BIOLOGICAL OVERVIEW

achaete is a proneural gene of the achaete-scute complex (AS-C). It is expressed in the presumptive neuroectoderm during embryonic development and is responsible for the allocation of specific cells (presumptive neuroblasts) to the neural fate. Neurogenesis is fostered by the interaction of several systems of gene expression. These include lateral inhibition (controlled by the Notch pathway) and proneural gene expression.

The process of allocation differs from the process of directing the specific fate of cells in a differentiation pathway. In the former process, cells are allocated, in effect, set aside and destined for a specific fate. This is the function of the achaete-scute complex. But the latter process brings other genes into play, to direct the specific fate, that is, neural development or Malphigian tubule development or any number of other potential fates.

One particularly intriguing aspect of development is the evolution of species-specific two dimensional patterns. For example, each species of insect has a specific arrangement of bristles and other sensory organs on the adult epidermis. These organs are neural in origin and require the involvement of the AS-C for their proper development. Observations of wing imaginal discs (larval sacs of cells that give rise to adult wings) reveal that achaete and scute are transcribed in a pattern that prefigures the future sites of neural elements.

What aspects of control establish this pattern of as-sc transcription? It is thought that genes that act earlier than the proneural genes establish a prepattern in the wing imaginal disc, and these genes in turn regulate the transcription of ac-sc according to the pattern they set. Candidate genes for this function are found early in development among pair-rule and segment polarity genes that establish the pattern of gene transcription in the embryo. A more proximal cause is the two recently discovered homeodomain proteins, Araucan and Caupolican that are transcribed in a prepattern that prefigures the future site of sensory elements and the wing veins as well (Gomez-Skarmeta, 1996).

An early step in the development of the large mesothoracic bristles (macrochaetae) of Drosophila is the expression of the proneural genes of the achaete-scute complex (AS-C) in small groups of cells (proneural clusters) of the wing imaginal disc. This is followed by a much increased accumulation of AS-C proneural proteins in the cell that will give rise to the sensory organ, the SMC (sensory organ mother cell). This accumulation is driven by cis-regulatory sequences, SMC-specific enhancers, that permit self-stimulation of the achaete, scute and asense proneural genes. Negative interactions among the cells of the cluster, triggered by the proneural proteins and mediated by the Notch receptor (lateral inhibition), block this accumulation in most cluster cells, thereby limiting the number of SMCs. In addition, proneural proteins trigger positive interactions among cells of the cluster that are mediated by the Epidermal growth factor receptor (Egfr) and the Ras/Raf pathway. These interactions, termed 'lateral co-operation', are essential for macrochaetae SMC emergence. Activation of the Efgr/Ras pathway appears to promote proneural gene self-stimulation mediated by the SMC-specific enhancers. Excess Egfr signaling can overrule lateral inhibition and allow adjacent cells to become SMCs and sensory organs. Thus, the Egfr and Notch pathways act antagonistically in notum macrochaetae determination (Culí, 2001).

The earliest stage in macrochaetae development is the formation of the proneural clusters of ac-sc expression. Accumulation of Sc in cells of proneural clusters located at the more central positions of the wing disc decreases upon reduction of the level of Egfr signaling. The effect is cell-autonomous, which indicates that reception of the signal is important for cells to express sc properly. In contrast, more marginally located clusters, like the notopleural or scutellar, are unmodified or slightly enhanced under conditions of insufficient Egfr signaling. It is known that expression of ac-sc in different proneural clusters depends on separate, functionally independent enhancers which are thought to respond to local, specific combinations of transcription factors (prepattern). The different, spatially restricted effects of the insufficiency of Egfr function may thus be due to interference in the deployment or function of particular factors expressed in the affected area. Interestingly, the expression of the homeobox genes of the iroquois complex, necessary for the expression of ac-sc in many notum proneural clusters, is especially sensitive to the expression of the Vein Egfr ligand in the central region of the notum. Alternatively, since Egfr function is a well known requisite for growth and patterning of imaginal discs, the reduced expression of sc may be due to a more general impairment of the patterning of the central area of the disc (Culí, 2001).

