org Interactive Fly, Drosophila scute: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - scute

Synonyms - Hairy-wing (HW), T4, sisterless-b (sis-b)

Cytological map position - 1B3

Function - transcription factor

Keywords - proneural

Symbol - sc

FlyBase ID:FBgn0004170

Genetic map position - 1-0.0

Classification - bHLH

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene | HomoloGene | UniGene
Recent literature
Kiparaki, M., Zarifi, I. and Delidakis, C. (2015). bHLH proteins involved in Drosophila neurogenesis are mutually regulated at the level of stability. Nucleic Acids Res 43(5): 2543-59.. PubMed ID: 25694512
Summary:
Drosophila Sc is a prototypical proneural activator that heterodimerizes with the E-protein Daughterless (Da) and is antagonized by, among others, the E(spl) repressors. This study determined parameters that regulate Sc stability in Drosophila S2 cells. Sc was a very labile phosphoprotein and its turnover took place via at least three proteasome-dependent mechanisms. (1) When Sc was in excess of Da, its degradation was promoted via its transactivation domain (TAD). (2) In a DNA-bound Da/Sc heterodimer, Sc degradation was promoted via an SPTSS phosphorylation motif and the AD1 TAD of Da; Da was spared in the process. (3) When E(spl)m7 was expressed, it complexed with Sc or Da/Sc and promoted their degradation in a manner that required the corepressor Groucho and the Sc SPTSS motif. Da/Sc reciprocally promoted E(spl)m7 degradation. Since E(spl)m7 is a direct target of Notch, the mutual destabilization of Sc and E(spl) may contribute in part to the highly conserved anti-neural activity of Notch. Sc variants lacking the SPTSS motif were dramatically stabilized and were hyperactive in transgenic flies. These results propose a novel mechanism of regulation of neurogenesis, involving the stability of key players in the process.

Quan, X. J., Yuan, L., Tiberi, L., Claeys, A., De Geest, N., Yan, J., van der Kant, R., Xie, W. R., Klisch, T. J., Shymkowitz, J., Rousseau, F., Bollen, M., Beullens, M., Zoghbi, H. Y., Vanderhaeghen, P. and Hassan, B. A. (2016). Post-translational control of the temporal dynamics of transcription factor activity regulates neurogenesis. Cell 164: 460-475. PubMed ID: 26824657
Summary:
Neurogenesis is initiated by the transient expression of the highly conserved proneural proteins, bHLH transcriptional regulators. This study discovered a conserved post-translational switch governing the duration of proneural protein activity that is required for proper neuronal development. Phosphorylation of a single Serine at the same position in Scute and Atonal proneural proteins governs the transition from active to inactive forms by regulating DNA binding. The equivalent Neurogenin2 Threonine also regulates DNA binding and proneural activity in the developing mammalian neocortex. Using genome editing in Drosophila, this study showed that Atonal outlives its mRNA but is inactivated by phosphorylation. Inhibiting the phosphorylation of the conserved proneural Serine causes quantitative changes in expression dynamics and target gene expression resulting in neuronal number and fate defects. Strikingly, even a subtle change from Serine to Threonine appears to shift the duration of Atonal activity in vivo, resulting in neuronal fate defects.
Li, Y., Pang, Z., Huang, H., Wang, C., Cai, T. and Xi, R. (2017). Transcription factor antagonism controls enteroendocrine cell specification from intestinal stem cells. Sci Rep 7: 988. PubMed ID: 28428611
Summary:
The balanced maintenance and differentiation of local stem cells is required for homeostatic renewal of tissues. In the Drosophila midgut, the transcription factor Escargot (Esg) maintains undifferentiated states in intestinal stem cells, whereas the transcription factors Scute (Sc) and Prospero (Pros) promote enteroendocrine cell specification. However, the mechanism through which Esg and Sc/Pros coordinately regulate stem cell differentiation is unknown. By combining chromatin immunoprecipitation analysis with genetic studies, this study shows that both Esg and Sc bind to a common promoter region of pros. Moreover, antagonistic activity between Esg and Sc controls the expression status of Pros in stem cells, thereby, specifying whether stem cells remain undifferentiated or commit to enteroendocrine cell differentiation. These data therefore reveal transcription factor antagonism between Esg and Sc as a novel mechanism that underlies fate specification from intestinal stem cells in Drosophila.


BIOLOGICAL OVERVIEW

scute is a proneural gene, one of the four genes that comprise the achaete-scute complex (AS-C). Like the achaete gene, scute plays a role in neurogenesis, but scute also functions in sex determination.

Males have only one X chromosome (haplo-X), while females are diploid for X (diplo-X). In 1916 it was reported that sex in Drosophila is determined by the number of sex chromosomes (Bridges, 1916); two X chromosomes signal female development, and a single X chromosome signals male development. scute is one of a number of factors on the X chromosome responsible for sex determination.

How does this work, biochemically? Females, with double the X chromosomes, have twice the level of scute found in males. In females, the dosage is high enough to activate transcription of the gene Sex lethal (Sxl). In males, sxl remains silent, since scute levels are too low for sxl activation. Sex ratios and sex determination can also be modified as other genes regulate scute. For example, Daughterless binds to and activates scute, while Extramachrochaete will inactivate scute. Through such modifications or mutations it is possible the genotype will code for one sex yet the phenotype will have developed into the opposite sex.

