Achaete protein first accumulates in the neuroectoderm in segmentally repeated clusters of 5 to 7 ectodermal cells in late stage 8. Two medial and two later clusters are found per hemisegment. One cell in each cluster comes to express achaete most intensely, and the future neuroblast delaminates. Next, the remaining cells lose achaete expression. Once the neuroblast has delaminated, and before it undergoes any divisions, ac expression in the neuroblast is extinguished. Thus four ac-positive cells on each side of the hemisegment delaminate and form a neuroblast. Only four of the first 10 neuroblasts express achaete. Neurogenic genes, including Notch and Delta, suppress ac expression in the non-segregating cells of each proneural cluster (Skeath, 1992a).

intermediate neuroblasts defective mutations were isolated by a mutagenesis screen for altered even-skipped (eve) expression in the CNS (J. Skeath and C.Q. Doe, unpubl.). The earliest ind mutant phenotype is observed in stage 7 embryonic neuroectoderm, when muscle segment homeobox expression occurs both in its normal locations in the dorsal columns and in the adjacent intermediate columns. Thus ind represses transcription of msh directly or indirectly within intermediate column neuroectoderm. Normally the ind and msh expression domains are adjacent but nonoverlapping, consistent with negative regulation of msh by ind. During the earliest stage of neurogenesis (stage 8 of development), wild-type embryos show expression of the proneural gene achaete in rows 3 and 7 of the neuroectoderm, with expression restricted to the ventral and dorsal columns and excluded from the intermediate column. ind expression in the intermediate column precisely abuts these clusters of achaete-expressing cells without overlapping them. In ind mutant embryos, derepression of achaete expression is observed within the intermediate column of neuroectoderm in rows 3 and 7 . This is consistent with a transformation of intermediate to dorsal neuroectoderm msh marker. It is concluded that ind represses msh and achaete gene expression directly or indirectly, and that ind is necessary for establishing proper intermediate-column identity within the neuroectoderm (Weiss, 1998).

The proneural genes achaete and scute and the segment polarity genes wingless and engrailed each have limited expression in only a few identifiable and stereotyped clusters of the head. For example, sc appears exclusively in a small part of the protocerebral domain, followed by transient expression in one to two protocerebral neuroblasts. wg is expressed in a total of three patches and engrailed is expressed in domains that are posterior and ventral to the adjacent wg domains. en is expressed in one patch in both the protocerebrum and the deuterocerebrum (Younossi-Hartenstein, 1996).

Loss of function mutations of the achaete-scute complex lead to a significant reduction of sensory bristles and glial cells. Genes within the complex affect gliogenesis with different strength and display some functional redundancy. Thus, neurogenesis and gliogenesis share the same genetic pathway. Despite these similarities, however, the mechanism of action of the achaete-scute complex seems to be different in the two processes. Neural precursors express products of the complex, therefore the role of these genes on neurogenesis is direct. However, markers specific to glial cells do not colocalize with products of the achaete-scute complex, showing that the complex affects gliogenesis indirectly. These observations lead to the hypothesis that gliogenesis is induced by the presence of sensory organ cells, either the precursor or its progeny (Giangrande, 1995).

Embryonic expression of achaete is also found in neural precursor cells of the gnathal region of the head: specifically, the procephalic region, the optic lobe, the clypeolabrum, and the stomodeum (Romani, 1987).

Achaete is required for the formation of the stomatogastric nervous system (SNS). The SNS consists of several peripheral ganglia which receive input from the brain and in turn innervate the muscles, pharynx, and gut. Precursors originate from the primordium of the foregut or stomodeum [Images]. Several subsets of precursors delaminate from the stomodeal epithelium as individual cells early in development. At later stages these precursors migrate to various locations where they differentiate as neurons. The function of achaete-scute and neurogenic genes is identical here to their function in the development of the ventral nerve cord (Hartenstein, 1996).

