InteractiveFly: GeneBrief

amos: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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).

Promoter

The regulation of proneural gene expression is an important aspect of neurogenesis. In the study of the Drosophila proneural genes, scute and atonal, several themes have emerged that contribute to the understanding of the mechanism of neurogenesis. First, spatial complexity in proneural expression results from regulation by arrays of enhancer elements. Secondly, regulation of proneural gene expression occurs in distinct temporal phases, which tend to be under the control of separate enhancers. Thirdly, the later phase of proneural expression often relies on positive autoregulation. The control of these phases and the transition between them appear to be central to the mechanism of neurogenesis. This study presents the first investigation of the regulation of the proneural gene, amos. Amos protein expression has a complex pattern and shows temporally distinct phases, in common with previously characterised proneural genes. GFP reporter gene constructs were used to demonstrate that amos has an array of enhancer elements up- and downstream of the gene, which are required for different locations of amos expression. However, unlike other proneural genes, there is no evidence for separable enhancers for the different temporal phases of amos expression. Using mutant analysis and site-directed mutagenesis of potential Amos binding sites, no evidence was found for positive autoregulation as an important part of amos control during neurogenesis. For amos, as for other proneural genes, a complex expression pattern results from the sum of a number of simpler sub-patterns driven by specific enhancers. There is, however, no apparent separation of enhancers for distinct temporal phases of expression, and this correlates with a lack of positive autoregulation. For scute and atonal, both these features are thought to be important in the mechanism of neurogenesis. Despite similarities in function and expression between the Drosophila proneural genes, amos is regulated in a fundamentally different way from scute and atonal (Holohan, 2006; full text of article).

In order to analyse amos regulation, its expression pattern was characterized. Expression begins at puparium formation in three distinct semicircles in the future antennal third segment. By 8 h after puparium formation (APF), this expression merges into a single large crescent and continues until 16 hours APF. This expression is responsible for the third wave of olfactory SOP specification that takes place in the third antennal segment, which forms the precursors of the sensilla basiconica and trichodea (Holohan, 2006).

In the embryo, amos RNA is expressed transiently in a segmentally repeated pattern of presumed proneural clusters and SOPs at stages 10/11. In the trunk, Amos protein expression begins at stage 10 in single small cluster in the dorsal ectoderm of each abdominal segment and of thoracic segments T2 and T3. This cluster is absent from T1. Shortly afterwards, Amos expression ceases here. At this time, in abdominal segments A1-A7 only, transient expression begins in a second small cluster of cells adjacent and ventral to the first cluster. Subsequent analysis shows that the first clusters give rise to the dbd neurons and the second clusters to the dmd1 neurons. Interestingly, the presumed SOPs that derive from both these clusters (but not the clusters) also expresses the related proneural protein, Atonal. The expression of Ato in these SOPs requires amos but the converse is not true, confirming that Amos provides the proneural function for these SOPs (Holohan, 2006).

In the stage 10 embryonic head, Amos is expressed in large ectodermal clusters in the antennal, maxillary, and labial segments. These are rather more ventral than the clusters in the trunk. Slightly later, expression appears in small clusters in the maxillary, mandibular and labial primordia. Expression also appears in small clusters that appear homologous to those in the trunk. The head expression suggests that amos may function in the formation of a variety of head sense organs (Holohan, 2006).

To identify the cis-regulatory sequences of amos, the intergenic regions upstream and downstream were tested for their ability to drive expression of a GFP reporter gene. A construct with a 3.5-kb upstream fragment (amos-3.5-GFP) supports GFP expression in the third antennal segment. The expression pattern was characterised by co-labelling with antibodies to Amos and Senseless (Sens) as a marker of SOPs. Over a period of 0-8 h APF, GFP expression was largely coincident with Amos protein, suggesting that the fragment contains a major enhancer for expression of Amos in the antennal disc. While generally co-expressed, GFP is not observed strongly in the cells in which Amos is most recently activated. This appears to represent a slower induction of GFP synthesis and maturation relative to endogenous proneural proteins. A subset of GFP-expressing cells (with deeper nuclei) also express Sens, thus representing the amos-dependent olfactory SOPs themselves. GFP-negative SOPs are also present, which are likely to represent the earlier waves of ato-dependent olfactory SOPs. Thus, amos-3.5-GFP contains a major enhancer that drives Amos expression in most or all cells of the olfactory PNCs. Strong expression in the resulting SOPs suggests that the fragment drives expression in both the PNCs and SOPs, although it is also possible that GFP expression in SOPs represents perdurance of expression driven by the PNC enhancer. Previously, the enhancer activity of a 56-bp larger fragment had been tested by cloning into a Gal4 expression vector. The expression pattern of amos-3.5-GFP described in this study differs from that described for amos-3.6-Gal4 in that the latter appeared largely SOP-specific. It is possible that this might represent a strongly delayed onset of GFP expression from this construct (Holohan, 2006).

amos-3.5-GFP also supports expression in the embryo. The pattern of GFP closely resembles that of Amos protein although the appearance of GFP is delayed. Owing to the transient nature of Amos expression, this means that relatively little overlap of Amos and GFP expression are observed. Between stages 10 and 11, GFP expression is detected in the same sequence of cell clusters as Amos in the head and trunk. Expression begins in the head antennal and maxillary segments. It is then observed in the other head clusters and the first thoracic and abdominal clusters. In the latter, GFP expression appears in the first clusters as Amos expression disappears from them and is replaced by expression in the second more ventral cluster in A1-7 (Holohan, 2006).

By stage 12, amos expression has been turned off. However, perdurance of GFP expression was used to follow the fate of the different amos clusters. There is a complex network of sensory neurons in the trunk of the embryo, but amos is responsible for only two of the multidendritic neurons, the dbd and dmd1 neurons. In the abdominal segments of late embryos, amos-3.5-GFP expression is observed strongly in the dorsally located dmd1 neuron in abdominal segments A1-7 [as marked by the sensory neuron marker 22C10 (anti-Futsch)]. Weaker and variable expression is observed in the dbd neuron and its associated glial cell in segments T2,3 and A1-8/9. In addition, some ectodermal cells also express GFP, which is consistent with perdurance in some of the PNC cells. GFP is not expressed in neurons in T1, which is consistent with observation that amos is not expressed in this segment. Interestingly, the lack of expression in T1 suggests that this segment does not possess a dbd neuron. Similarly, only segments A1-7 appear to have a GFP-expressing dmd1 neuron. Consistent with this, a marker of the dbd and dmd1 cells (anti-Pdm) detects no neurons in T1, and only the dbd neuron in T2,3. Interestingly, the dorsoventral locations of the dbd and dmd1 neurons appear reversed relative to their proneural clusters, suggesting that one or both neurons undergo migration (Holohan, 2006).

GFP expression also perdures in the head region. This is particularly associated with the complex clusters of sense organs that form the antennomaxillary complex and pharynx-associated sense organs of the larva. Perdurance of expression confirms that in the antennal segment amos contributes to the larval olfactory organ, the dorsal organ (do). The main expression in the maxillary segment contributes to the ventral organ (vo), and perhaps the papilla organ (pao), whose neurons are reported to resemble dbd neurons. Equivalent expression in the labial segment contributes to the labial sense organ (lbso) (Holohan, 2006).

amos-3.5-GFP is expressed in all locations of Amos expression except for three small areas dorsally in the maxillary and labial segments. To locate the enhancer sequences responsible for these areas, a 1-kb region downstream of amos was also tested for enhancer activity. This region supports GFP expression in these three groups of cells. Their location and GFP perdurance suggests that these appear to contribute neurons of the terminal organ (to), labial organ (lbo), and labial sense organ (lbso). Between them, the upstream and downstream flanking regions contain enhancers that can account for the entire amos expression pattern (Holohan, 2006).