The data support a key role for Egfr signaling in the emergence of the notum macrochaetae SMCs from proneural clusters. Indeed, expression of the Egfr inhibitory ligand Aos exclusively in proneural clusters, a condition that permits essentially wild-type Sc accumulation in these clusters, almost completely suppresses the appearance of SMCs and SOs. SMC emergence is also impaired in discs from heat-treated temperature sensitive Egfr larvae and in clones of cells expressing UAS-raf^DN2.1. Moreover, when the cells that accumulate Raf^DN2.1 occupy positions where SMCs normally appear, wild-type neighboring cells give rise to displaced SMCs. This phenomenon is reminiscent of and in accordance with the observation, made with mosaic individuals, that when the position of a dorsocentral bristle is in ac minus territory, this bristle does not develop, but a nearby ac plus cell can give rise to a dorsocentral bristle displaced from its wild-type position. The cell-autonomous effect of Raf^DN2.1 indicates that reception of the Egfr signal, mediated by the Ras/Raf/MAP kinase cassette, is essential for notum macrochaetae SMC determination. This was further substantiated by the cell autonomous induction of SMCs and bristles in clones of cells overexpressing a constitutively activated form of Ras. Taken together, these results indicate that reception of the Egfr signal promotes sc expression and SMC determination (Culí, 2001).

In the notum anlagen the expression of rho/ve occurs mainly in proneural clusters and this expression is dependent on ac-sc. Rho/ve facilitates the processing of Spitz, an activating ligand of Egfr. The soluble, active form of Spitz promotes ectopic sc expression and SMC emergence. Hence, these data suggest that, in proneural clusters, Ac-Sc promote expression of rho/ve, which by activating Spitz, would stimulate Egfr signaling in the cells of the cluster. (The Vein Egfr ligand probably does not specifically act in proneural clusters, because many of these lie outside of its expression domain). It is thus proposed that Egfr mediates a mutual positive signaling among cells of the proneural cluster, which promotes SMC emergence by probably reinforcing ac-sc expression. This positive signaling is called lateral cooperation. Evidently, this does not exclude an autocrine activation of the Egfr pathway in the cells that express AS-C proteins, but the lateral cooperation hypothesis is favored since it is well established in other systems that the Egfr pathway is used mainly for intercellular communication. This signaling should facilitate the acquisition of the SMC state by one or a few cells of a proneural cluster (Culí, 2001).

The SMC state is associated with greatly increased levels of proneural protein. These are accomplished by the self-stimulation of ac, sc and ase mediated by AS-C enhancers that activate these genes specifically in the cells that become SMCs. Since Ras1^V12 elicits the expression of both sc and SRV-lacZ, it is proposed that, in the extant proneural clusters, the SMC-specific enhancers are targets of Egfr signaling. Unidentified effector(s) of the Egfr/Ras pathway should facilitate the self-stimulation of the proneural genes mediated by the SMC-specific enhancers by, possibly, binding to these enhancers. Conclusive evidence in support of this role requires the identification of the signaling effector(s) and of their interaction with the enhancer. Interestingly, overexpression of the effector Pointed P1 promotes development of many extra macrochaetae on the notum and putative Ets-domain binding sites have been identified in the sc and ase SMC enhancers (GTGGAAAT and ACGGAAAC, respectively) (Culí, 2001).