Neither achaete nor lethal of scute can substitute for scute in sex determination, since only scute is transcribed early enough to function in this role. Sex-specific differences in SXL protein levels are already established in the syncytial blastoderm, where scute is uniformly transcribed. XX flies lacking scute die as embryos because they cannot activate their sxl gene. This phenomena explains the alternative name for scute: sisterless-b. Males survive because they undergo X hyperactivation in the absence of functional Sxl (Sxl blocks msl-2 translation, thus inhibiting dosage compensation in females).

The sensitivity of sex lethal transcription to gene dosage is remarkable, engendering a great deal of respect for the precision of transcription factors as regulators of the level of gene activation (Cline, 1988, Parkhurst, 1993, Steinmann-Zwicky, 1993, Erickson, 1993 and Deshpande, 1995).

The discussion now turns from sex determination to neurogenesis. To learn about the acquisition of neural fate by ectodermal cells, a very early sign of neural commitment in Drosophila has been analyzed, namely the specific accumulation of achaete-scute complex (AS-C) proneural proteins in the cell that becomes a sensory organ mother cell (SMC). An AS-C enhancer has been analyzed that directs expression specifically in SMCs. To delimit the sequences responsible for expression in SMCs, subfragments of a 3.7-kb fragment immediately upstream of scute were assayed for their ability to drive lacZ expression in wing discs. The necessary sequences are within a 356-bp fragment. This fragment specifically directed expression in SMCs. It also promotes expression in SMCs of other imaginal discs and of the embryonic PNS, but not in neuroectoderm neuroblasts. The SMC enhancer is shown to promote macrochaetae formation. Interspecific sequence comparisons and site-directed mutagenesis show the presence of several conserved motifs necessary for enhancer action, some of them binding sites for proneural proteins. The conserved sequences contain three E boxes: these are putative binding sites for bHLH proteins of the Achaete, Scute, and Daughterless (Da) type. The most proximal of the three is adjacent to an N box, a site that can be recognized by the E(spl)-C bHLH proteins. In addition, there are three copies of a motif reminiscent of a consensus binding site for the NF-kappaB family of transcription factors (named alpha1, alpha2, and alpha3), and three copies of a T-rich motif (termed beta1, beta2 and beta3) that does not fit with known protein-binding sequences. In spite of considerable effort, the NF-kappaB family member binding to the alpha motifs has not been identified (Culi, 1998).

To investigate the functional significance of these motifs, each was mutated, except for beta1, which has no clear counterpart in D. virilis, and the modified enhancers were assayed in vivo. Mutation of the E1 box and, to a slightly lower extent, the E2, alpha2, alpha3, and beta2 boxes greatly reduces enhancer function. In contrast, mutation of the E3, alpha1, beta3, or N boxes does not or only slightly modifies it. Simultaneous mutation of alpha2 and alpha3 does not decrease further the residual enhancer activity observed with only one mutated motif (Culi, 1998).

The fact that E boxes are required for the function of the SMC enhancer suggests that Sc and other proneural proteins bind to these sites and participate in sc activation. To examine this possibility, the ability of the Sc protein to bind to wild-type and mutated enhancers was analyzed. Binding occurs to the wild-type enhancer and it requires the bHLH protein Daughterless. At least two DNA/protein complexes with different mobilities are detected, which suggests that the enhancer has at least two binding sites for Sc/Daughterless heterodimers, consistent with the presence of two functional E boxes in the enhancer. In the absence of the E1 box, only one complex is detected, which indicates that E1 is a binding site for the Sc/Da heterodimer. Removal of the E2 box preferentially depletes the slower migrating complex. This suggests that E2 is also a binding site. Affinity for site E2 is lower than that for E1. Taken together, these results indicate that Sc/Da heterodimers interact with the E boxes important for enhancer activity. As proneural proteins promote transcriptional activation, it is most likely that the high accumulation of Sc in SMCs is attributable to sc self-stimulation. Given that Achaete/Da and Asense/Da dimers, also present in SMCs, recognize similar E boxes, they may also participate in sc activation in these cells (Culi, 1998).

Whether high levels of Sc are sufficient to trigger the SMC enhancer and drive sc self-stimulation in cells other than SMCs was also examined. This is not apparently the case, since strong, generalized accumulation of Sc provided by a UAS-sc gene driven by Gal4 line C-765 does not induce generalized expression of the SRV-lacZ transgene, not even in those cells of the proneural clusters located near SMCs that already contain elevated concentrations of endogenous Sc protein. beta-Galactosidase accumulation only occurs in isolated cells, which are most likely ectopic SMCs, as suggested by the many extra SOs that developed in adult flies. It is concluded that sc self-stimulation, mediated by the SMC enhancer, is specific to SMCs and has requirements in addition to a high level of Sc protein (Culi, 1998).