The complex nature of amos expression suggests that the 3.5-kb fragment may contain different enhancers for different aspects of pattern. The 3.5-kb fragment was subdivided into three smaller fragments (A, B, C) measuring 1.68 kb, 961 bp and 893 bp. amos-A-GFP is expressed solely in the trunk of the embryo: there is no GFP expression in either the antennal disc or the head region of the embryo. This suggests that an enhancer element responsible for amos expression in the dbd and dmd clusters is present in fragment A. In late embryos, perduring GFP was seen in the trunk in a similar pattern to that of amos-3.5-GFP (Holohan, 2006).

amos-B-GFP also supported expression in the embryo but not the antennal disc. In this case, GFP was observed in the head region in a pattern similar to that supported by amos-3.5-GFP. Thus, an enhancer(s) for amos expression in the head is present in fragment B, and this appears to be required for most of the components of the amos head pattern, as confirmed by 22C10 staining of late embryos. amos-B-GFP is also expressed in a pattern of ectodermal clusters in the trunk, but this expression appears to be ectopic: the clusters are more ventral than those for amos-3.5-GFP or for Amos itself, and the expression does not perdure into cells associated with the PNS. Interestingly, this ectopic expression appears to resemble in its segmental location the amos-B-GFP pattern observed in the head. This suggests that the ectopic trunk pattern represents the inappropriate activity of the head enhancer present in B. In contrast, a construct combining fragments A and B (amos-AB-GFP) shows correct head and trunk expression. There may therefore be an inhibitory sequence in A that normally restricts the activity of the B enhancer to the head (Holohan, 2006).

amos-C-GFP does not support expression in the embryo at the time that amos is normally expressed. However, as the neurons start to differentiate, GFP expression is switched on in an inconsistent subset of cells marked by 22C10. This presumably represents artefactual expression. In contrast, fragment C drives expression in the antennal imaginal disc. GFP expression is present in the amos dependent SOPs and in some cells of the proneural cluster. There is no discernible difference between the location of GFP expression as driven by the amos-3.5-GFP fragment and that driven by amos-C-GFP, although expression from the latter is generally weaker (Holohan, 2006).

In summary, distinct enhancer sequences are required for amos expression in the embryo and antennal disc. More than one enhancer is responsible for amos expression in the embryo, and head and trunk enhancers appear to be separate. The presence of enhancer modules for different expression locations is consistent with the findings of other proneural genes. However, this analysis found only one enhancer for each location of amos expression (Holohan, 2006).

For sc and ato, experiments investigating transgene rescue of sensillum loss in mutants showed that substantial phenotypic rescue is achieved if a transgene includes enhancers for both phases of expression. In contrast, a transgene driven by a single enhancer (for either the first or second phase of expression) rescues poorly. In the case of amos, no separation was found of enhancers for temporal phases. Although distinct phases of amos expression can be discerned, they appear to be driven by a single element in each location. It seems unlikely that further subdivision will reveal such separable enhancers, nor that other enhancers exist farther up- or downstream of amos. It was therefore determined whether the 3.5-kb region contained all elements responsible for amos regulation in the antennal disc in a rescue experiment. The amos-3.6-Gal4 line was used to drive UAS-amos expression in amos mutant flies, and antennae from such flies were examined for types and numbers of sensilla on the third segment. Mutation of amos results in the loss of all sensilla trichodea and sensilla basiconica, as well as the appearance of ectopic sensory bristles. Expression of amos driven by the amos-3.6-Gal4 line resulted in a substantial rescue of this defect. Ectopic bristles were almost completely suppressed. Sensilla trichodea were present in numbers close to that expected in wildtype. Substantial numbers of sensilla basiconica were also present, although less than half the number expected for wildtype. Although quantitatively not complete, the degree of rescue suggests that all major patterning elements necessary for amos expression in the antenna are present in amos-3.6-Gal4 and also, by inference, in the 3.5-kb fragment. Lack of complete rescue may reflect the delay in onset of Gal4-driven expression in this system. Interestingly, the numbers of ato-dependent sensilla coeloconica are reduced compared to wildtype. Such reduction is also seen when amos is misexpressed in the wildtype antenna. It is possible that perdurance of Gal4-driven expression of amos interferes with endogenous ato function (Holohan, 2006).

Where known for other proneural genes, the second phase of expression involves direct autoregulation, with proneural/Daughterless protein heterodimers binding to E box sequences within an autoregulatory enhancer. It was asked whether autoregulation is also important in amos regulation, concentrating on the antennal disc expression. If amos is autoregulatory, one might expect functional E box binding sites to be present within the amos-C antennal regulatory region. Four potential E box sequences (CANNTG) are present in this fragment, two of which are conserved between Drosophila melanogaster and D. pseudoobscura. None of these sequences (ttCAAGTGa, aaCAATTGt, gtCATATGg, gtCATTTGg) conform completely to the consensus sequences reported for Sc (gCAG(G/C)TG(g/t)) or Ato (a(a/t)CA(G/T)GTG(g/t). However, although Amos protein is predicted to function via E box binding, no such site has yet been characterised. Therefore, whether any of these E box sequences are important for amos-C enhancer function was investigated by mutating all four E boxes within the amos-C-GFP construct (amos-Cmut-GFP). However, no clear reduction in expression was observed for amos-Cmut-GFP compared with the unmutated construct. In case autoregulation lies outside fragment C, the whole of the 3.5-kb sequence was scanned for E box sequences. No further E boxes matching the known consensus sequences for Sc or Ato were found. The closest match is a site of atCAGGTGa (differing from the Ato consensus sequence in its 3' flanking base). This sequence is conserved in D. pseudoobscura. However, when mutated within amos-3.5-GFP, no difference in GFP expression pattern was observed in the antenna or embryo (Holohan, 2006).

Autoregulation may occur indirectly via the regulation of an intermediate factor. To find evidence for indirect autoregulation, it was determined whether misexpression of amos results in ectopic induction of amos-3.5-GFP. In the embryo, no ectopic expression was observed from amos-3.5-GFP when UAS-amos was driven in the ectoderm by a sca-Gal4 driver. Using the Gal4^109-68 line in imaginal discs in third instar larvae, no ectopic amos-3.5-GFP expression was observed upon amos misexpression, except for a small number of GFP-expressing cells in antennal discs. However, variable numbers of these cells were also visible in control antennal discs that lacked the UAS-amos, and so appear to represent a genetic background effect (Holohan, 2006).

amos-3.5-GFP expression was also examined in amos mutant embryos to look for loss of GFP expression that might indicate the need for autoregulation. In such embryos, no clear difference from wild type was observed in the GFP expression pattern. In mutant antennal discs, ectodermal GFP expression appeared unchanged, although SOP expression was lost as would be expected from the absence of such cells. In summary, no part of the amos expression pattern could clearly be seen to depend on endogenous amos expression (Holohan, 2006).