Defects in single minded mutants are characterized by the loss of the gene expression required for the proper formation of the ventral neurons and epidermis, and by a decrease in the spacing of longitudinal and commissural axon tracks. Molecular and cellular mechanisms for these defects were analyzed to elucidate the precise role of the CNS midline cells in proper patterning of the ventral neuroectoderm during embryonic neurogenesis. These analyses have shown that the ventral neuroectoderm in the sim mutant fails to carry out its proper formation and characteristic cell division cycle. This results in the loss of the dividing neuroectodermal cells that are located ventral to the CNS midline. The CNS midline cells are also required for the cell cycle-independent expression of the neural and epidermal markers. This indicates that the CNS midline cells are essential for the establishment and maintenance of the ventral epidermal and neuronal cell lineage by cell-cell interaction. Nevertheless, the CNS midline cells do not cause extensive cell death in the ventral neuroectoderm. This study indicates that the CNS midline cells play important roles in the coordination of the proper cell cycle progression and the correct identity determination of the adjacent ventral neuroectoderm along the dorsoventral axis (Chang, 2000).

sim is required for the proper development of ventral epidermis. This was demonstrated by the fact that the expression of the ventral ectodermal markers, enhancer trap line BP28 and otd and pnt genes, is missing in the sim mutant. Thus, this study focuses on the role of the CNS midline cells in the formation and identity determination of the ventral NBs during early neurogenesis. Initial NB formation and identity determination depend on the function of the achaete-scute (ac-sc) complex of proneural genes to provide a group of neuroectodermal cells with the competence to become a NB. To investigate whether the CNS midline cells affect the expression of a proneural gene that is essential for the initial NB formation and identity determination, ac expression pattern was analyzed by in situ hybridization. In wild-type stage 9 embryos, ac is expressed in the MP2 and S1 NBs 3-5, 7-1, and 7-4 in each hemisegment. In sim embryos, ac expression is absent in more than 90% of the examined hemisegments. This result indicates that the CNS midline cells are required for the expression of the proneural genes in the medial and lateral S1 NBs from the initial stage of neurogenesis (Chang, 2000).

Robustness and flexibility and the role of lateral inhibition in the neurogenic network

Many gene networks used by developing organisms have been conserved over long periods of evolutionary time. Why is that? A model is presented of the core neurogenic network in Drosophila. This model exhibits at least three related pattern-resolving behaviors that the real neurogenic network accomplishes during embryogenesis in Drosophila. Furthermore, the model exhibits these behaviors across a wide range of parameter values, with most of its parameters able to vary more than an order of magnitude while it still successfully forms these test patterns. With a single set of parameters, different initial conditions (prepatterns) can select between different behaviors in the network's repertoire. Two new measures are introduced for quantifying network robustness that mimic recombination and allelic divergence and these were used to reveal the shape of the domain in the parameter space in which the model functions. Lateral inhibition yields robustness to changes in prepatterns and a reconciliation of two divergent sets of experimental results is suggested. Finally, it is shown that, for this model, robustness confers functional flexibility. It is concluded that the neurogenic network is robust to changes in parameter values, which gives it the flexibility to make new patterns. The model also offers a possible resolution of a debate on the role of lateral inhibition in cell fate specification (Meir, 2002).

The experimental literature includes both support for, and refutation of, an important role for lateral inhibition in neural determination. The results of this analysis can account for both sets of experiments. If the prepattern that initiates neuroblast selection is well tuned, the prepattern plus a constant level of inhibition could select the winner, absent lateral inhibition. But lateral inhibition buffers the patterning against perturbations in the initial prepatterning (e.g., due to genetic or environmental variation, or 'developmental noise'). Seugnet (1997) reported that, with only constant production of Dl, 80% of proneural clusters developed normally, but 20% produced an extra NB. These experiments are interpreted to say that the prepattern is well tuned in most proneural clusters, but in 20%, either a poorly tuned prepattern or noise causes errors in the absence of lateral inhibition. This is a testable idea. One could remove lateral inhibition as Seugnet did. It would then be predicted that the embryo would be much more sensitive to hyper- and hypo-morphs in prepatterning genes such as extramachrochaete and hairy. It would also be predicted that such embryos would be more sensitive to mutations in genes within the network itself, such as missing or extra copies of Dl or N. The latter prediction is made because those mutations should change the threshold to which the prepattern is tuned. In the absence of lateral inhibition, a prepattern that was well tuned to the former threshold could not also be well tuned to the new threshold (Meir, 2002).