The above findings suggest that in order to promote transcription, the SMC enhancer requires, besides proneural proteins, either additional activating factors or the removal of inhibitors. Activating factors might interact with the alpha and beta boxes necessary for efficient enhancer action. To identify the minimum number of different motifs sufficient to constitute an SMC-specific enhancer, the enhancer activity of a synthetic oligonucleotide containing two E1 boxes and one alpha2 box were examined. It promotes beta-galactosidase accumulation only in SMCs, although a weak one. A four tandem repeat of the same oligonucleotide drives much stronger lacZ activity and this also occurs exclusively in SMCs. In contrast, a four tandem repeat with E1 boxes, but without alpha2 boxes, drives strong expression in many cells of proneural clusters. A four tandem repeat of alpha2 boxes without E1 boxes fails to drive expression. Hence, both E and alpha boxes are sufficient, in the context of the minienhancer, to constitute an SMC-specific enhancer (Culi, 1998).

Promoters of other genes share similar enhancer motifs. The asense sequences that direct expression in SMCs contain several E boxes necessary for optimal expression in SMCs. The corresponding DNA from D. virilis was sequenced and compared with that of D. melanogaster. Similar to the sc SMC-enhancer, the stretches of D. virilis conserved DNA contain E boxes, one N box, two alpha boxes, and one beta box, supporting the relevance of these boxes for SMC enhancer function. Moreover, the neurogenic gene Bearded, which is expressed in proneural clusters and SMCs, contains in its regulatory region one E box, necessary for its expression, and one motif identical to the alpha2 box. An evolutionarily conserved alpha box is also found within the regulatory region of rough, a homeobox gene important for restricting photoreceptor R8 specification (Culi, 1998).

Notch signaling prevents more than one of a proneural cluster from becoming SMCs. When the N pathway is not operative, as for instance in Su(H) larvae or in larvae harboring a Nts allele raised at a nonpermissive temperature, Ac and Sc proteins accumulate in many cells of proneural clusters at levels higher than in the wild type. The extra accumulation of Ac and Sc might be mediated by the cluster-specific enhancers, by the SMC enhancer (which under insufficient N signaling may promote expression in many cells of the proneural cluster as they become SMCs), or by both. To distinguish among these alternatives, an examination was carried out of the activity promoted by each type of enhancer, in both wild-type and in Nts discs. N inactivation allows the SMC enhancer to drive expression in many cells of proneural clusters. Expression can occur in contiguous cells, indicating the failure of lateral inhibition. In contrast, N inactivation does not modify the activity of the enhancer that drives expression in the vein L3 and TSM (twin sensilla of the wing margin) proneural clusters, although the accumulation of Sc in these clusters is increased. Hence, the SMC enhancer is responsible for most of the increased levels of proneural protein that occur in proneural clusters under insufficient N function (Culi, 1998).

N signaling, triggered by Ac-Sc in the emitter cell, promotes in the receptor cell the accumulation of E(spl)-C proteins, the main effectors of this signal. E(spl)-C proteins are detectable in proneural cluster cells, except for the SMCs. This correlates with the SMCs being the cells that signal maximally and inhibit their neighbors from acquiring the neural fate, while the SMCs themselves are not inhibited. Ectopic accumulation of E(spl)-C protein prevents SMCs from emerging, as detected by a neuralized enhancer trap line and the consequent absence of SOs in the adult fly. Overexpression of UAS-E(spl)-m8 or UAS-E(spl)-m7 transgenes driven by da-GAL4 or the C-253 GAL4 lines block the activity of the SMC enhancer and the development of the corresponding SOs. In contrast, either of these overexpressions allowed normal accumulation of Ac and Sc in proneural clusters despite the high levels of ectopic E(spl)-m8 mRNA, which are severalfold higher than those in the wild type. However, overexpression with presumably stronger GAL4 drivers does interfere with ac-sc expression in proneural clusters. Taken together these results indicate that the function of the SMC enhancer is more sensitive to E(spl)-C inhibition than are the proneural cluster enhancers, and suggest that the SMC enhancer is the main target of lateral inhibition mediated by the N pathway (Culi, 1998).

Does E(spl)-m8 bind to the SMC enhancer, given the inhibition of SMC enhancer function by E(spl)-C? E(spl)-m8 binds to the N box and, unexpectedly, also protects a broad region of the enhancer (nucleotides 142-182), which does not contain sequences that fit the E(spl)-C consensus binding site. Binding to an enhancer with a mutated N box is weaker, and binding to an enhancer without the N box and the second E(spl)-m8-binding site is undetectable. Remarkably, the removal of one or both binding sites does not modify the SMC specificity of the enhancer, as might be expected if these binding sites mediated the repression of enhancer function in response to N signaling. E(spl)-m8 is unable to bind to the synthetic SMC-specific minienhancer. These results were extended to other E(spl)-C proteins by verifying that [similar to E(spl)-m8] E(spl)-m5 binds to an oligonucleotide with the E1-N sequence, but not to oligonucleotides containing only E2 or E3 boxes. Thus, it is concluded that the E(spl)-C proteins restrict enhancer function to SMCs by a mechanism that does not require direct interaction with enhancer DNA. Thus the Enhancer of split bHLH proteins block the proneural gene self-stimulatory loop, possibly by antagonizing the action on the enhancer of the NF-kappaB-like factors or the proneural proteins. These data suggest a mechanism for SMC committment (Culi, 1998).