Transcriptional Regulation

lozenge (lz) functions in eye, antennal, and tarsal claw development. In leg and antennal discs, the pattern of lz expression strongly resembles that of amos, although amos expression begins later than lz. These observations suggest that lz might regulate amos expression during the process leading to the formation of basiconic and trichoid SOPs in the antenna and perhaps in the tarsal claw. Therefore, changes in the expression pattern of amos in lz mutants were sought. Strong lz alleles (including lz¹, lz³, and lz³⁴) almost completely lack sensilla basiconica and exhibit up to a 50% reduction in sensilla trichodea. The number of sensilla coeloconica is reported to be unaffected. In these strong alleles, AMOS mRNA is absent from the middle of all three antennal bands. For band 3, the affected region corresponds to the area fated to form sensilla basiconica SOPs. The correlation between this loss of amos expression and the loss of sensilla basiconica is therefore consistent with a requirement for amos in sensillum basiconica formation. In addition, it may be deduced that the middle regions of the other two bands give rise to those sensilla trichodea that are missing in strong lz mutants. Conversely, lz-independent sensilla trichodea may arise from the lz-independent tips of the amos-expressing bands. Topologically, SOPs from the band tips will end up on the lateral edge of the antenna after metamorphosis, which is where the sensilla trichodea are concentrated. Interestingly, comparison with the ato expression pattern suggests that amos is also expressed in regions of sensillum coeloconica formation in bands 1 and 2. Since these SOPs are not lost in lz mutants, the loss of amos expression from the middle of these regions provides evidence that amos is not required at least for many sensilla coeloconica (Goulding, 2000).

In weaker lz alleles (such as lz^g), amos expression appears patchy but spatially normal, suggesting that SOP selection itself is not strongly altered. This would be consistent with observation that the major phenotype of weak lz alleles is one of subtype transformation from basiconic to trichoid fate rather than sensillum loss. This is postulated to result from a role of lz in subtype specification, such that higher levels are required for SOPs to take on basiconic fate while lower levels are sufficient for trichoid fate. It was determined in a complementary experiment whether ectopic lz expression could induce ectopic amos expression. When ubiquitous lz expression is activated in pupae containing a heatshock-inducible lz construct (hs-lz). lz misexpression also results in ectopic amos expression in pupal wings and legs. These experiments show that lz is both necessary for much of amos's expression pattern and also sufficient to drive ectopic amos activation in many other locations (Goulding, 2000).

To investigate further the relationship between lz and amos, it was determined whether amos gene dosage reduction would modify the number of sensilla formed in lz mutants. In the intermediate allele, lz^g, the number of basiconica is reduced to 28% of wild-type. Removing one copy of the chromosomal region containing amos results in a further 70% reduction in this number. The number of sensilla trichodea is unaltered, probably because these are not affected in this intermediate lz allele. To gauge the effect on sensilla trichodea, amos's modification of a strong lz allele, lz³, was examined. In addition to a total lack of sensilla basiconica, lz³ exhibits a strong reduction of sensilla trichodea. In the absence of one copy of amos, sensilla trichodea are reduced by a further 54% in lz³, to 24% of wild-type (Goulding, 2000).

From the genetic and expression analyses, it has been concluded that amos transcription is partly downstream of lz and that its loss of expression may explain the loss of sensilla basiconica and trichodea in lz mutants. It was therefore tested whether experimentally induced amos expression could rescue the loss of sensilla basiconica in strong lz mutants. Using hsGal4 as a driver, UAS-amos was misexpressed in lz³ pupal antennae. Such misexpression results in a significant recovery of sensilla basiconica when compared with lz³ alone. This rescue is still far short of wild-type levels, perhaps because amos is not optimally expressed using hsGal4. Alternatively, lz might need to activate other genes required for basiconic fate in addition to amos (i.e., amos alone cannot replace all the functions of lz). Significantly, ato is unable to direct any rescue under the same conditions, even though the number of sensilla coeloconica is increased. Therefore, amos, but not ato, can partially bypass the requirement for lz. Interestingly, many of the rescued basiconica were located in the lateral region of the antenna (Goulding, 2000). Such a distribution was also observed upon rescue of lz mutants by hs-lz (Gupta, 1998).

Since the expression of ato overlaps with the inner two bands of amos expression, it is possible that one gene may be dependent on the other. However, no defect in amos expression was observed in ato mutant antennal discs. Furthermore, ato expression is not dependent on lz, and therefore by inference ato does not depend on amos, at least not in the medial antennal region. It is concluded that the two olfactory proneural genes, ato and amos, are largely independent of each other. Furthermore, ato shows no interaction with lz. Thus, lz³; ato¹/ato¹ double mutants exhibit a complete absence of sensilla basiconica and coeloconica, as expected from the loss of lz and ato functions, respectively. However, the number of sensilla trichodea is not reduced below that observed in lz³ mutant flies. This suggests that there is no redundancy between lz and ato in formation of the remaining sensilla trichodea, which are instead likely to require the lz-independent part of amos's expression (Goulding, 2000).

Protein Interactions

The ability of the Amos and Da proteins to form complexes in the presence of E boxes has been examined. Since the DNA-binding domain of Amos is almost identical to that of Ato, E box-containing oligonucleotides, E1 and E4, which represent high-affinity binding sites for the Da/Ato protein complex, were examined. The Sc/Da complex was used as a positive control since this complex also binds to these two E boxes. E1 and E4 boxes are well bound by Amos/Da. In contrast, this shifted complex including Amos and Da is undetectable with either Amos or Da alone. The Amos/Da complex is supershifted by addition of anti-GST antibody, which recognizes the GST-Da protein used in this assay. This binding of the Amos/Da complex to E boxes is efficiently completed by addition of excess E1 and E4 cold probes but not by two corresponding mutant E boxes. These data suggest that Amos and Da form a heterodimer when bound to E boxes and that the binding activities are sequence specific. To further analyze the interaction between amos and da in vivo, the effects of amos misexpression were examined in different da genetic backgrounds. The number of neurons were counted in sca-GAL4/UAS-amos embryos carrying different da gene dosages. When a moderate level of amos is induced in wild-type embryos with two copies of da⁺, some ectopic Elav-positive cells are observed. The ectopic neurons are suppressed in embryos carrying only one copy of da⁺. When amos and da are simultaneously misexpressed, numerous Elav-positive cells are induced. The strong neuralization by amos and da has also been revealed by the staining of MAb 22C10, which labels the neuronal morphology. These ectopic neurons include MD neurons that express lacZ from E7-2-36 insertion. As a control, misexpression of da causes only a minor effect on the number of neurons in this assay. These results suggest that the ectopic neuron formation elicited by amos is very sensitive to the gene dosage of da (Huang, 2000).

DEVELOPMENTAL BIOLOGY

amos is expressed very transiently and dynamically during embryogenesis. AMOS mRNA is present in a small cluster of ectodermal PNS precursor cells in each thoracic and abdominal segment during stage 10. Later, in stage 11, expression is restricted to a single cell per segment. This cell quickly ceases to express amos, first in the thoracic segments and then in the abdomen. As with SOP formation by ato and the AS-C, this restriction of amos expression suggests that lateral inhibition functions within a proneural cluster defined by amos. Consistent with this, amos expression fails to resolve to single cells in Notch mutant embryos, which lack lateral inhibition. Significant amos expression is also observed in developing head segments (including antennal, mandibular, and labial segments) in areas that correspond to the anlage of the olfactory sense organs of the larval antennomaxillary complex. The expression pattern of amos thus makes this gene a likely candidate for the AS-C- and ato-independent larval sense organs. In addition to the above, AMOS mRNA was also transiently detected at the cellular blastoderm stage of embryogenesis in a dorsoventral band in the posterior of the embryo and during oogenesis in nurse cells, the centripetal follicle cells, and the oocyte itself (Goulding, 2000).

amos expression is extremely restricted during adult development. Very transient amos expression is detected in distal leg discs at approximately 0-4 hr after puparium formation (APF), correlating with the anlage of the innervated tarsal claw. The main site of expression initiates in the antennal disc at approximately puparium formation (PF) in three semicircular bands on the medial side of the developing third segment. These three bands correspond to sites from which olfactory sensillum precursors are selected. The two inner bands partly coincide with ato expression in the antennal disc. The outer band widens by 4-8 hr APF and persists until at least 21 hr APF, during which time the two inner bands appear to fuse to give a single inner region of expression. Double labeling of antennal discs from the A101 enhancer-trap line, which marks all SOPs, shows that SOPs indeed arise from the amos-expressing ectodermal bands. The expression pattern thus identifies amos as a candidate proneural gene for olfactory sensilla. Furthermore, it can be inferred that amos may be required for any or all three types of olfactory sensillum (Goulding, 2000).