From these results, it is deduced that E(spl) greatly reduces the percentage of random parameter sets that enable lateral inhibition. It is believed this is because E(spl) acts as a homeostat. As the expression levels of the proneural genes (ac and sc) rise, their products activate E(spl). E(spl) then downregulates the proneural genes. As with the thermostat in a house, this negative feedback loop tends to keep the proneural genes at an intermediate level rather than allowing them to switch to either a high or low state. Both E(spl) autoinhibition, and to a lesser extent cis-Dl inhibition of N activation, help overcome this homeostat. On the face of it, this seems a strange design. The ac/sc network itself is a bistable switch that tends to go in the direction it is pushed and remain there. The switch and homeostat mechanisms are exact opposites. Removing the homeostat (the reduced model) makes it easier to find parameter sets that pass various tests (which all involve throwing the switch). Why incorporate counteracting mechanisms in the same circuit (Meir, 2002)?

It is, of course, possible that this is simply a vestige of the network's evolutionary history, with no design rationale. But electrical engineering suggests one possible advantage. An op-amp is a famous circuit that amplifies the difference between two inputs. Good op-amps can amplify a voltage difference more than a million-fold. Usually, though, engineers add a negative feedback circuit (that is, a homeostat). This greatly attenuates the gain but makes the amplifier much more stable; noise generated internally inside the op-amp will not affect the output signal. Reducing function to gain stability is common in other electrical circuits as well. These electrical circuits do not make good direct analogies to genetic networks, but the concept of adding negative feedback to increase stability might still apply. Perhaps the E(spl) homeostat reduces the network's sensitivity to developmental noise such as stochastic changes in transcription or translation rates, in the prepattern, or in the concentrations of modulators such as Da and Emc (Meir, 2002).

A related design benefit might be that the E(spl) homeostat prevents the network from switching individual cells on or off before the prepattern has a chance to decree the winner. A simple bistable switch consisting of ac and sc alone could not help but be thrown in one direction or the other by noise (as apparently takes place in C. elegans anchor cell specification). Adding E(spl) leads to a new, neither-on-nor-off steady state, which could enable the proneural switch to procrastinate until some extrinsic cue forces the system to choose one or the other switched state (Meir, 2002).


Both Achaete and Scute accumulate in well defined proneural clusters in imaginal discs. Their expression patterns largely overlap (Gomez-Skarmeta, 1995).

The spatial organization of epidermal structures in the embryo and adult fly constitutes a classicically modelled system for understanding how the two dimensional arrangement of particular cell types is generated. Adult legs are covered with sensory bristles, arranged in longitudinal rows in most segments. Two regulatory genes, hairy andachaete are chiefly responsible for setting up this periodic bristle pattern. achaete is expressed during pupal leg development in a dynamic pattern that develops into narrow longitudinal stripes, 3-4 cells in width, each of which represents a field of cells (proneural field) from which bristle precursor cells are selected. This pattern of gene expression foreshadows the adult bristle pattern established in part through the function of the hairy gene.

In pupal legs, hairy is expressed in four longitudinal stripes, located between every other pair of achaete stripes. In the absence of hairy function, achaete expression expands into the interstripe regions that normally express hairy, fusing the two achaete stripes and resulting in extra-wide stripes of achaete expression. This misexpression of achaete alters the fields of potential bristle precursor cells. This leads to the misalignment of bristle rows in the adult. hairy's role in patterning achaete expression is distinct from that in the wing, in which Hairy suppresses late expression of achaete but has no effect on the initial patterning of achaete expression. Thus, the leg bristle pattern is apparently regulated at two levels: a global regulation of hairy and achaete expression patterns partitioning the leg epidermis into striped zones, and later, a local regulation that involves refinement steps that may control the alignment and spacing of bristle cells within these zones (Orenic, 1993).

Effects of Mutation or Deletion

Loss of function in genes of the achaete-scute complex causes neural hypoplasia (too few cells). A lower than normal proportion of neuroblasts delaminate from the neuroectoderm and there is increased cell death at later stages. Increasing the copy number of AS-C alleles leads to an opposite effect: neural hyperplasia (Jimenez, 1990). Note that the effect of mutation and overexpression of proneural genes is exactly the opposite of that observed with the neurogenic genes Delta and Notch.