scute is required for development of enteroendocrine cells which in turn support intestinal stem-cell-mediated homeostasis in Drosophila

Intestinal stem cells in the adult Drosophila midgut are regulated by growth factors produced from the surrounding niche cells including enterocytes and visceral muscle. The role of the other major cell type, the secretory enteroendocrine (EE) cells, in regulating intestinal stem cells remains unclear. This study shows that newly eclosed scute loss-of-function mutant flies are completely devoid of enteroendocrine (EE) cells. These enteroendocrine cell-less flies have normal ingestion and fecundity but shorter lifespan. Moreover, in these newly eclosed mutant flies, the diet-stimulated midgut growth that depends on the insulin-like peptide 3 expression in the surrounding muscle is defective. The depletion of Tachykinin-producing enteroendocrine cells or knockdown of Tachykinin leads to a similar although less severe phenotype. These results establish that enteroendocrine cells serve as an important link between diet and visceral muscle expression of an insulin-like growth factor to stimulate intestinal stem cell proliferation and tissue growth (Amcheslavsky, 2014).

Previous evidence shows that adult midgut mutant clones that have all the AS-C genes deleted are defective in EE formation while overexpression of scute (sc) or asense (ase) is sufficient to increase EE formation (Bardin, 2010). Moreover, the Notch pathway with a downstream requirement of ase also regulates EE differentiation. To study the requirement of EEs in midgut homeostasis, attempts were made to delete all EEs by knocking down each of the AS-C transcripts using the ISC/EB driver esg-Gal4. The results show that sc RNAi was the only one that caused the loss of all EEs in the adult midgut. The esg-Gal4 driver is expressed in both larval and adult midguts, but the esg > sc RNAi larvae were normal while the newly eclosed adults had no EEs. Therefore, sc is likely required for all EE formation during metamorphosis when the adult midgut epithelium is reformed from precursors/stem cells (Amcheslavsky, 2014).

The sc6/sc10-1 hemizygous mutant adults were also completely devoid of midgut EEs, while other hemizygous combinations including sc1, sc3B, and sc5 were normal in terms of EE number. Df(1)sc10-1 is a small deficiency that has both ac and sc uncovered. sc1 and sc3B each contain a gypsy insertion in far-upstream regions of sc, while sc5 and sc6 are 1.3 and 17.4 kb deletions, respectively, in the sc 3' regulatory region. The sc6/sc10-1 combination may affect sc expression during midgut metamorphosis and thus the formation of all adult EEs (Amcheslavsky, 2014).

The atonal homolog 1 (Atoh1) is required for all secretory cell differentiation in mouse. However, esg-Gal4-driven atona; (ato) RNAi and the amorphic combination ato1/Df(3R)p13 showed normal EE formation. Nonetheless, older ato1/Df(3R)p13 flies exhibited a significantly lower increase of EE number, suggesting a role of ato in EE differentiation in adult flies (Amcheslavsky, 2014).

In sc RNAi guts, the mRNA expression of allatostatin (Ast), allatostatin C (AstC), Tachykinin (Tk), diuretic hormone (DH31), and neuropeptide F (NPF) was almost abolished, consistent with the absence of all EEs. On the other hand, the mRNA expression of the same peptide genes in heads showed no significant change. Even though the EEs and regulatory peptides were absent from the midgut, the flies were viable and showed no apparent morphological defects. There was no significant difference in the number of eggs laid and the number of pupae formed from control and sc RNAi flies, suggesting that the flies probably have sufficient nutrient uptake to support the major physiological task of reproduction. However, when the longevity of these animals was examined, the EE-less flies after sc RNAi showed significantly shorter lifespan. In addition, when the number of EEs was increased in adult flies by esgGal4;tubGal80ts (esgts)-driven sc overexpression, an even shorter lifespan was observed. These results suggest that a balanced number of EEs is essential for the long-term health of the animal. Moreover, there may be important physiological changes in these EE-less flies that are yet to be uncovered, such as reduced intestinal growth described in detail below (Amcheslavsky, 2014).

One of the phenotypic changes found for the sc RNAi/EE-less flies was that under normal feeding conditions, their midguts had a significantly narrower diameter than that of control midguts. When reared in poor nutrition of 1% sucrose, both wild-type (WT) and EE-less flies had thinner midguts. When reared in normal food, WT flies had substantially bigger midgut diameter, while EE-less flies had grown significantly less. The cross-section area of enterocytes in the EE-less midguts was smaller, suggesting that there is also a growth defect at the individual cell level (Amcheslavsky, 2014).

A series of experiments showed that ingestion of food dye by the sc RNAi/EE-less flies was not lower than control flies. The measurement of food intake by optical density (OD) of gut dye contents also showed similar ingestion. The measurement of excretion by counting colored deposits and visual examination of dye clearing from guts showed that there was no significant change in food passage. The normal fecundity also suggested that the mutant flies likely had absorbed sufficient nutrient for reproduction. Nonetheless, another phenotype that was detected was a substantial reduction of intestinal digestive enzyme activities including trypsin, chymotrypsin, aminopeptidase, and acetate esterase. These enzyme activities exhibit strong reduction after starvation of WT flies. The EE-less flies therefore have a physiological response as if they experience starvation although they are provided with a normal diet (Amcheslavsky, 2014).