amos expression during embryonic development was analyzed by in situ hybridization. Before cellularization, maternally contributed amos transcripts are ubiquitously present. The possible function of amos during these early stages is not clear. During gastrulation, from stage 9 to early stage 12, amos mRNA is expressed in a spatiotemporally regulated pattern. The earliest zygotic signal is detected in the procephalic region and gradually in the segments of head, thorax, and abdomen. The signal appears in clusters of cells in these regions. There is one cluster in every thoracic and abdominal hemisegment; three clusters in the maxillary and the labial hemisegment, and three clusters in the procephalic region, which contains the antenno-maxillary complex. Since the sensory neurons in abdominal segments are well studied, analyses have focused on this region. In each abdominal hemisegment, from A1 to A7, the amos transcript is present in a cluster that contains about 10 to 12 cells at early stage. Slightly later, the number of cells that express amos is reduced; the dorsal portion of each cluster diminishes, with occasionally one or two cells that maintain expression, while the ventral portion of the cluster continues amos expression. At late stage 11, the ventral cluster is restricted to one cell in each abdominal segment (Huang, 2000)

Programmed cell death and context dependent activation of the EGF pathway regulate gliogenesis in the Drosophila olfactory system: cells in the Amos lineage are fated to die

In the Drosophila antenna, sensory lineages selected by the basic helix-loop-helix transcription factor Atonal are gliogenic while those specified by the related protein Amos are not. What are the mechanisms that cause the two lineages to act differentially? Ectopic expression of the Baculovirus inhibitor of apoptosis protein (p35) rescues glial cells from the Amos-derived lineages, suggesting that precursors are removed by programmed cell death. In the wildtype, glial precursors express the extracellular-signal regulated kinase (phosphoERK) transiently, and antagonism of Epidermal growth factor pathway signaling compromises their development. It is suggested that all sensory lineages on the antenna are competent to produce glia but only those specified by Atonal respond to EGF signaling and survive. These results underscore the importance of developmental context of cell lineages in their responses to non-autonomous signaling in the choice between survival and death (Sen, 2004).

Several lines of investigation have ascertained that the first cells to divide in the sensory lineages are the secondary progenitors: PIIa, PIIb and PIIc. The numbers of sensory cells undergoing division at different times in the developing antenna were estimated by staining mitotic nuclei with antibodies against phosphorylated histone. A peak of cell division was observed between 16 and 24 h after puparium formation (APF). It has been considered that only in those sensory lineages specified by Ato, PIIb produces a glial cell and a tertiary progenitor, PIIIb, which in turn divides to form the sheath cell and a neuron. In Amos dependent lineages, PIIb is believed to directly give rise to a neuron and a sheath cell. The difference between the two lineages could be entirely dependent on the nature of the proneural genes activated; Amos, for example, could direct a non-gliogenic lineage. Alternatively, the two proneural genes could specify similar division patterns but the glial cell precursor in Amos-lineages could be removed by PCD, resulting in non-gliogenic lineages (Sen, 2004).

To test the latter possibility, cell death profiles were examined in developing pupal antennae using the terminal transferase assay (TUNEL) and attempts were made to correlate the timing of PCD with cell division profiles discussed above. The appearance of TUNEL-positive cells peaked between 22 and 24 h APF consistent with the occurrence of PCD immediately after division of secondary progenitors (Sen, 2004).

TUNEL reactions were performed on 22-24 h APF antennae from lz-Gal4; UAS-lacZnls and ato-Gal4; UAS-lacZnls animals. Double labeling with antibodies against ß-galactosidase marked sensory cells arising from the Lz and Ato lineages. Lz::lacZ overlaps the regions of the antennal disc where amos expression occurs and labels all the basiconic and trichoid sensilla in the mature (36 h APF) antenna. Hence for the purpose of this study, all cells in which lz-Gal4 expresses will be considered to belong to the Amos-dependent lineages. ato-Gal4 drives reporter activity in proneural domains of the disc and subsequently in all cells of the coeloconic sense organs (Sen, 2004).

Most of the apoptotic nuclei observed during olfactory sense organ development co-localized with Lz::LacZ suggesting that death occurred mainly within the 'Amos-dependent' sensory clusters. Only very few TUNEL-positive cells were detected in regions where ato-lacZ expressed and these did not co-localize with the reporter expression. If PCD is the mechanism used to remove glial precursors from Amos lineages, then their rescue would be expected to result in additional peripheral glia in the antenna (Sen, 2004).

The GAL4/UAS system was used to target ectopic expression of baculovirus inhibitor of apoptosis protein (p35) to different cell types within the developing antennal disc. distalless⁹⁸¹-Gal4 (henceforth called dll-Gal4), which drives expression in all cells of the antennal disc, resulted in the formation of >300 glial cells as compared to ~100 in the wildtype. Other sensory cells--neurons, sheath, socket and shaft cells--within sense organs were unaffected. Ectopic expression of p35 specifically in Ato lineages (ato::p35) did not alter glial number. This means that the `additional' glial cells rescued in dll::p35 must arise from lineages other than Ato. Mis-expression of p35 in Amos-dependent lineages using lz-Gal4, on the other hand, resulted in a significant increase in glial number. While other explanations are possible, it is believed that the somewhat lower number of glia obtained in lz::p35 as compared to dll::p35 could be accounted for by the strength of the P(Gal4) driver (Sen, 2004) (Sen, 2004).

In order to identify the cell within the Amos lineage that is fated to die, the cellular events during development of sense organs were re-examined. At approximately 12-14 h APF, most sensory cells are associated in clusters of secondary progenitors. Two cells in each cluster -- PIIb and PIIc -- express the homeodomain protein Prospero (Pros). pros-Gal4;UAS-GFP recapitulates Pros expression at this stage and marks PIIb and PIIc and their progeny in all olfactory lineages. In the wildtype, a Repo-positive cell was associated with only a few of the total sensory clusters, these were all located within the coeloconic domain of the antenna. Targeted expression of p35 using pros-Gal4 increased glial number indicating that cells which are the progeny of either PIIb or PIIc could be rescued from apoptosis. In the pros-Gal UAS-2XEGFP/UAS-p35 genotype, a glial cell was associated with most clusters at 18 h APF rather than in Ato lineages alone (Sen, 2004).

In order to directly visualize the cell undergoing apoptosis, 22-24 h APF antenna from the neu^A101 strain were stained with antibodies against ß-galactosidase to mark the sensory cells and with TUNEL. Sensory clusters located in basiconic and trichoid domains of the pupal antenna each had a single associated TUNEL positive cell. Since TUNEL reactivity data does not reflect the initiation of the death program, developing antennae were also stained at different time points with an antibody that recognized the activated caspase -- Drice. At 20 h APF, a single Drice-positive cell was found within each sensory cluster within the basiconic and trichoid domains of the pupal antenna. This cell also expressed low levels of Pros suggesting that it could arise from either PIIb or PIIc. This means that the PIIb/c in Amos lineages, like that in Ato, divides to give rise to a PIIIb and its sibling. The sibling in the former lineage was not previously detected because it expresses only low levels of Pros and soon dies. Since this cell is capable of expressing the glial-identity gene repo when rescued from death, it is denoted as a glial precursor (Sen, 2004).