Early in development the Drosophila endoderm segregates into three non-neural cell types: the principle midgut epithelial cells, the adult midgut precursors, and the interstitial cell precursors. This process requires proneural and neurogenic genes. In neurogenic mutants the principle midgut epithelial cells are missing and the other two cell types develop in great excess. Consequently, the midgut epithelium does not form. In achaete-scute complex and daughterless mutants the interstitial cell precursors do not develop and the number of adult midgut precursors is strongly reduced. Development of the principle midgut epithelial cells and formation of the midgut epithelium is restored in neurogenic proneural double mutants. The neurogenic/proneural genes, in contrast to the neuroectoderm, are not expressed in small clusters of cells but initially homogeneously in the endoderm suggesting that no prepattern exists which determines the position of the segregating cells (Tepass, 1995).

To test whether proneural genes not normally expressed in a specific precursor can function in the specification of neural precursor identity in the CNS, proneural proteins not normally expressed in the MP2 precursor cell were ectopically expressed in MP2 precursors. Unlike other CNS precursors, MP2 normally expresses achaete and scute only. MP2 is unique in several respects: in its localization of Prospero protein to the nucleus soon after delamination, in the subsequent expression of the Fushi tarazu protein, and in the expression of the 22C10 antigen shortly before the MP2 division. lethal of scute, atonal and asense, each not normally expressed in MP2, were tested for their ability to specifiy MP2 when ectopically expressed in the MP2 precursor. All proneural proteins are similarly able to promote the segregation of a neural precursor at the MP2 position but show distinct capabilities in its specification. achaete/scute mutants have CNS precursors in only 17% of the hemisegments, as determined with antibodies against Hunchback, a general neuroblast marker. A significant fraction of the remaining precursors do not express nuclear Prospero or Fushi tarazu, and none of the precursors express appreciable amounts of 22C10 antigen. Targeted expression of either achaete or scute is sufficient to sustain formation of normal MP2 precursors. Targeted expression of either l'sc, ase or ato promotes delamination of a neural precurosor at the MP2 position, however this putatitive MP2 precursor expresses FTZ only when its development is prompted by l'sc or ato; expression of 22C10 occurs only in the case of ato. Thus Ato seems to be an efficient replacement for Ac/Sc. Targeted Ato results in both progeny neurons projecting to the anterior, instead of the normal situation in which one progeny neuron projects to the anterior and the second projects to the posterior. Thus Ato causes the misspecification of the MP2 lineage. It is concluded that totally normal specification of the MP2 fate can only be attained by the proneural genes achaete or scute (Parras, 1996).

klumpfuss shows genetic interactions with achaete, scute, lethal of scute and asense. l'sc is able to activate klu expression, but apparently only in the wing disc. There appears to be only a weak influence of the AS-C genes on klu expression, restricted to the wing area of the wing disc. However, the overall expression pattern of klu is largely independent of proneural genes. The assumption that SOPs enter apoptosis in klu mutants is supported by the observation of abundant cell death in other developing organs of klu mutants, like the legs. At certain bristle positions, such as that of the anterior sternopleura, klu is required during early bristle development immediately after proneural gene function, in order to allow a particular epidermal cell to develop as a SOP. It is suggested that klu is required only for initiation of bristle development, being downregulated once specification takes place (Klein, 1997).

The magnitude of segregating variation for bristle number in Drosophila exceeds that predicted from models of mutation-selection balance. To evaluate the hypothesis that genotype-environment interaction (GEI) maintains variation for bristle number in nature, the extent of GEI was quantitated for abdominal and sternopleural bristles among 98 recombinant inbred lines, derived from two homozygous laboratory strains, in three temperature environments. There is considerable GEI for both bristle traits. A genome-wide screen was conducted for quantitative trait loci (QTLs) affecting bristle number in each sex and temperature environment, using a dense (3.2-cM) marker map of polymorphic insertion sites of roo transposable elements. Nine sternopleural and 11 abdominal bristle number QTLs were detected. Significant GEI is exhibited by 14 QTLs, but there was heterogeneity among QTLs in their sensitivity to thermal and sexual environments. To further evaluate the hypothesis that GEI maintains variation for bristle number, estimates of allelic effects across environments at genetic loci affecting the traits are required. This level of resolution may be achievable for Drosophila bristle number because candidate loci affecting bristle development often map to the same location as bristle number QTL, including achaete-scute, scabrous, hairy, and Delta (Gurganus, 1998).

In mutants lacking both the Achaete-Scute Complex (ASC) 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 absent MD neurons and olfactory sensilla (amos). Both the ASC and Atonal physically interact with the protein Daughterless (Da). Since the solo-MD neurons that exist in ASC;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).

