clear that the GFP-expressing subset are the sensilla trichodea and basiconica. This confirms that early SOPs form sensilla coeloconica whereas late SOPs produce sensilla trichodea and basiconica. Interestingly, these GFP-expressing sensilla differentiate late, because there is no overlap with the 22C10 marker until late in development. Thus, the timing of neuronal differentiation reflects the timing of SOP birth. These findings correlate with the differing effects of proneural genes on fasciculation as described above: the first-born ato-dependent cells organize the nerves, and the later amos-dependent ORNs follow passively (zur Lage, 2003).

Loss of SOPs is one of the defining characteristics of proneural gene mutations. SOP formation was examined in amos mutants relative to wild type by examining Sens expression. As expected from the expression analysis, the first two waves of SOP formation show little discernible difference in pattern between amos mutant antennal discs and wild-type discs. This is consistent with these early SOPs expressing and requiring ato, and they indeed express ato in a pattern indistinguishable from wild type. This shows that ato expression does not depend on amos function. After this, the later arising SOPs do not appear to form between rows of ato-dependent SOPs, corresponding to those cells shown to express amos. A few precursors do not express ato, and these may represent precursors of the ectopic bristles. This is supported by an analysis of Cut expression, which is the key molecular switch that must be activated to allow SOPs to take a bristle fate, and whose expression correlates with bristle SOPs. In the wild-type antenna, Cut is not expressed during olfactory SOP formation, although later it is expressed in differentiating cells of all olfactory sensilla. This expression normally appears after 16 hours and does not overlap with Amos. In amos mutant antennae, expression begins earlier than normal in a subset of SOPs that appear to correspond to the ones identified above (zur Lage, 2003).

By 16 hours APF, there is a large loss of Sens staining in amos mutants. The remaining cells tend to be in clusters as would be expected for the early ato-dependent sensilla, but otherwise the identity of these cells cannot be determined. Detection of Amos protein in amos¹ mutant antennal discs also shows that although the Amos domains are still present, the deeper Amos/Sens-expressing nuclei are absent. Thus, at least a large number of amos-associated SOPs are not formed in the amos mutant (zur Lage, 2003).

The processes and lineages by which olfactory SOPs lead to the differentiated cells of the olfactory sensillum are not entirely known. The limited information available comes from analysis of the early wave of SOPs, which represent the ato-dependent sensilla. After a SOP is selected there appears in its place a cluster of 2-3 cells expressing the A101 enhancer trap [the pre-sensillum cluster (PSC)]; this is apparently caused not by division of the SOP but perhaps by recruitment by the SOP, although the evidence for this is indirect. These PSC cells then divide to form the cells of the sensillum, including the outer support cells (hair and socket cells), inner support cells (sheath cells) and 1-4 neurons. For the early subset of SOPs, formation of the PSC occurs at a time in which amos is still expressed in the epithelial domains, and so amos could influence the development of these cells. Using A101 as a marker of the PSC cells, it was determined that amos is not expressed in recognisable PSCs at 8 or 16 hours APF. Moreover, there is also no apparent co-labelling of Amos and Pros (a marker of one of the PSC cells). This suggests either that early PSC cells do not derive from amos-expressing cells or that amos is switched off rapidly when cells join a PSC (zur Lage, 2003).

The situation appears different for the cells derived from amos-dependent SOPs. Surprisingly at 24 hours and beyond, the amos enhancer drives GFP expression in most or all cells of the differentiating sensilla basiconica and trichodea, including most or all of the neurons (recognised by Elav expression); the sheath cell (recognised by Pros expression), and the outer support cells (recognised by the higher expression of Cut). This suggests that the late PSC cells do derive from amos-expressing cells and that activation of an enhancer within the 3.6 kb regulatory fragment (possibly separate from the SOP enhancer) is part of their specification process, although amos expression itself may not be long lived in these cells (zur Lage, 2003).

amos mutant antennae have Cut-expressing SOPs, but, although cut expression decides SOP subtype fate, it does not specify ectodermal cells as SOPs de novo. To investigate the involvement of other proneural genes, it was first determined whether the bristles depended on ato, since ato is expressed in close proximity to the emerging bristle SOPs. Clones of amos¹ mutant tissue were induced in ato¹ mutant antennae. In such clones, all olfactory sensilla were absent, as expected, but ectopic bristles were still formed. Therefore the bristles do not depend on ato function (zur Lage, 2003).

Cut expression normally follows from ac/sc proneural function, and so the ectopic bristle SOPs might depend on these proneural genes. Indeed, mutation of ac and sc greatly reduces the number of ectopic bristles in amos¹ flies. By contrast, mutation of the non-proneural ASC gene asense (ase) has no effect alone. This suggests that in the absence of amos, ac/sc function, to a large extent, causes the formation of bristle SOPs (zur Lage, 2003).