How is apoptosis of a specific cell within the lineage regulated? In Drosophila three genes [reaper (rpr), grim and head involution defective (hid)] which all map under the Df(3L)H99 are necessary for the initiation of the death program. Heterozygotes of Df(3L)H99 show a small but significant increase in glial number over that of normal controls. hid-lacZ was used to follow expression during antennal development; reporter activity occurs at low levels ubiquitously including in glial cells. Levels of reporter expression indicate somewhat higher hid transcription in glia rescued by p35 mis-expression. The presence of Hid in the 'normal' glial precursors suggests a mechanism dependent on possible trophic factors to keep cells alive. In several other systems signaling, mainly through the EGFR pathway, results in an antagonism of Hid action and transcription. The sustained levels of hid transcription in the rescued glia, is not unexpected since inhibitors of apoptosis act by antagonizing a downstream event of caspase activation, rather than on Hid itself (Sen, 2004).

The peripheral olfactory glia ensheath the axonal fascicles as they project towards the brain. Antennae from animals deficient for ato (ato¹/Df(3R)p¹³), lack a large fraction of glia; the ~30 which remain, appear to arise elsewhere and migrate into the third segment of the antenna somewhat later during development (Sen, 2004).

Is death the default fate for all glial precursors and are there signals that keep Ato-glia alive? Several studies have provided compelling evidence for the role of receptor tyrosine kinase signaling in glial survival. In order to test this in the olfactory glia, 14-16 h APF pupal antennae were stained with antibodies against Pros and the phosphorylated form of ERK (pERK). The PIIb and PIIc cells are recognized by expression of Pros, which becomes asymmetrically localized in PIIb during mitosis. At this stage sensory cells do not express pERK. pERK was detected in the daughter of the first secondary progenitor to divide (probably PIIb). Staining with anti-Repo antibodies shows that this cell is glial. Clusters showing pERK expressing cells were observed only in regions of the pupal antenna from where the coeloconic sensilla originate and not in the Amos-lineage sensory clusters. pERK expression decays rapidly as Repo rises; only occasional cells can be detected that show immunoreactivity to both (Sen, 2004).

The presence of pERK in nascent glial precursors is evidence for receptor tyrosine kinase pathway signaling in these cells. Since EGFR activation is a well recognized signal for glial survival, the role of this pathway in glial cell development was tested. In order to demonstrate a role for EGF signaling during glial formation in the antenna, a hs-Gal4 strain and carefully timed heat-pulses were used to drive transient expression of various antagonists of the pathway. Ectopic expression of a dominant negative form of human Ras (DN-RasN17) between 14 and 15 h APF, severely reduced the numbers of antennal glia. Similarly, expression of the inhibitory ligand Argos or a dominant negative construct of Drosophila EGFR (DN-EGFR) compromises glial development. Since abrogation of signaling could, in principle, affect other developmental events, antenna from 36 h APF pupae of relevant genotypes were stained with support cell and neuronal markers to ascertain that these treatments did not affect sense organ development generally (Sen, 2004) (Sen, 2004).

Interference with EGFR activity affects glial number, suggesting that signaling is required for glial development and/or survival. Cells in the Amos-lineage also produce glial precursors, which do not express pERK. This implies that Amos-lineage glial precursors fail to experience EGF signaling and are fated to die. This hypothesis could, in principle, be tested by constitutively activating the pathway in Amos lineages. These experiments were not possible to carry out since activation of EGFR at the time when Amos-dependent secondary progenitors were undergoing division resulted in pupal lethality. The spatial and temporal expression of Vein-lacZ and Sprouty-lacZ (Sty-LacZ) supports a role in development of glia and requires further genetic analysis. Vein-LacZ was first detected at 18 h APF and expression was elevated at 20 h APF in a domain of the pupal antenna occupied mainly by coeloconic sensilla. The fact that this is the region of the antenna from where most glia originate, is interesting in the light of data from other systems that demonstrate that Vein acts as gliotrophin (Sen, 2004).

The spatial and temporal pattern of Vein and Sty expression is consistent with a role for these ligands in glial development. The region where Vein is expressed shows high pERK activity suggesting activation of the EFGR pathway in the coeloconic (Ato) domains of the developing antenna. Sty, however, is present in the basiconic and trichoid (Amos) domains of the antenna. At 10-14 h, when the secondary progenitors have not yet divided, Sty expressing cells lie adjacent to the PIIb and PIIc; these cells are labeled by Pros. This localization is consistent with a role for Sty in antagonizing EGF activity in the secondary progenitors (Sen, 2004) (Sen, 2004).

This work provides one more instance where PCD plays a crucial role in the selection of a specific population of cell types during development although the mechanisms employed still need to be elucidated. How does the development context of lineages of cells within a single epidermal field together with non-autonomous cues result in distinct consequences? The Drosophila antenna is a valuable system to address these issues because of the diversity of morphologically and molecularly distinct cell types located in a highly stereotyped architecture and the wealth of reagents available for study (Sen, 2004).

Effects of Mutation or Deletion

SOP formation is very sensitive to simultaneous reduction in copy number of proneural genes and da, which encodes their common heterodimer partner. For instance, simultaneously removing one copy of the AS-C and da genes (i.e., transheterozygotes) results in adults with a proportion of missing sensory bristles. Likewise, reducing the dosage of ato and da genes results in reduction in the number of chordotonal organs and sensilla coeloconica. Although there is no specific mutation of amos, lethal chromosomal deficiencies have been investigated that delete amos (Df(2L)M36F-S5 and Df(2L)M36F-S6) for genetic interaction with da. Olfactory sensillum numbers were analyzed in flies heterozygous for an amos deficiency either alone or in combination with the loss of one copy of da (Df(2R)da^KX136/+). In flies with a single copy of each gene (abbreviated as amos^+/-:da^+/-), the number of sensilla basiconica is significantly reduced (by 30%) compared with wild-type, amos^+/-, da^+/-, or ato^+/-:da^+/- flies. This genetic interaction suggests functional cooperation between da and a bHLH gene in the amos genomic region. Given that amos is the only bHLH-encoding gene in this region, these data are consistent with the function of Amos/Da heterodimers during the formation of sensilla basiconica. Sensilla trichodea are also significantly reduced in amos^+/-:da^+/- flies, but a reduction in amos^+/- flies, when compared with wild-type was also observed. Therefore, although consistent with a requirement for amos in trichodea formation, such a requirement seems to be less sensitive to da gene dosage. Although sensillum coeloconica numbers are rather variable, amos^+/-:da^+/- flies have only slightly fewer sensilla coeloconica than flies with either mutation alone, whereas there are significantly fewer sensilla coeloconica in ato^+/-:da^+/- flies as expected. In summary, these data support a role for the chromosomal region containing amos in sensillum basiconica formation and are suggestive of a role during sensillum trichodea development (Golding, 2000).

One shared characteristic of proneural genes is that ectopic expression leads to ectopic sense organ formation, which is consistent with the proneural gene competence function. Moreover, specific subtypes of ectopic organ are formed, demonstrating that different proneural genes regulate distinct neuronal subtype properties . When AS-C genes are overexpressed, they each give a similar phenotype of ectopic external sense organs. Misexpression of ato results largely in the formation of ectopic chordotonal organs. Gal4/UAS misexpression experiments were carried out to determine whether amos can function as a proneural gene and assess its subtype-determining properties. Using Gal4^OK384 to drive expression in the larval eye-antennal disc, misexpression of UAS-ato causes increased formation of sensilla coeloconica. Upon misexpression of UAS-amos, a dramatic increase in olfactory sensilla is observed. The antenna itself is bloated, perhaps a secondary effect of housing more sensilla. Accurate counting of sensilla is difficult because of the antennal morphology and uncertainty in assigning sensilla to specific classes. Nevertheless, sensillum counts suggest that there is an increase of 24% to 44% for all sensillum types. Thus, unlike ato, amos seems capable of directing formation of sensilla basiconica and trichodea. In addition, extra sensillum coeloconica formation may result from an ability of amos to substitute functionally for (mimic) ato. Interestingly, a proportion of sensilla appeared to be of intermediate basiconica/trichodea morphology (Golding, 2000). This has also been reported as an effect of lz misexpression (Gupta, 1998).