Amos is a novel bHLH protein and is expressed in proneural clusters. This expression is later restricted to SOP cells. 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 ASC;atonal mutants. These results suggested that amos is required for the formation of dbp and some dmd neurons, the neurons, which remain in ASC;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 ASC, atonal and amos play a role in the induction of different types of sensory neurons (Huang, 2000).

Changes in cell shape in the ventral neuroectoderm of Drosophila melanogaster depend on the activity of the achaete-scute complex genes

In the embryonic ventral neuroectoderm of Drosophila the proneural genes achaete, scute, and lethal of scute are expressed in clusters of cells from which the neuroblasts delaminate in a stereotyped orthogonal array. Analyses of the ventral neuroectoderm before and during delamination of the first two populations of neuroblasts show that cells in all regions of proneural gene activity change their form prior to delamination. Furthermore, the form changes in the neuroectodermal cells of embryos lacking the achaete-scute complex, of embryos mutant for the neurogenic gene Delta, and of embryos overexpressing l'sc, suggest that these genes are responsible for most of the morphological alterations observed (Stollewerk, 2000).

Almost all neuroectodermal cells are larger than the cells of the dorsal epidermal anlage (DEA). In comparison with the cells of the DEA in early stage 8 embryos the dorsoectodermal cells of mid-stage 8 embryos are clearly smaller. A comparison of the neurogenic region in early and mid-stage 8 embryos shows that the medial and intermediate regions of the ventral neuroectoderm (VNE) do not increase further in size whereas the lateral region enlarges considerably during this time. Due to these morphological changes the VNE can now be subdivided in relation to the cell sizes into three longitudinal regions on both sides of the midline: medial, intermediate, and lateral regions. In contrast to the cells of the medial and lateral regions, which now have approximately the same average values, the intermediate cells are smaller. Only 20% of all cells in the intermediate region are larger than the average, whereas 63% of the medial and 64% of the lateral cells exceed the average value. Most of the enlarged cells have a cuboidal shape. In every hemisegment the apical surfaces of two to four cells in the medial and lateral regions are very small (12-16 µm2) but expand basally to cover an area of 65-80 µm2. One or two cells of this shape are also located in the intermediate regions but are smaller basally (48-58 µm2) than the medial and lateral cells. The number and position of these cells suggest that they correspond to the delaminating neuroblasts; this was confirmed by staining the embryos with anti-Hunchback antibody, an early marker for neuroblasts (Stollewerk, 2000).

During delamination of the SI neuroblasts the neuroectodermal cells gradually decrease in size, with the exception of a few cells located close to the midline. The cells that remain enlarged are either elongated perpendicularly to the midline or have a rounded appearance. Basally, between neuroectoderm and mesoderm, large round cells are located that lose contact with the apical surface at about 60% EL. On the basis of their position and arrangement, as well as the analysis of embryos stained for Hunchback, these cells can be identified as the SI neuroblasts. Before delamination of the SII neuroblasts, cells in the intermediate region of the neuroectoderm increase in size. Most of the SII neuroblasts delaminate from this region, whereas only a few neuroblasts arise from the medial region, where enlarged cells can also be detected. After delamination of the SII neuroblasts the enlarged cells shrink once again, as revealed by double staining with anti-Hunchback antibody and phalloidin. Cells in all regions of the VNE increase in size again prior to delamination of the SIII neuroblasts. Thus, the VNE of wild-type embryos becomes morphologically distinguishable from the DEA shortly before delamination of the SI neuroblasts. At this point the cells of the DEA have already divided, and about two-thirds of all cells in the medial and the lateral regions have become enlarged so that the DEA and the VNE are clearly distinguishable due to differences in cell size. In addition, almost all cells of the intermediate region increase in size prior to delamination of the SII neuroblasts. These data are at odds with claims that only the neuroblasts enlarge prior to delamination, both in grasshopper and Drosophila (Stollewerk, 2000).