To determine how amos might normally repress bristle formation, the pattern of sc mRNA was examined in the pupal antenna. Significantly, a weak stripe of sc expression is observed in the wild-type antenna. This stripe coincides with amos expression, and consists of ectodermal cells and SOPs. In the amos mutant antenna, sc mRNA expression is stronger and more clearly correlates with SOPs. This suggests that sc is expressed in olfactory regions of the wild-type antenna but that its function is repressed by the presence of amos. sc functional activity in the antenna was investigated by analyzing the expression of specific sc target genes as indicators of Sc protein function. First, Ac protein, whose expression is ordinarily activated by Sc function as a result of cross regulation, was examined. Ac protein is present in some SOPs in amos mutant antennae, but is not present in wild-type antennae. A similar result was observed for sc-SOP-GFP, which is a reporter gene construct that is directly activated by sc upon SOP formation. This reporter showed GFP expression in some SOPs in amos mutant antennae but not in wild-type antennae. Finally, sc-E1-GFP, a reporter gene construct comprising GFP driven solely by a sc-selective DNA binding site, was examined. This reporter is invariably activated in all cells containing active Sc protein (including PNCs and SOPs). As with the other target genes, this reporter was only expressed in amos mutant antennae. Thus, it is concluded that sc mRNA is expressed in the wild-type pupal antenna, and amos normally must repress either the translation of this RNA or the function of the Sc protein produced. This conclusion is supported by misexpression experiments. When amos is misexpressed in sc PNCs of the wing imaginal disc (109-68Gal4/UAS-amos), there is a dramatic reduction in bristle formation, even though endogenous sc RNA levels are unaffected (zur Lage, 2003).

The transcription factor encoded by lozenge (lz) plays a number of roles in olfactory sensillum development, including activating amos expression. Mutants therefore show a loss of many amos-dependent sensilla. Interestingly flies mutant for both lz and amos (lz³⁴; amos¹/Df(2L)M36F-S6) have third antennal segments that bear only sensilla coeloconica, and so the ectopic bristles of amos mutants are dependent on lz function. Correlating with this, the expression of sc mRNA in the third antennal segment is much reduced in a lz mutant compared with wild type. Thus, lz appears at least partly responsible for the expression of sc in the antenna (zur Lage, 2003).

It is concluded that amos is the proneural gene for the precursors of two classes of olfactory sensilla. These precursors are absent in amos mutants, resulting in highly defective antennae lacking all sensilla basiconica and trichodea. Unusually, this is not the only phenotype of amos mutants. Unique among Drosophila proneural genes, mutation of amos results in the appearance of new sense organs: mechanosensory bristles are now formed on the third antennal segment. Evidence that amos must normally repress sc-promoted bristle specification in addition to promoting olfactory neurogenesis. Significantly, inhibitory interactions between bHLH genes have recently been reported during mouse neurogenesis, where discrete domains of bHLH transcription factor expression are set up partly by mutual cross-inhibition combined with autoregulation. As with amos, cross-inhibition occurs between members of different bHLH families: Mash1 (ASC homolog), Math1 (ato homolog), and neurogenin1 (tap homolog) (zur Lage, 2003).

On misexpression evidence, it has been argued that neuronal subtype specification involves repression of bristle fate by ato during chordotonal SOP formation and by amos during olfactory precursor formation. In this light, the ectopic bristles in amos mutants are of significant interest. They represent the first loss-of-function evidence that an ato-type proneural gene suppresses bristle fate during the normal course of its function. However, how this relates to amos function is complex. In misexpression experiments, bristle suppression by amos is most strongly observed using a PNC- and SOP-specific Gal4 driver line. Yet paradoxically, misexpression of amos more generally in the ectoderm, but only weakly in SOPs, yields dramatically different results: in such cases amos produces ectopic bristles very efficiently. This bristle formation does not require the function of endogenous ac/sc genes, but probably reflects the intrinsic SOP-specifying function of amos in situations that are not conducive to its subtype-specifying (and bristle suppressing) function. It appears therefore that bristle suppression particularly requires amos expression in SOPs (zur Lage, 2003).

What does amos repress in the antenna? It appears that sc is expressed within the wild-type amos expression domain during olfactory SOP formation. Clearly amos must prevent the function of sc, since sc expression in ectoderm usually results in bristle specification. It may be significant that some of the sc RNA is in olfactory SOPs in the wild-type antenna, suggesting that the SOP may be a major location of repression by amos, as indicated by misexpression experiments. However, some bristle formation is maintained in ac/sc; amos mutants. This may be due to redundancy with other genes in the ASC: certainly wild-type bristle formation outside the antenna is not completely abolished in the absence of ac/sc. An alternative possibility is that some bristle SOPs result from other proneural-like activity in the antenna. Direct proneural activity of lz is a possibility, although misexpression of lz elsewhere in the fly (using a hs-lz construct) is not sufficient to promote bristle formation (zur Lage, 2003).

The amos² hypomorph appears to represent a different situation. In such flies, a number of amos-dependent SOPs appear to have mixed olfactory/bristle fate. This suggests that on occasions the mutant Amos² protein is able to specify SOPs, but is less able to impose its subtype function (and so this, to some extent, resembles more the outcome of some misexpression experiments). amos² may therefore be a useful tool for exploring these two functions. For example, if subtype specification requires interaction of Amos with protein cofactors, then these interactions may be specifically impaired in the amos² mutant (zur Lage, 2003).