The subtypes of ectopic sense organ produced by misexpression of ato are dependent on the tissue. Thus, ato directs sensilla coeloconica formation on the third antennal segment but chordotonal formation in most other sites. To see how amos function might be modulated outside the antenna, amos expression was induced in many proneural clusters in all imaginal discs using Gal^109-68. With this driver, UAS-amos misexpression results in a mixture of ectopic sense organs. Part of this phenotype resembles that obtained from ato misexpression. (1) Ectopic chordotonal organs are formed in the scutellum and third wing vein. In the scutellum, these are similar in number to those induced by strong ato misexpression, although much more disorganized. Therefore, amos can indeed mimic ato outside its normal developmental context. (2) Some extra external sense organs are formed, mostly along the third wing vein. In the case of ato, such bristle formation has been interpreted as an artifact resulting from incomplete subtype determination of ectopic SOPs under the conditions of misexpression, external sense organ apparently being a default fate for such SOPs. (3) Strikingly, amos misexpression also produces a third phenotype: on the thorax, head, and along the third wing vein unusual organs form that appear morphologically similar to olfactory sensilla, particularly sensilla trichodea. (4) Moreover, at high expression levels, some of the sensory bristles on the thorax are transformed toward similar olfactory-like morphology. While wild-type thoracic bristles are always innervated by a single sensory neuron, a small proportion of these amos-converted sensilla have two or more sensory neurons, a characteristic of olfactory sensilla. Thus, amos can form olfactory-like sensilla outside its normal developmental context of the antenna. It also appears able to impose olfactory sensillum fate on external sense organs in a manner similar to the chordotonal fate transformation imposed by ato. The ability of amos to form nonantennal olfactory sensilla is apparently not shared by ato, since strong ato misexpression results in no or very few ectopic olfactory-like sensilla, and no olfactory-like transformation of thoracic bristles (Golding, 2000).

To investigate whether amos is required for dbp neuron formation, the double-stranded RNAi technique was used. The dbp neuron can be unambiguously recognized by its specific location and characteristic neuronal processes by stage15. After injection of amos dsRNA into wild-type embryos, the formation of dbp neurons was eliminated. Closer to the injection site (25% to 50% of the embryo-length at the ventral side), the lack of dbp neurons affected by amos dsRNA is more prominent. In the bHLH domains of amos and ato, there is a high level of DNA sequence similarity (70% identity). Thus, ato dsRNA was injected as a control. In embryos injected with ato dsRNA, CH neurons in the lch5 clusters are eliminated, while dbp neurons remain. In contrast, injection of amos dsRNA has no affect in CH neuron formation. Therefore, amos and ato dsRNAs interfere with the formation of distinct types of sensory neurons. In addition to the dbp neuron, one or two dmd neurons remain in each hemisegment in AS-C;ato mutants. To examine if these dmd neurons are also controlled by amos, sc^B57 mutant embryos were used for injection. The sc^B57 allele removes AS-C, and the mutant embryos lack ES and ES-dependent MD neurons. These neurons are also unaffected in ato mutants. In the sc^B57 embryos injected with amos dsRNA, these remaining dbp and dmd neurons are eliminated. In summary, amos is required for the formation of dbp neurons and some dmd neurons that belong to the type of solo-MD neurons (Huang, 2000).

amos maps to a chromosomal location near the boundary of 36E and 36F of the second chromosome. Several deficiency strains with a chromosomal deletion near this region have been examined for amos mRNA expression. The deficiencies TW203 and M36F-S6, when homozygous, fail to express amos during stage 9-11. When stained with anti-Elav antibody, which stains the neuronal nuclei, the dorsal group of neurons is well isolated from other neurons in the same segment and can be easily scored in the deficiency embryos. Two to three Elav-positive neurons of the dorsal group are controlled by a chromosomal region that includes the amos locus. When stained with MAb 22C10, which reveals the neuronal morphology, the homozygous or transheterozygous embryos show disorganization of the PNS and this prevents the observation of phenotypes. However, no dbp neurons were present, to judge from their specific location and morphology (Huang, 2000).

To examine the ability of amos to induce sensory neuron formation, UAS-amos transgenic flies have been generated. The UAS-amos flies were crossed to flies carrying a hairy-GAL4 transgene, which drives target gene expression in odd parasegments of embryos. Embryos of the genotype hairy-GAL4;UAS-amos shows additional neurons, as recognized by anti-Elav antibody and MAb 22C10. In the ventral-most region, five isolated MD neurons (vmd5) were unambiguously recognized in the segment that does not misexpress amos. In the segment that misexpresses amos, six or more MD neurons are frequently observed. Furthermore, ectopic CH neurons in the lateral region and ectopic neurons in the dorsal group are also induced. However, the dorsal cluster normally consists of both ES and MD neurons, thus making it difficult to differentiate between these two types of neurons (Huang, 2000).

The anti-Pickpocket (Ppk) antibody labels three of the 21 MD neurons (one of dmd6, v'ada, and one of vmd5) in each abdominal hemisegment. In sca-GAL4;UAS-amos embryos, ectopic MD neurons appear near the original Ppk-positive cells. The ppk gene encodes an ion channel subunit that is expressed in late embryonic and larval stages and may play a role in sensory function, suggesting that ectopic MD neurons promoted by amos are likely functional (Huang, 2000).

The ability of the Amos and Da proteins to form complexes in the presence of E boxes has been examined. Since the DNA-binding domain of Amos is almost identical to that of Ato, E box-containing oligonucleotides, E1 and E4, which represent high-affinity binding sites for the Da/Ato protein complex, were examined. The Sc/Da complex was used as a positive control since this complex also binds to these two E boxes. E1 and E4 boxes are well bound by Amos/Da. In contrast, this shifted complex including Amos and Da is undetectable with either Amos or Da alone. The Amos/Da complex is supershifted by addition of anti-GST antibody, which recognizes the GST-Da protein used in this assay. This binding of the Amos/Da complex to E boxes is efficiently completed by addition of excess E1 and E4 cold probes but not by two corresponding mutant E boxes. These data suggest that Amos and Da form a heterodimer when bound to E boxes and that the binding activities are sequence specific. To further analyze the interaction between amos and da in vivo, the effects of amos misexpression were examined in different da genetic backgrounds. The number of neurons were counted in sca-GAL4/UAS-amos embryos carrying different da gene dosages. When a moderate level of amos is induced in wild-type embryos with two copies of da⁺, some ectopic Elav-positive cells are observed. The ectopic neurons are suppressed in embryos carrying only one copy of da⁺. When amos and da are simultaneously misexpressed, numerous Elav-positive cells are induced. The strong neuralization by amos and da has also been revealed by the staining of MAb 22C10, which labels the neuronal morphology. These ectopic neurons include MD neurons that express lacZ from E7-2-36 insertion. As a control, misexpression of da causes only a minor effect on the number of neurons in this assay. These results suggest that the ectopic neuron formation elicited by amos is very sensitive to the gene dosage of da (Huang, 2000).