Is there a correlation between the activity of the ASC genes and the observed morphological changes? The results presented indicate that the ASC genes are not the only ones responsible for the morphological changes that occur before delamination of the SI neuroblasts. Although the number of enlarged cells corresponds closely to the number of cells that express the ASC genes at this time point, the lack of the ASC does not result in all cells remaining the same size. Whereas in the medial region of the VNE of Df (1)260-1 embryos (that is, those lacking the ASC) only about 50% of the cells are smaller in size than in the wild type, and the lateral region is most strongly affected in comparison to the medial and intermediate regions. Therefore the enlargement of the neuroectodermal cells depends to a varying degree on the activity of the ASC genes and is additionally influenced by other factors. However, a clear correlation can be seen prior to delamination of the SII neuroblasts. At this time almost all cells of the intermediate region increase in size, which coincides with the expression of l'sc in this region. Furthermore, analysis of the VNE of embryos lacking the ASC reveals that the intermediate cells do not become enlarged prior to delamination of the SII neuroblasts, suggesting that the observed morphological changes are due to the activity of the ASC genes at this point. In addition, the shrinkage of the cells that had enlarged during delamination of the SI and SII neuroblasts is correlated with the decrease in ASC gene expression in the VNE at these time points (Stollewerk, 2000).

Analysis of wild-type and Delta mutant embryos also suggests that the ASC genes are important for the maintenance of the morphology of the neuroectodermal cells. Despite the fact that the total area of the intermediate region does not change significantly between early and mid-stage 8, cell size changes can be detected in this region shortly before delamination of the SI neuroblasts. While 20% of the intermediate cells remain larger than the average, the cells that had an average cell size in the VNE of early stage 8 embryos now split into groups of smaller cells. The fact that the number of cells that remain larger than the average corresponds to the number of cells that express the ASC genes in the intermediate region suggests that the proneural genes are required to keep these cells enlarged. This view is confirmed by analyses of the VNE of Delta mutant embryos. In Delta mutant embryos all cells of a proneural cluster continue to express the proneural genes and become neuroblasts. This altered gene expression causes all cells of a proneural cluster to remain enlarged until proneural gene expression is turned off (Stollewerk, 2000).

A correlation between increase in cell size and ASC gene expression has also been shown by the analysis of embryos labeled for ac protein and embryos overexpressing l'sc. Area measurements reveal that 85% of all cells that express ac are enlarged in these embryos. The fact that not all ac-expressing cells are larger than the average at the time point analyzed may be due to the rapidity of the morphological changes (enlargement and shrinkage) that occur immediately before and during delamination of the neuroblasts. A clear influence of a proneural gene on the cell sizes in the VNE can be seen in embryos overexpressing l'sc: 45% more cells become enlarged in the intermediate region in comparison to the wild type. Only a minor increase in the numbers of enlarged cells can be seen in the medial and lateral regions, because two-thirds of these cells already express proneural genes. In addition, the high proneural gene activity in the VNE of embryos overexpressing l'sc causes the future neuroblasts to change their morphologies: they expand not only their basal but also their apical surfaces. These data clearly show that the ASC genes have an influence on the morphologies of the neuroectodermal cells (Stollewerk, 2000).

achaete, but not scute, is dispensable for the peripheral nervous system of Drosophila

The achaete-scute complex of Drosophila has been the focus of extensive genetic and developmental analysis. Of the four genes at this locus, achaete and scute appear to act redundantly to specify the peripheral nervous system. They share cis-regulatory elements and are co-expressed at the same locations. A mutation removing scute activity has been previously described; it causes a loss of some sensory bristles. Thus, when Scute is absent, the activity of achaete allows formation of the remaining bristles. However, all existing achaete mutants are rearrangements affecting regulatory sequences common to both achaete and scute. To determine the level of redundancy between the two genes, a P element approach was used to generate a null allele of achaete, which leaves scute and all cis-regulatory elements intact. The peripheral nervous system of achaete null mutant larvae and imagos lacks any detectable phenotype. However, when the levels of Scute are limiting, then some sensory organs are missing in achaete mutant flies. achaete and scute are thought to have arisen from a duplication event about 100 Myr ago. The difference between achaete and scute null flies is surprising and raises the question of the retention of both genes during the course of evolution (Marcellini, 2005; full text of article).