Because the proneural proteins are normally transcriptional activators, it is unlikely that Amos/Ato proteins directly inhibit gene expression during bristle suppression. The presence of sc RNA in amos-expressing cells in the wild-type antenna is consistent with this. The involvement of protein interactions is to be suspected. An interesting parallel is found in vertebrates, where neurogenin1 promotes neurogenesis and inhibits astrocyte differentiation. The glial inhibitory effect could be separated from the neurogenesis promoting effect: whereas neurogenesis promotion depends on DNA binding and activation of downstream target genes, astrocyte differentiation is inhibited through a DNA-independent protein-protein interaction with CBP/p300. In the case of amos, an interesting possibility is that inhibition of bristle formation may involve the sequestering of Sc protein by Amos protein. Such a mechanism would have to be sensitive to the level or pattern of amos, since general misexpression does not mimic this activity (zur Lage, 2003).

Apart from giving rise to separate classes of olfactory precursor, there are interesting differences in the way that ato and amos are deployed in the antenna. Three waves of olfactory precursor formation were characterized. The first and second waves are well defined, giving rise to well-patterned sensilla coeloconica of the sacculus and the antennal surface, respectively. These precursors express and require ato. The third wave of precursors is much more extensive and has little obvious pattern; it gives rise to the much more numerous sensilla basiconica and trichodea. This wave expresses and requires amos. For the early waves, ato is expressed according to the established paradigm: it is expressed in small PNCs, each cluster giving rise to an individual precursor. The pattern of the PNCs is very precise and prefigures the characteristic pattern of precursors. amos expression is dramatically different. It is expressed in large ectodermal domains for an extended period of time. Densely packed precursors arise from this domain continuously without affecting the domain expression. This shows that singling out does not necessarily require shut down of proneural expression, and therefore has implications for how singling out occurs. In current models, it is assumed that PNC expression must be shut down to allow a SOP to assume its fate. The amos pattern better supports the idea that a mechanism of escaping from or becoming immune to lateral inhibition is more likely to be important generally. One prediction would be that amos and ato (and ac/sc) differ in their sensitivities to Notch-mediated lateral inhibition, a situation that has been noted for mammalian homologs (zur Lage, 2003).

Why are the proneural genes deployed so differently? One possibility is simply that there are very many more sensilla basiconica and trichodea than coeloconica. All the coeloconica precursors can be formed by ato action in a precise pattern in two defined waves. This would not be possible for the large number of basiconica and trichodea precursors, and so precursor selection has been modified for amos. Indeed, amos appears to be a particularly 'powerful' proneural gene when misexpressed. This may make amos a useful model of other neural systems in which large numbers of precursors must also be selected (zur Lage, 2003).

For most insects, the antenna is the major organ of sensory input. It is not only the site of olfaction, but also of thermoreception, hygroreception, vibration detection and proprioception, as well as of touch. Patterning the sensilla is therefore complex and three types of proneural gene are heavily involved to give different SOPs. It is clear that the study of antennal sensilla will provide a useful model for exploring the fate determining contribution of intrinsic bHLH protein specificity and extrinsic competence factors (zur Lage, 2003).

GENE STRUCTURE

Open reading frame - 0.6 kb

Bases in 5' UTR - 294

Bases in 3' UTR - 266

PROTEIN STRUCTURE

Amino Acids - 198

Structural Domains

In a yeast two-hybrid screen used to identify proteins that interact with Numb, 12 independent clones were isolated including the amos cDNA. This interaction was also observed in an in vitro, GST pull-down assay. The function of this interaction between Numb and Amos in respect to sensory organ development awaits further study. The Amos protein has a bHLH domain at the extreme carboxyl terminus. The Amos protein is highly homologous to the Ato family; their bHLH domains share 67%-89% identity. Amos is most similar to the Ato homolog, Tath1 of Tribolium (89% identity), suggesting that Amos and Tath1 may represent a new subtype of the Ato protein family. When Amos is compared to two other Drosophila proneural gene products, Achaete (Ac) and Scute (Sc), the conservation in the bHLH domains is much lower, with only 44% identity. The predicted protein sequences of Amos and Ato are 74% identical over the entire bHLH region and share an identical basic domain except for an R to K conservative change. This compares with ~70% identity between bHLH domains of the AS-C and ~40% between Amos and Scute. It is suggested that an ato-amos gene duplication occurred after the invertebrate-vertebrate split, so that vertebrate ato-like genes may exhibit functional similarity with either amos or ato. The gene duplication is nevertheless an ancient event, because Ato and Amos share no similarity outside the bHLH domains. As might be expected from its sequence identity with Ato, Amos has the conserved residues required for correct folding of the bHLH domain and interaction with DNA. Such conservation also leads to the prediction that Amos functions as a heterodimer with Da protein. Indeed, Amos has been shown to bind to DNA as a heterodimer with Da in vitro (Huang, 2000 and Goulding, 2000).

date revised: 7 February 2000

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