A previous report has established that newly eclosed flies respond to nutrient availability by increasing ISC division that leads to a jump start of intestinal growth. When newly eclosed flies were fed on the poor diet of 1% sucrose, both WT and sc RNAi/EE-less guts had a very low number of p-H3-positive cells, which represent mitotic ISCs because ISCs are the only dividing cells in the adult midgut. When fed on normal diet, the WT guts had significantly higher p-H3 counts, but the sc RNAi/EE-less guts were consistently lower at all the time points. The sc6/sc10-1 hemizygous mutant combination exhibited a similarly lower mitotic activity on the normal diet (Amcheslavsky, 2014).

When possible signaling defects were investigated in the EE-less flies,in addition to other gut peptide mRNAs, the level of Dilp3 mRNA was also found to be highly decreased in these guts while the head Dilp3 was normal. This is somewhat surprising, because Dilp3 is expressed not in the epithelium or EEs but in the surrounding muscle. Dilp3 promoter-Gal4-driven upstream activating sequence (UAS)-GFP expression (Dilp3 > GFP) was used to visualize the expression in muscle. Both control and sc RNAi under this driver showed normal muscle GFP expression, demonstrating that sc does not function within the smooth muscle to regulate Dilp3 expression. The esg-Gal4 and Dilp3-Gal4, and the control UAS-GFP samples showed the expected expression in both midgut precursors and surrounding muscles. When these combined Gal4 drivers were used to drive sc RNAi, the smooth muscle GFP signal was clearly reduced. These guts also exhibited no Prospero staining and overall fewer cells with small sizes as expected from esg > sc RNAi (Amcheslavsky, 2014).

A previous report showed an increase of Dilp3 expression from the surrounding muscle in newly eclosed flies under a well-fed diet. This muscle Dilp3 expression precedes brain expression and is essential for the initial nutrient stimulated intestinal growth. The EE-less flies show similar growth and Dilp3 expression defects, suggesting that EE is a link between nutrient sensing and Dilp3 expression during this early growth phase (Amcheslavsky, 2014).

WT and AS-C deletion (scB57) mutant clones in adult midguts did not exhibit a difference in their cell numbers. Moreover, esgts > sc RNAi in adult flies for 3 days but did not undergo a decrease of mitotic count or EE number. Together, these results suggest that sc is not required directly in ISC for proliferation, and they imply that the ISC division defects observed in the sc mutant/EE-less flies is likely due to the loss of EEs. To investigate this idea further, the esgts > system to was used to up- and downshift the expression of sc at various time points, and the correlation of sc expression, EE number, and ISC mitotic activity were measured. The overexpression of sc after shifting to 29°C for a few days correlated with increased EE number, expression of gut peptides, and increased ISC activity. Then, flies were downshifted back to room temperature to allow the Gal80ts repressor to function again. The sc mRNA expression was quickly reduced within 2 days and remained low for 4 days. Although there was no working antibody to check the Sc protein stability, the expression of a probable downstream gene phyllopod showed the same up- and downregulation, revealing that Sc function returned to normal after the temperature downshift. Meanwhile, the number of Pros+ cells and p-H3 count remained higher after the downshift. Therefore, the number of EEs, but not sc mRNA or function, correlates with ISC mitotic activity (Amcheslavsky, 2014).

Another experiment that was independent of sc expression or expression in ISCs was performed. The antiapoptotic protein p35 was driven by the pros-Gal4 driver, which is expressed in a subset of EEs in the middle and posterior midgut. This resulted in a significant albeit smaller increase in EE number and a concomitant increase in mitotic activity, which was counted only in the middle and posterior midgut due to some EC expression of this driver in the anterior region. Therefore, the different approaches show consistent correlation between EE number and ISC division (Amcheslavsky, 2014).

Dilp3 expression was significantly although modestly increased in flies that had increased EE number after sc overexpression, similar to that observed in fed versus fasted flies. Whether Dilp3 was functionally important in this EE-driven mitotic activity was tested. Flies were generated that contained a ubiquitous driver with temperature controlled expression, i.e., tub-Gal80ts/UAS-sc; tub-Gal4/UAS-Dilp3RNAi. These fly guts showed a significantly lower number of p-H3+ cells than that in the tub-Gal80ts/UAS-sc; tub-Gal4/+ control flies. These results demonstrate that the EE-regulated ISC division is partly dependent on Dilp3. The expression of an activated insulin receptor by esg-Gal4 could highly increase midgut proliferation, and this effect was dominant over the loss of EEs after scRNAi, which is consistent with an important function of insulin signaling in the midgut (Amcheslavsky, 2014).