Senseless and Daughterless confer neuronal identity to epithelial cells in the Drosophila wing margin: Achaete and Scute are required for the survival of the mechanosensory neuron and support cells in these lineages

The basic helix-loop-helix (bHLH) proneural proteins Achaete and Scute cooperate with the class I bHLH protein Daughterless to specify the precursors of most sensory bristles in Drosophila. However, the mechanosensory bristles at the Drosophila wing margin have been reported to be unaffected by mutations that remove Achaete and Scute function. Indeed, the proneural gene(s) for these organs is not known. This study shows that the zinc-finger transcription factor Senseless, together with Daughterless, plays the proneural role for the wing margin mechanosensory precursors, whereas Achaete and Scute are required for the survival of the mechanosensory neuron and support cells in these lineages. Evidence is provided that Senseless and Daughterless physically interact and synergize in vivo and in transcription assays. Gain-of-function studies indicate that Senseless and Daughterless are sufficient to generate thoracic sensory organs (SOs) in the absence of achaete-scute gene complex function. However, analysis of senseless loss-of-function clones in the thorax implicates Senseless not in the primary SO precursor (pI) selection, but in the specification of pI progeny. Therefore, although Senseless and bHLH proneural proteins are employed during the development of all Drosophila bristles, they play fundamentally different roles in different subtypes of these organs. The data indicate that transcription factors other than bHLH proteins can also perform the proneural function in the Drosophila peripheral nervous system (Jafar-Nejad, 2006).

In 1978, García-Bellido and Santamaria reported that ac and sc are required for the generation of the majority of the Drosophila bristles. The large body of work that followed this discovery led to the realization that Ac and Sc are members of the bHLH proneural protein family, which are involved in early steps of neurogenesis in flies and vertebrates. Later, two other bHLH genes, atonal and amos, were shown to play the proneural role for almost all SOs that did not depend on Ac and Sc function, with the notable exception of the wing margin (WM) mechanosensory bristles (Garcia-Bellido, 1978). This study shows, based on multiple lines of evidence, that Sens plays the proneural role for these bristles: sens expression in the WM begins before the selection of mechanosensory pIs in a proneural cluster, similar to other proneural proteins; sens expression is upregulated in presumptive pIs and is downregulated in ectodermal cells, just like ac and sc expression is refined to pIs in thoracic proneural clusters; loss and gain of sens function result in loss and gain of SOs in the wing; and Sens synergizes with the Da protein in vivo and in transcription assays, and binds Da in a GST pull-down assay. Unexpectedly, overexpression of the anti-apoptotic protein P35 in the WM results in the generation of a large number of neurons along the PWM, uncovering the neural identity of the PWM bristle precursors. Similar to the AWM (anterior wing margin), the expression pattern and loss of-function phenotype of sens in the PWM (posterior wing margin) indicate a proneural role for sens for the PWM bristles as well. However, the neural potential of the PWM bristles is not realized in the wild-type situation because of apoptosis of the pI progeny, providing an example of the role of apoptotic machinery in diversifying the various sensory lineages. In summary, Sens satisfies all the genetic and developmental criteria for being a proneural protein for the WM bristles, and is the only zinc finger protein shown to play a proneural role in SO development in flies (Jafar-Nejad, 2006).

As for other proneural proteins, the proneural function of Sens requires the function of Da. Da serves as the binding partner for the bHLH proneural proteins to bind E-box sequences and is also able to bind DNA as homodimers. No function has been assigned to Da homodimers in Drosophila, largely because of the identification of tissue-specific bHLH proteins in most contexts in which Da functions. In the WM mechanosensory precursors, however, none of the known tissue-specific bHLH proneural proteins is expressed, suggesting a proneural role for Da homodimers. One might argue that there is probably an unknown dimerization partner for Da in these sensory precursors, and this possibility cannot be excluded. However, two groups have independently identified all Drosophila genes encoding bHLH proteins using database searches of the complete Drosophila genome and none of the newly identified bHLH proteins are predicted to be a transcriptional activator of the Ac-Sc or Atonal families. Also, none of these genes shows an embryonic expression pattern compatible with a proneural function for the CNS. Because da is required for mechanosensory organ formation, and as it can efficiently generate bristles in the absence of ASC, it is proposed that Da homodimers cooperate with Sens to endow neural identity to AWM mechanosensory organs and PWM bristle precursors. The physical interaction of these two proteins and the strong transcriptional synergy between them strongly favors a role in activating key target genes in SO development (Jafar-Nejad, 2006).