The Tufted¹ (Tft¹) dominant mutation promotes the generation of ectopic bristles (macrochaetae) in the dorsal mesothorax of Drosophila. Tft¹ corresponds to a gain-of-function allele of the proneural gene amos that is associated with a chromosomal aberration at 36F-37A. This causes ectopic expression of amos in large domains of the lateral-dorsal embryonic ectoderm, which results in supernumerary neurons of the PNS, and in the notum region of the third instar imaginal wing, which gives rise to the mesothoracic extra bristles. Revertants of Tft¹, which lack ectopic neurons and bristles, do not show ectopic expression of amos. One revertant is a loss-of-function allele of amos and has a recessive phenotype in the embryonic PNS. These results suggest that both normal and ectopic Tft¹ bristles are generated following similar rules, and both are subjected to Notch-mediated lateral inhibition. The ability of Tft¹ bristles to appear close together may be due to amos having a stronger proneural capacity than that of other proneural genes like asense and scute. This ability might be related to the wild-type function of amos in promoting development of large clusters of closely spaced olfactory sensilla (Villa-Cuesta, 2003).

It has been reported that the Tft phenotype is suppressed by the further removal of the ase gene. The expression of proneural genes was examined in Tft¹ wing discs to analyze possible regulatory interactions between the proneural genes and amos. ac, sc, and ase are ectopically expressed in the area where the Tft¹ SOPs appear, but their expressions occurs only in single, isolated cells. Not even in younger discs did expression of these genes in that area occur in a proneural-like cluster. The isolated cells were most likely SOPs, since these three genes are expressed in singled-out bristle precursor cells due to self-stimulatory loops. The pattern of expression of ase in single cells, as opposed to the homogeneous large domain of ectopic amos, suggests that it is the presence of Amos that triggers the expression of ase and not vice versa. Indeed, the ectopic expression of amos is not removed in Tft¹/+ discs simultaneously mutant for the null ase1 allele. Taken together, these results indicate that the ectopic expression of amos in a relatively large area of the presumptive notum creates an oversized proneural cluster from which many SOPs emerge. These then express ac, sc, and ase, similarly to the SOPs of other external sensory organs. Moreover, amos needs the panneural function of ase to single out SOPs from this proneural cluster. The expression of amos is normal in ase¹ embryos and in ase¹ adults, the olfactory SOs of the antenna appear unaffected. This further indicates that the expression of amos does not depend on ase and that this gene is dispensable for the generation of the olfactory SO (Villa-Cuesta, 2003).

Tufted is a classical Drosophila mutant characterized by a large number of ectopic mechanosensory bristles on the dorsal mesothorax. Unlike other ectopic bristle mutants, Tufted is epistatic to (lies downstream of) achaete and scute, the proneural genes that normally control the development of these sensory organs. Genetic and molecular evidence is presented that Tufted is a gain-of-function allele of the proneural gene amos that ectopically activates mechanosensory neurogenesis. The ability of the various proneural bHLH proteins to cross-activate each other has been systematically examined: their ability to do so is in general relatively limited, despite their common ability to induce the formation of mechanosensory bristles. This phenomenon seems instead to be related to their shared ability to activate Asense and Senseless (Lai, 2003).

Role of proneural genes in the formation of the larval olfactory organ of Drosophila

This study addressed the role of proneural genes in the formation of the dorsal organ in the Drosophila larva. This organ is an intricate compound comprising the multineuronal dome-the exclusive larval olfactory organ-and a number of mostly gustatory sensilla. The numbers of neurons and of the different types of accessory cells in the was determined dorsal organ. From these data, it was concluded that the dorsal organ derives from 14 sensory organ precursor cells. Seven of them appear to give rise to the dome, which therefore may be composed of seven fused sensilla, whereas the other precursors produce the remaining sensilla of the dorsal organ. By a loss-of-function approach, the roles were examined of atonal, amos, and the achaete-scute complex (AS-C), which in the adult are the exclusive proneural genes required for chemosensory organ specification. atonal and amos are necessary and sufficient in a complementary way for four and three of the sensory organ precursors of the dome, respectively. AS-C, on the other hand, is implicated in specifying the non-olfactory sensilla, partially in cooperation with atonal and/or amos. Similar links for these proneural genes with olfactory and gustatory function have been established in the adult fly. However, such conserved gene function is not trivial, given that adult and larval chemosensory organs are anatomically very different and that the development of adult olfactory sensilla involves cell recruitment, which is unlikely to play a role in dome formation (Grillenzoni, 2007).

Functional distinctness of closely related transcription factors: A comparison of the Atonal and Amos proneural factors

Using the well-characterised paradigm of Drosophila sensory nervous system development, the functional distinctness was examined of the Amos and Atonal (Ato) proneural transcription factors, which have different mutant phenotypes but share very high similarity in their signature bHLH domains. Using misexpression and mutant rescue assays, it was shown that Ato and Amos proteins have abundantly distinct intrinsic proneural capabilities in much of the ectoderm. The eye, however, is an exception: here both proteins share the capability to direct the R8 photoreceptor fate choice. Therefore, functional distinctness between these closely related transcription factors vary with developmental context, indicating different molecular mechanisms of specificity in different contexts. Consistent with this, the structural basis for their distinctness also varies depending upon the function in question. In previous studies of neural bHLH factors, specificity invariably mapped to the bHLH domain sequence. Similarly, and despite their high similarity, much of the Amos’ specificity relative to Ato maps to Amos-specific residues in its bHLH domain. For Ato-specific functions, however, the Amos bHLH domain can substitute for that of Ato. Consequently, Ato’s specificity relative to Amos requires the non-bHLH portion of the Ato protein. Ato provides a powerful precedence for a role of non-bHLH sequences in modulating bHLH functional specificity. This has implications for structural and functional comparisons of other closely related transcription factors, and for understanding the molecular basis of specificity (Maung, 2007).

This study analyses the functional specificities of two proneural proteins, Ato and Amos, as examples of how highly related transcription factors achieve functional distinctness. This analysis is complex, partly because each proneural protein is multifunctional in neural development, each being required for different neural fates in different developmental contexts. Nevertheless, the results point to two general conclusions. Firstly, closely related transcription factors can exhibit high functional distinctness, but this distinctness can vary strongly with developmental context. Secondly, the function of the bHLH domain itself can depend on the protein context: most notably, chimeric proteins reveal that the Amos bHLH domain can largely support Ato-specific functions when in the context of the Ato protein. Unravelling the molecular and structural bases underpinning these observations is likely to be complex, but an important conclusion is that, for a given factor, different functional specificities will have different protein structural requirements, pointing to different underlying molecular mechanisms (Maung, 2007).

Within the antennal funiculus, ato and amos have very distinctive specificities. In this context they also display mutual antagonism in misexpression experiments, although it is not clear how this might relate to their physiological functions. One possibility is that unproductive Ato/Amos heterodimers are formed. Outside the funiculus they also show specificity, but this is only revealed in the absence of endogenous ato function. The most likely explanation is that amos’s specificity is masked by its ability to activate the endogenous ato gene in this context. This ability might be an aberrant reflection of a true regulatory relationship: it is known that ato is downstream of amos in certain defined embryonic locations (Holohan, 2006; Grillenzoni, 2007; Maung, 2007 and references therein).

The comparison of Ato and Amos provides a useful precedent for other pairs/groups of closely related bHLH factors, such as Math1/Math5, Mash1/Mash2, Ngn1/Ngn2. Very little is known of the functional distinctness of these. In such circumstances, an apparent lack of specificity may simply indicate that the appropriate context or cell fate has not been assayed. Moreover, although misexpression phenotypes provide useful specificity assays, mutant rescue may be a more discerning assay for closely related proteins (Maung, 2007).

