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Gene name - atonal Synonyms - Cytological map position - 84F Function - transcription factor Keywords - proneural - eye and peripheral nervous system | Symbol - ato
FlyBase ID:FBgn0010433 Genetic map position - 3-[48] Classification - bHLH Cellular location - nuclear |
Drosophila has four types of sensory elements: external sense organs, multiple dendritic neurons, chordotonal neurons, and photoreceptors. The latter two do not require the proneural genes of the achaete-scute complex (AS-C) in order to develop and function properly, but they do need atonal, a neurogenic gene that behaves in many ways like the genes of AS-C. atonal gets its name from the disruptive effects the gene's mutation has on chordotonal neuron differentiation. Mutants are completely difficient in this lateral sense organ (Jarman, 1995).
Early in embryonic development, atonal is expressed in all chordotonal organ progenitor cells, but its later expression is restricted to only a particular set of precursors, through a process of lateral inhibition. Notch does not seem to be required for this process in chordotonal organs, and the mechanism is not well understood. Mutation in atonal also disrupts eye development (Jarman, 1995).
Atonal protein is produced in all cells just anterior to the morphogenic furrow. Expression is downstream of hedgehog, an important gene involved in regulating the progression of the furrow. As the furrow progresses, most cells that express atonal in front of the furrow lose that capacity. atonal expression behind the furrow is confined to the R8 progenitors, whose fate atonal determines. atonal is produced within the context of the furrow, which is only rudimentary in atonal mutants. atonal is a neurogenic gene, functioning in the place of achaete-scute complex bHLH genes, both in the eye and in chordotonal organs.
The function of Atonal is best illustrated by its role in chordotonal organ development. A scolopidium, the basic unit of chordotonal organs, consists of four cells: a neuron with a single dendrite, the scolopale cell, cap cell and ligament cell. The scolopale cell (a glial cell) forms a sheath around the dendrite, while the cap cell and ligament cell mediate the attachment of the chordotonal organ to the body cell. Expression of atonal is restricted to a subset of atonal-requiring chordotonal precursors, called founder precursors. In atonal mutants, all chordotonal organs are absent except for one scolopidium of Ich5, the abdominal pentascolopidial organ (Ich5 consists of five scolopidia organized in a linear array). This one scolopidium formed in atonal mutants is atonal independent. The atonal independent precursor corresponds to the earliest chordotonal precursor (precursor C1) and corresponds to the P cell which gives rise to the anterior-most scolopidium of Ich5 (zur Lage, 1997).
EGF receptor signaling is required in neural recruitment during formation of Drosophila chordotonal sense organ clusters. A total of five neural precursors express atonal in abdominal segments, and this number is too few to explain the formation of the eight scolopidia in each abdominal segment. Of the five precursors, C-1, C2 and C3 contribute to Ich5, C4 migrates slightly anterodorsally and gives rise to v'ch1, the dorsal most scolopidium, which is solitary. C5 contributes to vchAB, a more ventral pair of scolopidia. The remaining precursors require Egf-R signaling for their selection. Signaling by the founder precursors is initiated by atonal activating (directly or indirectly) rhomboid expression in the founder cells. It should be noted that in some developmental processes, rhomboid appears to function in the signal-receiving cells, such as in the patterning of ovarian follicle cells. It is not believed that this is the case in chordotonal-precursor formation, because rho is expressed in precursors that do not require rhomboid function (C1-C5 are formed even in rhomboid mutants). Signaling by these founder precursors, presumably through the EGF receptor ligand Spitz, then provokes a response in the surrounding ectodermal cells, as shown by the activation of expression of the Egf-R target genes pointed and argos. The signal and response then leads to recruitment of some of the ectodermal cells to the chordotonal precursor cell fate. Egf-R hyperactivation by misexpression of rhomboid results in excessive chordotonal precursor recruitment. Argos functions in a feedback mechanism to prevent the excess recruitment of additional ectodermal cells. The increase in the number of scolopidia caused by Egf-R hyperactivation is confined to an enlargement of existing cluster sizes: no new chordotonal clusters are formed. A two step mechanism is postulated for the formation of clusters of chordotonal precursors. In the first step, precursors C1-C5 are selected as founder precursors by the conventional route of proneural gene expression and lateral inhibition. In a distinct second phase, these precursors then signal to adjacent ectodermal cells via the Egf-r pathway, inducing some of them to become chordotonal precursors (secondary or recruited precursors). This two-step process is strongly reminiscent of the way atonal acts in neurogenesis in compound eyes. Here, atonal expression is initially refined by lateral inhibition, until atonal is expressed in only the founding R8 precursor, which then recruits R1-R7 in a mechanism that does not require the activation of atonal in these cells (zur Lage, 1997).