Normally hatched flies did not lower their EE number after esgts > sc RNAi, perhaps due to redundant function with other basic-helix-loop-helix proteins in adults. The expression of proapoptotic proteins by the prosts-Gal4 also could not reduce the EE number. Thus other drivers were screened and a Tk promoter Gal4 (Tk-Gal4) was identifed that had expression recapitulating the Tk staining pattern representing a subset of EEs. More importantly, when used to express the proapoptotic protein Reaper (Rpr), this driver caused a significant reduction in the EE number, Tk and Dilp3 mRNA, and mitotic count. The Tk-Gal4-driven expression of another proapoptotic protein, Hid, caused a less efficient killing of EEs and subsequently no reduction of p-H3 count. The knockdown of Tk itself by Tk-Gal4 also caused significant reduction of p-H3 count. A previous report revealed the expression by antibody staining of a Tk receptor (TkR86C) in visceral muscles, and the knockdown of TkR86C in smooth muscle by Dilp3-Gal4 or Mef2-Gal4 showed a modest but significant decrease in ISC proliferation. There was a concomitant reduction of Dilp3 mRNA in guts of all these experiments, while the head Dilp3 mRNA had no significant change in all these experiments. As a comparison, TkR99D or NPFR RNAi did not show the same consistent defect (Amcheslavsky, 2014).

In conclusion, this study has shown that among the AS-C genes, sc is the one essential for the formation of all adult midgut EEs and is probably required during metamorphosis when the midgut is reformed. In newly eclosed flies, EEs serve as a link between diet-stimulated Dilp3 expression in the visceral muscle and ISC proliferation. Depletion of Tk-expressing EEs caused similar Dilp3 expression and ISC proliferation defects, although the defects appeared to be less severe than that in the sc RNAi/EE-less guts. The results together suggest that Tk-expressing EEs are part of the EE population required for this regulatory circuit. The approach reported in this study has established the Drosophila midgut as a model to dissect the function of EEs in intestinal homeostasis and whole-animal physiology (Amcheslavsky, 2014).


GENE STRUCTURE

Bases in 5' UTR - 111 and 116 There are two transcription start sites, five nucleotides apart.

Exons - one

Bases in 3' UTR - 290


PROTEIN STRUCTURE

Amino Acids - 345

Structural Domains

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


EVOLUTIONARY HOMOLOGS

The lin-32 gene of C. elegans is an achaete-scute homolog, sufficient for specification of neuroblast fate (Zhou, 1995). Chicken achaete-scute homolog (cach-1) is one element in a multiple parallel pathway involving notochord or floor plate-derived signals for the specification and development of chick sympathetic neurons (Groves, 1995). Xenopus achaete-scute homolog (xash-3), when expressed with the promiscuous binding partner XE12, specifically activates the expression of neural genes in naive ectoderm (Ferreiro, 1994). Zebrafish achaete-scute homologs Zash-1a and Zash-1b are expressed in defined regions of the developing central nervous system. Their patterns of expression are modified by the cyclops mutant (Allende, 1994).

The study of achaete-scute (ac/sc) genes is a paradigm to understand the evolution and development of the arthropod nervous system. The ac/sc genes have been identified in the coleopteran insect species Tribolium castaneum. Two Tribolium ac/sc genes have been identified -- 1) a proneural achaete-scute homolog (Tc-ASH) and 2) asense (Tc-ase), a neural precursor gene that reside in a gene complex. These genes reside 55 kb apart from each other and thus define the Tribolium ac/sc complex. Focusing on the embryonic central nervous system it is found that Tc ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies it has been shown that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. These studies, however, do not support a role for Tc-ASH in specifying the individual fate of neural precursors, suggesting that the ability of ac and sc to separately regulate this process may represent a recent evolutionary specialization within the Diptera. Furthermore, it is found that Tc-ase is expressed in all neural precursors, suggesting an important and conserved role for asense genes in insect nervous system development. This analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development (Wheeler, 2003).

Homologs of ac/sc genes have been described in a number of insect and non-insect species. These data support and augment the model in which the last common ancestor of arthropods contained a single prototypical ac/sc gene that carried out both proneural and asense functions. In support of this model, the sole Hydra ac/sc gene, CnASH, does not group with either the proneural or asense genes in phylogenetic analysis and contains motifs indicative of both the proneural and asense genes. In addition, phylogenetic analysis of the two ac/sc genes found in a spider, the chelicerate Cupiennius salei, indicates these genes are more closely related to each other than any other ac/sc genes. These data raise the possibility that a single ancestral ac/sc gene underwent independent duplication events in chelicerates and insects. Given this possibility, it is interesting that one of the Cupiennius ac/sc genes, Cs-ASH1, exhibits a proneural-like expression pattern and appears to carry out a proneural-like function and the other, Cs-ASH2, exhibits an asense-like expression pattern and appears to carry out an asense-like function. These data suggest that independent duplications of an ancestral ac/sc gene have independently given rise to proneural-like and asense-like functions in the chelicerate and insect groups. Alternatively, phylogenetic analysis may inappropriately partition chelicerate ac/sc genes from insect ac/sc genes because of evolutionary selection for species-specific amino acid changes in chelicerate as compared to insect proteins (Wheeler, 2003).

Within the insects, it has become clear that serial duplications of a single proneural ac/sc gene gave rise to multiple proneural ac/sc genes in the more derived groups. For example, Tribolium and the basal dipteran Anopheles each contain a single proneural ac/sc gene. However, Ceratitis, a more derived dipteran, contains two proneural ac/sc genes. Thus, a duplication of the ancestral proneural ac/sc gene occurred within the dipteran lineage after the divergence of Ceratitis and Anopheles. The presence of three proneural ac/sc genes in Drosophila, a highly derived genus of dipterans, identifies a second duplication event. The simplest explanation for these data is that the second duplication occurred after the divergence of Drosophila and Ceratitis. However, comparative sequence analysis suggests this duplication may have preceded the divergence of Drosophila and Ceratitis and that Ceratitis has either lost an ac/sc homolog or it has yet to be identified (Wheeler, 2003).