The bHLH domain sequence is clearly important for specificity, and this is reflected in its strong sequence conservation in orthologues across species. For example, the sequence identity in the bHLH domain between orthologues from D. melanogaster and D. virilis is 100% for Ato and 97% for Amos. This strongly implies that most bHLH residues have functional significance, either for general bHLH properties or for specificity. A proportion of bHLH residues (19/60) are highly conserved across Class II bHLH domains because they function in dimerisation or in contact with the generic E box sequence. The potential importance of the remaining conserved (i.e., consistently different) residues can be inferred by correlating functional properties with cross-species patterns of sequence similarity (Maung, 2007).

Seventeen amino acid residues differ between Amos and Ato; most of them consistently differ in orthologues. The experiments suggest that these differences may be more important for the specificity of Amos than for Ato. Interestingly, seven residues are unique to Drosophila orthologues of Amos, consistent with some functional specialisation relative to a putative Ato/Amos ancestor. Only four residues are unique to Drosophila Ato (Maung, 2007).

The functional distinctness of the Ato-like proteins provides an explanation for why highly conserved Amos and Ato orthologues have been maintained in the genomes of diverse insects. Yet Amos and Ato retain high bHLH sequence similarity. Twelve amino acid residues are shared between Amos and Ato (but not Sc). This is most striking in the almost complete identity of their basic regions. The retention of these residues in both proteins implies a common functional constraint that has prevented greater bHLH divergence. Indeed the two proteins may have several shared functions. One is the ability to repress endogenous bristle formation. Shared features might also be responsible for common aspects of the function of both proteins in olfactory sensillum development (albeit of different subtypes) (Maung, 2007).

While these functions are physiologically relevant, the constraint they provide may also have had two ‘accidental’ consequences. The shared bHLH residues may be important in R8 determination, thereby explaining why Amos (but not Sc) inappropriately shares Ato’s ability to drive R8 formation. Perhaps this has not been selected against because Amos is not expressed in the context in which this R8 ability is manifest (the eye). Another accidental consequence would be that the Amos bHLH domain shares the capability of Ato for driving chordotonal formation. However this is not seen in the Amos protein as a whole because chordotonal capability also requires other non-bHLH Ato sequences, i.e., a specific protein context (Maung, 2007).

These studies demonstrate that an Ato-like bHLH domain is necessary for Ato specificity, but is not sufficient. Ato sequences outside the bHLH domain are required to modulate its function, enabling Ato to attain its specificity relative to Amos. Comparison of the orthologous Ato proteins from three Drosophila species shows that they share regions of strong amino acid sequence conservation outside the bHLH domain. Overall there is 59% identity between the three, with many areas of greater than 70% identity. The N terminus shows particularly high identity over 99 amino acids (84%). Interestingly, the non-bHLH sequences of the three orthologous Amos proteins share less similarity. Overall identity is only 29%, with the highest identity of 62.5% found in a region of 32 amino acids. This supports a scenario in which the non-bHLH sequence of Ato has a strong role in promoting Ato specificity, whereas Amos's non-bHLH sequence appears to have a more accessory role for Amos specificity (Maung, 2007)

These findings contrast strongly with all previous studies on neural bHLH proteins in both Drosophila and vertebrates, which have overwhelmingly highlighted the bHLH domain as the sole determinant of protein specificity. For Ato, while the bHLH domain is clearly important for distinguishing Ato from Sc, it is the non-bHLH sequences that distinguish Ato from Amos. Similar structure-function comparisons between other closely related pairs of proteins may also reveal the importance of non-bHLH sequences. However, such comparisons would require a much greater understanding of the functional distinctness of such closely related factors (Maung, 2007).

In summary, proneural specificity in different contexts or for different neural fates requires different underlying amino acid sequence elements. Moreover, the pattern of specificity is complicated by the simultaneous need for shared and diverged functions. Strong constraint for shared functions may have left little room for functional specialisation via divergence of the bHLH domain. To some extent, bHLH specialisation was achieved during Amos's evolution, but for Ato, functional divergence required the acquisition of a modulatory role by its non-bHLH sequences (Maung, 2007).

Proneural specificity must entail differences in target gene regulation that result from differences in interaction specificity either with DNA binding sites or with cofactor proteins (or both). Differential interaction with cofactor proteins is widely favoured as an explanation for specificity, partly because context dependence is most easily explained by interaction with different spatially restricted cofactors. This would be consistent with the conclusion that the different sequence motifs mediate different aspects of specificity, since they might provide different protein interaction interfaces. In addition to specific cofactor interactions, it has recently been demonstrated that Ato-specific and Sc-specific target genes have E box binding sites that conform to distinct (Ato- and Sc-specific) consensus sequences. It is open to speculation whether the functional differences between Ato and Amos have a similar molecular basis. Since Amos's basic region is identical to that of Ato, it seems possible that Ato and Amos utilise similar DNA binding sites. The in vivo binding site preference of Amos, however, is currently unknown. It remains an important priority in the study of this group of transcription factors to determine the molecular basis of their functional specificity (Maung, 2007).

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 (Cachero, 2009).

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 refered 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 (Cachero, 2009).

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 (Cachero, 2009).

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 (Cachero, 2009).

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 (Cachero, 2009).

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 (Cachero, 2009).

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 (Cachero, 2009).

REFERENCES

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

Grillenzoni, N., et al. (2007). Role of proneural genes in the formation of the larval olfactory organ of Drosophila. Dev. Genes Evol. 217: 209-219. PubMed citation: 17260155

Gupta, B. P. and Rodrigues, V. (1997). atonal is a proneural gene for a subset of olfactory sense organs in Drosophila. Genes Cells 2: 225-233. PubMed Citation: 9189759

Gupta, B. P., Flores, G. V., Banerjee, U. and Rodrigues, V. (1998). Pattterning an epidermal field: Drosophila Lozenge, a member of the AML/Runt family of transcription factors, specifies olfactory sense organ type in a dose-dependent manner. Dev. Biol. 203: 400-411. PubMed Citation: 9808789

Holohan, E. E., zur Lage, P. I. and Jarman, A. P. (2006). Multiple enhancers contribute to spatial but not temporal complexity in the expression of the proneural gene, amos. BMC Dev. Biol. 6: 53. Medline abstract: 17094800

Huang, M.-L., Hsu, C.-H. and Chien, C.-H. (2000). The proneural gene amos promotes multiple dendritic neuron formation in the Drosophila peripheral nervous system. Neuron 25: 57-67. Medline abstract: 10707972

Lai, E. C. (2003). Drosophila Tufted is a gain-of-function allele of the proneural gene amos. Genetics 163: 1413-1425. 12702685

Maung, S. M. T. W. and Jarman, A. P. (2007). Functional distinctness of closely related transcription factors: A comparison of the Atonal and Amos proneural factors Mech. Dev. 124: 647-656. PubMed citation: 17709231

Reddy, G.V., Gupta, B., Ray, K., and Rodrigues, V. (1997). Development of the Drosophila olfactory sense organs utilizes cell-cell interactions as well as lineage. Development 124: 703-712. PubMed Citation: 9043085

Sen, A., Kuruvilla, D., Pinto, L., Sarin, A. and Rodrigues, V. (2004). Programmed cell death and context dependent activation of the EGF pathway regulate gliogenesis in the Drosophila olfactory system. Mech. Dev. 121: 65-78. 14706701

Villa-Cuesta, E., et al. (2003). Tufted is a gain-of-function allele that promotes ectopic expression of the proneural gene amos in Drosophila. Genetics 163: 1403-1412. 12702684

zur Lage, P. I., et al. (2003). The Drosophila proneural gene amos promotes olfactory sensillum formation and suppresses bristle formation. Development 130: 4683-4693. 12925594

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

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date revised: 7 February 2000

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