The selection of Drosophila sense organ precursors (SOPs) for sensory bristles is a progressive process: each neural equivalence group is transiently defined by the expression of proneural genes (proneural cluster), and neural fate is refined to single cells by Notch-Delta lateral inhibitory signalling between the cells. Unlike sensory bristles, SOPs of chordotonal (stretch receptor) sense organs are tightly clustered. It has been shown that for one large adult chordotonal SOP array (the adult femoral chordotonal sense organ), clustering results from the progressive accumulation of a large number of SOPs from a persistent proneural cluster. This is achieved by a novel interplay of inductive epidermal growth factor- receptor (EGFR) and competitive Notch signals. EGFR acts in opposition to Notch signaling in two ways: it promotes continuous SOP recruitment despite lateral inhibition, and it attenuates the effect of lateral inhibition on the proneural cluster equivalence group, thus maintaining the persistent proneural cluster. SOP recruitment is reiterative because the inductive signal comes from previously recruited SOPs (zur Lage, 1999).
The adult femoral chordotonal sense organ arises from a group of some 70-80 SOPs. A developmental analysis of Ato expression has revealed that these SOPs accumulate over an extended period of time in the dorsal region of each leg imaginal disc during the third larval instar and early pupa. The continued expression of Ato implies a sustained requirement for proneural function throughout the process of SOP accumulation. Unusually, Ato is persistently expressed in a group of ectodermal cells identified as the proneural cluster (PNC). From this PNC, cells are funnelled inward into a cavity formed by the folding of the disc. This invagination later becomes visible as a distinctive 2-cell wide intrusion, which is referred to as the 'stalk'. Cells at the deepest end of the stalk undergo shape changes to form an amorphous inner SOP mass. Invaginating cells are characterised by upregulation of Ato expression, a characteristic of SOP commitment. Surprisingly, SOP markers (Ase protein and the A101 enhancer trap line) are not expressed in all the stalk SOPs. Instead, these markers are only apparent in older cells, particularly at the time when they become part of the inner mass (which is therefore referred to as mature SOPs). Despite this, entry into the stalk seems to mark SOP commitment, since both the stalk and the mature SOPs are absent in discs from ato mutant larvae. This apparent intermediate stage may not have a counterpart in external sense organ precursor formation, although there is some evidence for multiple steps between the uncommitted cell and the SOP (the so-called pre-sensory mother cell state). Initially, Ato remains activated in all invaginated SOPs. This extended period of proneural gene expression is unusual since AS-C proneural expression is typically switched off in SOPs shortly after commitment. Later, at approximately 6 hours before puparium formation (BPF), Ato expression is switched off synchronously in the mature SOPs, although expression remains in the stalk SOPs and the PNC. At this point there is very little overlap between Ato and Ase or A101 (zur Lage, 1999).
The process of chordotonal SOP formation described above is at odds in several respects with the well-known paradigm of SOP selection for sensory bristles. In the latter, the solitary SOP expresses Delta, which triggers expression in the PNC of genes of the E(spl)-C, thereby preventing further SOP commitment and forcing loss of AS-C expression and neural competence. In the case of the femoral chordotonal organ, newly committed cells from the PNC are in contact with previously committed SOPs in the stalk, but are apparently not receiving (or not responding to) lateral inhibition signals from these to prevent their commitment. Likewise, the presence of committed SOPs does not switch off ato expression in the PNC. Nevertheless, components of the N-Dl pathway are expressed in patterns consistent with lateral inhibition. The newly formed SOPs express Dl, suggesting that they send inhibitory signals, while the PNC expresses mgamma, a member of the E(spl)-C, suggesting that these cells are responding to the Notch-Delta signal. Indeed, mgamma is coexpressed with ato in the PNC throughout the development of the SOP cluster. Chordotonal SOP formation is shown to be sensitive to N inhibitory signaling. Strong activation of N signaling or its effectors can inhibit chordotonal SOP formation. Thus, N signaling has an important role to play: it acts to limit the process of SOP selection from the PNC. Some mechanism, however, must prevent N signaling from completely inhibiting multiple SOP formation (zur Lage, 1999).
The progressive accumulation of chordotonal SOPs suggests that a recruitment mechanism could explain the clustering of SOPs. The Drosophila Egfr signaling pathway is involved in a number of recruitment processes in development, and a role for Egfr signaling has been demonstrated in the induction of embryonic chordotonal precursors (zur Lage, 1997). Although there appear to be significant differences in the process of SOP formation in imaginal discs, as compared with the embryo, it was asked whether Egfr signaling is also involved in forming the femoral chordotonal cluster. To address this question, the pathway was conditionally disrupted by expressing a dominant negative form of Egfr protein. Expression of UAS-Egfr DN results in a dramatic loss of chordotonal SOPs in late third instar imaginal leg discs (as judged by Ase protein expression or the A101 enhancer trap line). This demonstrates that Egfr signaling is required for the process of femoral chordotonal SOP formation. In contrast, the appearance of bristle SOPs is unaffected, arguing against the possibility of a nonspecific effect on SOPs in general (zur Lage, 1999).