In contrast to the plasticity in proneural ac/sc genes within insects, asense genes appear to be well conserved. A single asense gene exists in Tribolium and Anopheles as well as in the derived dipteran species Ceratitis and Drosophila. In addition, Cupiennius contains a single non-orthologous ac/sc gene with asense-like properties (Cs-ASH2). Thus, the potential that the asense function evolved independently in insects and chelicerates suggests an important role for the asense function in arthropod neural development (Wheeler, 2003).

The existence of ac/sc genes in complexes in Drosophila, Anopheles and Tribolium suggests that this genomic arrangement has been conserved in most if not all holometabolous insects. Shared cis-regulatory regions probably explain why proneural ac/sc genes remain linked in insects and perhaps other species. However, this does not explain why asense is retained in the ac/sc complex as the regulation of asense expression is distinct from that of the proneural ac/sc genes. This phenomenon may be explained by the presence of proneural ac/sc gene cis-regulatory regions surrounding the asense gene. In this model, chromosomal rearrangements that separate asense from the ac/sc complex would probably disrupt proneural ac/sc gene expression and neural precursor formation, thus leading to decreased viability. Consistent with this idea, cis-regulatory regions that drive proneural ac/sc gene expression in the Drosophila PNS appear to flank the ase gene. Thus, the modular cis-regulatory regions that control proneural ac/sc gene expression may also be responsible for the evolutionary conservation of the ac/sc complex. Alternatively, other as yet unidentified genomic forces may preserve the linkage between asense and proneural ac/sc genes (Wheeler, 2003).

These findings raise a number of interesting points. (1) They highlight the potential for evolutionary plasticity of ac/sc genes. Significant changes in ac/sc gene number and expression have occurred over relatively short evolutionary distances and have been correlated with modifications to neural pattern and/or gene function. For example, alterations to ac/sc gene expression in Diptera appear to account for the different patterns of sensory organs found on dipteran species. In addition, data on the role of proneural genes in MP2 fate specification suggest that the increase in ac/sc gene number in Drosophila appears to have facilitated the evolution of new developmental roles for ac and sc in this lineage. (2) The possibility that independent duplication events in chelicerates and insects each gave rise to proneural-like and asense-like genes, indicates that dividing these genetic functions between two genes may be developmentally advantageous. (3) The hypothesis that the last common ancestor of all arthropods contained a single ancestral ac/sc gene suggests it may be possible to identify direct descendants of the prototypical ac/sc gene in extant basal members of each arthropod group. The recent emphasis on the development of genomic resources in non-model organisms should greatly aid progress along this line of inquiry. Thus, continued analysis of ac/sc gene expression, organization and function in arthropods should provide additional insight into the genetic basis of the development and evolution of nervous system pattern (Wheeler, 2003).

The work presented in this paper together with studies on ac/sc gene function in Drosophila provide strong evidence that serial duplications of proneural ac/sc genes in the dipteran lineage led to the diversification of proneural ac/sc gene function in Drosophila. In Drosophila, ac and sc carry out functions distinct from l'sc in specifying the individual fate of the MP2 precursor. Tc-ASH can function in Drosophila as a proneural gene but like Drosophila l'sc fails to specify efficiently the MP2 fate in the CNS. Together these results suggest the ability of ac and sc to specify MP2 fate in Drosophila arose after the divergence of Drosophila and Tribolium. These data provide an example whereby a subset of duplicated genes has evolved a new genetic function while the entire set of duplicate genes has retained the ancestral function (Wheeler, 2003).

In addition to functional changes, the generation of multiple proneural ac/sc genes in the insects was paralleled by modifications to the expression profiles of these genes. In Anopheles (a basal dipteran), and Tribolium a single proneural ac/sc gene is expressed in all CNS proneural clusters. In more derived Diptera the presence of multiple ac/sc genes allows for more complex proneural ac/sc gene expression patterns. For example, Ceratitis contains two proneural ac/sc genes, l'sc and sc; l'sc is expressed in all CNS proneural clusters while sc is expressed in a subset of these clusters. In Drosophila, ac and sc are expressed in the identical pattern of proneural clusters and their expression is largely complementary to that of l'sc. The sum of proneural ac/sc expression in each species then marks all CNS proneural clusters despite differences in the expression pattern of individual proneural ac/sc genes. Thus, in Drosophila, the complete expression pattern of proneural ac/sc genes is divided between the largely complementary expression profiles of ac and sc relative to l'sc. The division of labor between proneural ac/sc genes in Drosophila has resulted in mutually exclusive expression patterns for ac and sc relative to l'sc in proneural clusters like MP2. This spatial separation of proneural gene expression probably facilitated the potential for ac and sc to acquire developmental functions distinct from l'sc (Wheeler, 2003).