Egfr-mediated lateral cooperation should tend to activate the SMC-specific enhancers in many cells of the proneural clusters. This, however, is prevented by N signaling, which is activated by Ac and Sc in the cells of the cluster. This signaling, by means of the bHLH proteins of the E(spl)-C, blocks the ac-sc-ase self-stimulatory loop promoted by the SMC-specific enhancers. However, within a proneural cluster the cells of the proneural field accumulate greater amounts of Ac-Sc proteins. As it has been hypothesized that cells that signal the most are the least inhibited by their neighbors, eventually, a cell of the proneural field will be released from the inhibitory loop and its levels of E(spl)-C bHLH protein will become minimal. This cell will turn on the ac-sc-ase self-stimulation and become an SMC. The SMC signals maximally to its neighbors and prevents them from following the same fate (lateral inhibition). These results add to this scenario the requirement for Egfr-mediated signaling for one cell of the proneural field to turn on the ac-sc-ase self-stimulatory loops and become an SMC. According to this model, Ac-Sc activate both the N-and Egfr-mediated signaling pathways, with their SMC-suppressing and SMC-promoting abilities, respectively, and both signaling systems appear to act on the same SMC-specific enhancers (Culí, 2001).

AC is not only involved in neurogenesis but also in a number of other morphogenetic events that essentially resemble the actions of AC in neurogenesis. The action of AC in the development of Malphigian tubules is a good example. These excretory organs take the form of four protuberances connected to the proctodeum. Achaete is necessary for cell allocation, but not cell commitment. Achaete function here is identical in manner to its function in neural differentiation. In this case Krüppel directs the differentiation pathway of the allocated cells. Expression of Krüppel is initially weak throughout a broadly diffuse region that with time grows progressively more concentrated, in fewer and fewer cells. Krüppel expression is controlled by forkhead, which in turn is induced by the activity of tailless and huckebein.

By stage 10, Krüppel has become more strongly stained, in still fewer cells (a subset of only 6-8 cells in each primordium), and by stage 12 just the tip cell expresses Krüppel. This single cell is required to direct all cell proliferation in the tubule for which it is a part. Through cell proliferation and migration, lead by the tip cell, the Malpighian tubules elongate until they reach full size. The restriction of Krüppel requires expression of neurogenic genes Delta, Notch and neuralized. In the absence of members of the AS-C, tip cells fail to segregate. Without the activity of the proneural genes, both the mitogenic capacity and the neuronal characteristics of the tip cell are lost. Tip cell allocation would seem to be dependent on the same kinds of genetic interactions required for neurogenesis (Hoch, 1994).

achaete's status as a proneural gene is solidly implanted in the literature. But paradoxically, it is nearly impossible to think of a single gene in the neurogenic pathway that requires achaete for expression, that is, a gene whose promoter binds AC and is activated by that binding. The reason for this dearth of information is the redundancy of the four AS-C genes. Mutations in any one gene produces little phenotypic effect. A good indication of genes regulated by Achaete can be found at the daughterless site. Mutations in daughterless, which codes for Achaete's dimerization partner, eliminate expression of AC target genes.

AC does bind to the promoter of Enhancer of split complex genes and activates their transcription. Since E(spl)-C genes inhibit neurogenesis such binding presents yet another paradox. It should be remembered that achaete is expressed in cells that by default will remain in the epidermal cell layer, inhibited from adopting the neural fate. Thus achaete has well defined neurogenic (anti-neural) effects.

The neurogenic effects of achaete can be contrasted with its proneural effects, representing the primary function of achaete. achaete expression is enhanced in cells which will delaminate from the neuroectoderm and become neuroblasts. What genes are regulated by this enhanced expression of achaete? It is probably true that achaete is only required transiently, that is, it is required to stimulate the proneural fate by allocating cells to that fate but is not required to carry out the development of that fate. It remains for future achaete research to unravel what its positive effects are on neurogenesis, and for that matter, on the other developmental pathways in which it is involved.

GENE STRUCTURE

Bases in 5' UTR - 63

Exons - one

Bases in 3' UTR - 246; there are three polyadenylation signals

PROTEIN STRUCTURE

Amino Acids - 201

Structural Domains

Achaete has a central bHLH domain and a C-terminal acidic domain (Villares, 1987).

date revised: 2 June 2001 

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