To determine whether Egfr signaling controls SOP number, expression of components of the Egfr pathway that determine the level of signaling was forced, thus resulting in hyperactivation of the pathway. pointed (pnt) is an effector gene that encodes a transcription factor and is activated in cells responding to Egfr signaling. Both rho and pnt are expressed during chordotonal SOP formation. Indeed, forced expression of rho or pnt increases chordotonal SOP formation. Egfr could promote SOP formation by stimulating the commitment of PNC cells or by stimulating proliferation of SOPs. Both functions would be consistent with known Egfr roles, but the current investigations favour the former. Analysis of Ato expression in leg discs in which rho has been misexpressed reveals a large invagination of cells and a smaller PNC. Shrinking of the PNC was confirmed by the reduced extent of mgamma expression. These observations are consistent with an increased rate of SOP commitment upon Egfr hyperactivation. Moreover, this effect is reminiscent of the effect of N loss of function on Ato expression, suggesting that Egfr signaling supplies the mechanism that interferes with lateral inhibition of SOP commitment (zur Lage, 1999).
Although it seems that cells of the PNC and stalk are held in a state of mitotic quiescence throughout the time that SOP fate decisions are being made, BrdU is incorporated in the older (mature) SOPs. The experiments so far have indicated that Egfr signaling affects SOP commitment from the PNC. To determine more precisely the spatial patterning of Egfr activity required for SOP clustering and N antagonism, the expression patterns of key components of the pathway were characterized. Localized expression of rho appears to play a central role in spatial restriction of Egfr activity in cases where Spi is the ligand; in these cases it appears to mark the cells that are a source of signaling. During development of the femoral chordotonal organ, rho is expressed in a very restricted pattern: RHO mRNA is only detected in the SOPs, becoming confined in the late third instar larva to the youngest SOPs at the top of the stalk. To identify the cells responding to rho-effected signaling, an antibody that detects the dual-phosphorylated (activated) form of the ERK MAP kinase (dp-ERK) was used. In leg imaginal discs, dp-ERK is detected in a confined area corresponding to the uppermost (youngest) stalk SOPs. Thus, like rho, dp-ERK is expressed in the newly formed stalk SOPs. Double labelling for RHO RNA and dp-ERK confirms this, but also suggests that the overlap in expression is not complete: dp-ERK is detected above the uppermost rho-expressing cells of the stalk, probably in one or a few cells of the proneural cluster as they funnel into the stalk. This suggests that Egfr promotes SOP commitment as a consequence of direct signaling from previous SOPs to overlying PNC cells. Since rho expression is itself activated upon SOP commitment, this process occurs cyclically: the newly recruited SOPs are in turn able to signal to further overlying PNC cells. That is, recruitment is reiterative. Egfr signaling via Spitz has been shown to help to maintain neural competence by attenuation of Notch directed lateral inhibition. The opposing forces of Notch and Egfr signaling are thought to be played out through direct Notch and Egfr signaling between the epidermal proneural cells, which bear Notch, and the SOP, which sends inhibitory signals through the Delta ligand, and stimulatory signals through the Spitz ligand (zur Lage, 1999).
Reiterative recruitment alone cannot entirely explain the accumulation of SOPs. Such an accumulation also relies on the persistence of the competent pool of PNC cells from which SOPs can be recruited. For AS-C PNCs, this does not occur, because the mutual inhibition required for continued competence is unstable and resolves quickly to a state of lateral inhibition once the SOP emerges from the PNC. This results in rapid shutdown of AS-C expression and hence competence within the PNC. It is possible that the members of E(spl)-C that are expressed in the PNC (notably mgamma and mdelta) are less aggressive inhibitors of proneural gene expression than the E(spl)-C members expressed in AS-C PNCs (m5 and m8). The results obtained in the femoral SOP suggest, however, that Egfr has a role to play in maintaining the PNC by partially attenuating lateral inhibition on a PNC-wide scale. Thus, the PNC is not completely shut off by inhibition from SOPs, but instead kept in check, allowing continued mutual inhibition and maintenance of competence but not allowing general SOP commitment. Since neither rho nor dp-ERK are detected in the PNC as a whole, this function of Egfr could be indirect and achieved through partial attenuation of Dl signaling from the stalk SOPs themselves. The trans- or auto-activation of EGFR signaling between the stalk SOPs (as suggested by the co-expression of dp-ERK and rho) might be an indicator of this function. It is also possible, however, that Egfr signaling is direct and that the dp-ERK antibody is not sensitive enough to detect expression in the PNC cells (zur Lage, 1999).
Bases in 5' UTR - 334
Exons - two
Bases in 3' UTR - 222
The bHLH domain is at the C-terminus. Preceding this is an acidic sequence that also contains a PEST sequence which aids in protein degradation (Jarman, 1993).
date revised: 3 MAR 97
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