These data also reveal that Ac and Sc promote the survival of the WM mechanosensory neurons and support cells independently of pI selection. The more severe loss of neurons compared with support cells associated with the loss of Ac and Sc in sc10-1 suggests either that the neurons (or their precursors) are more sensitive to the lack of ac and sc function, or that the loss of support cells is secondary to the neuronal death. The observation that adding or removing one copy of wild-type sens strongly modifies the sensory lineage apoptosis observed in sc10-1 animals indicates that, in addition to a proneural function, Sens also plays an anti-apoptotic role in these cells; this is in agreement with many reports on the role of sens and its homologues in mammals and C. elegans in preventing apoptosis. It is interesting to note that although Ac and Sc are not detected in the PWM by antibody staining, P35 overexpression rescues many more neurons in the PWM of wildtype flies than in sc10-1 animals. This indicates a requirement for Ac and Sc in these cells (Jafar-Nejad, 2006).

During the third instar larval period, low levels of Sens are expressed in the proneural clusters along the AWM that will give rise to the pI cells of the AWM chemosensory bristles. Using in vivo and in vitro assays, it has been shown that low levels of Sens repress, and high levels of Sens activate, ac and sc expression in these proneural clusters, and thereby that Sens is involved in pI selection. Given the similar low-level expression of Sens in thoracic microchaetae proneural clusters and the severe loss of microchaetae in adult sens clones, it had been hypothesized that Sens also functions during proneural upregulation and in the selection of the microchaetae pIs. It was therefore surprising to find that microchaetae pI selection does not require Sens function. Data has been presented on the function of the adaptor protein Phyllopod and its relationship with Sens in microchaetae development. Sens was shown to be required for the function of Phyllopod in the pIs, as well as for timely downregulation of phyllopod expression in epidermal cells. This suggests a dual role for Sens in pIs and surrounding epidermal cells, in agreement with the binary switch model. In contrast, phyllopod expression can still be upregulated in single cells in sens mutant clones, suggesting that pI selection is not disrupted. This study now presents evidence that microchaetae pIs are indeed selected in sens clones and that they divide to generate progeny. However, the mutant pIs exhibit an abnormal division pattern, and a pIIa-to-pIIb transformation is observed, as evidenced by a gain of neurons at the expense of support cells. These data indicate that Sens regulates several aspects of microchaetae precursor development after the pIs are selected (Jafar-Nejad, 2006).

In summary, the normal development of all adult bristles in flies relies on the function of Ac and Sc, Da and Sens. The data indicate that despite the structural and functional similarities between various adult bristles, sens functions at four distinct steps in different lineages. First, in the WM mechanoreceptor and noninnervated lineages, very high levels of Wingless induce the expression of Sens, which assumes a true proneural role and specifies SO fate independently of the typical proneural proteins Ac and Sc. Second, in the WM chemosensory lineages, for which ac and sc are the proneural genes, Sens is required for pI selection, as evidenced by the observation that it represses proneural gene expression in ectodermal cells and activates proneural gene expression in presumptive pIs. Third, even though gain-of-function studies show that Sens is able to induce pI formation in the thorax in the absence of Ac and Sc function, it normally plays a later role in specification of the pIIa versus the pIIb of microchaetae lineages. Fourth, Sens is required for the survival of the pI progeny in the WM mechanosensory lineages. It was also found that ac and sc prevent apoptosis in this lineage independently of pI specification. Finally, the data suggest that a typical Da heterodimeric complex is not required during the formation of the WM mechanosensory and noninnervated bristle pIs. Hence, the cooperation between the same group of genes is adapted in different ways to ensure the proper development of various SOs (Jafar-Nejad, 2006).

The Sens homolog Gfi1 plays important roles in several developmental processes, including inner ear hair cell development, hematopoietic stem cell self-renewal rate, intestinal cell fate specification and neutrophil differentiation. Moreover, Gfi1 has an oncogenic potential and has been implicated in several human diseases, such as hereditary neutropenia, spinocerebellar ataxia type 1 and small cell lung carcinoma. Therefore, given the structural and functional similarities between Gfi1 and Sens, further analysis of the various aspects of Sens function in Drosophila SO development will continue to help unravel the mechanisms of Gfi1 function in health and disease (Jafar-Nejad, 2006).

achaete: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Targets of activity | Protein Interactions and Post-transcriptional Regulation | Developmental Biology | References

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

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