Together this work and that of others on arthropod ac/sc genes highlights the utility of studying ac/sc genes in elucidating the genetic basis of the development and evolution of arthropod nervous system pattern. These studies illustrate the dynamic nature of ac/sc gene number, expression and function over a relatively short evolutionary time. Based on this, future work on ac/sc genes in additional arthropod species should continue to provide insight into the molecular basis of the evolution of arthropod nervous system development (Wheeler, 2003).

The stereotyped positioning of sensory bristles in Drosophila has been shown to result from complex spatiotemporal regulation of the proneural achaete-scute genes, that relies on an array of cis-regulatory elements and spatially restricted transcriptional activators such as Pannier. Other species of derived schizophoran Diptera have equally stereotyped, but different, bristle patterns. Divergence of bristle patterns could arise from changes in the expression pattern of proneural genes, resulting from evolution of the cis-regulatory sequences and/or altered expression patterns of transcriptional regulators. Described in this study is the isolation of achaete-scute homologs in Ceratitis capitata, a species of acalyptrate Schizophora whose bristle pattern differs slightly from that of Drosophila. At least three genes, scute, lethal of scute and asense have been conserved, thus demonstrating that gene duplication within the achaete-scute complex preceded the separation of the families Drosophilidae and Tephritidae, whose common ancestor goes back more than 100 million years. The expression patterns of these genes provide evidence for conservation of many cis-regulatory elements as well as a common origin for the stereotyped patterns seen on the scutum of many Schizophora. Some aspects of the transcriptional regulation have changed, however, and correlate in the notum with differences in the bristle pattern. The Ceratitis pannier gene was isolated and displays a conserved expression domain in the notum (Wulbeck, 2000).

The pattern on the scutum of many species of schizophoran flies is thought to be derived from a basic arrangement of four rows of bristles that appear to be in homologous positions in different flies. This suggests that an ancestor, common to most of today's species, already possessed these four rows. While the Calyptrata generally bear rows of macrochaetes extending the full length of the scutum, the Acalyptrata display only a subset of bristles from some or all rows, a feature that is thought to be derived. Therefore, another possibility is that the positional enhancer elements of the D. melanogaster AS-C originate from ancient regulatory elements whose function may have been to drive ac-sc expression in four stripe-like domains corresponding to the four rows of notal bristles. The fact that the two dorsocentral precursors in D. melanogaster arise sequentially from a single cluster may reflect their common origin from the same row. The acrostichal row is absent in D. melanogaster, but is represented by a single bristle, the prescutellar, in Ceratitis. The precursor for this bristle forms within a discrete dorsally located PNC, clearly separate from the DC cluster, consistent with the hypothesis that this bristle has a different origin (Wulbeck, 2000).

Many families of Schizophora retain the prescutellar bristle, so if the prescutellar PNC relies on a discrete regulatory element this is likely to be ancient. It may not be present in D. melanogaster. However, this particular bristle is of special interest because it is conserved even within the family Drosophilidae itself. Indeed the two subfamilies of Drosophilidae, the Steganinae and the Drosophilinae, are classified on the basis of the presence or absence respectively, of this bristle. Absence of the prescutellar bristle, in the Drosophilinae, is attributable to a loss during the course of the evolutionary history of this taxon. Interestingly, one genus of the Drosophilinae, Scaptodrosophila, does carry a prescutellar bristle, the presence of which is considered to be a secondary gain. If so, then the information required for its differentiation, perhaps including a discrete regulatory sequence, may have been retained in a latent form in some species of Drosophilinae (Wulbeck, 2000).

The Drosophila gene pannier (pnr) has been assigned to a new class of selector genes. It specifies pattern in the dorsal body. On the dorsal notum it is expressed in a broad medial domain and directly regulates transcription of the achaete-scute (ac-sc) genes driving their expression in small discrete clusters within this domain at the sites of each future bristle. This spatial resolution is achieved through modulation of Pnr activity by specific co-factors and by a number of discrete cis-regulatory enhancers in the ac-sc gene complex. Homologs of pnr and ac-sc have been isolated in Anopheles gambiae, a basal species of Diptera that diverged from Drosophila melanogaster (Dm) about 200 million years ago, and their expression patterns were examined. An ac-sc homolog of Anopheles, Ag-ASH, is expressed on the dorsal medial notum at the sites where sensory organs emerge in several domains that are identical to those of the pnr homolog, Ag-pnr. This suggests that activation of Ag-ASH by Ag-Pnr has been conserved. Indeed, when expressed in Drosophila, Ag-pnr is able to mimic the effects of ectopic expression of Dm-pnr and induce ectopic bristles. These results are discussed in the context of the gene duplication events and the acquisition of a modular promoter, that may have occurred at different times in the lineage leading to derived species such as Drosophila. The bristle pattern of Anopheles correlates in a novel fashion with the expression domains of Ag-pnr/Ag-ASH. While precursors for the sensory scales can arise anywhere within the expression domains, bristle precursors arise exclusively along the borders. This points to the existence of specific positional information along the borders, and suggests that Ag-pnr specifies pattern in the medial, dorsal notum, as in Drosophila, but via a different mechanism (Wülbeck, 2002).

Ag-ASH appears closest to Drosophila l'sc. Sequence analysis has revealed that 81% of the amino acids in the bHLH domain are identical to those of the Drosophila l'sc protein. Outside of this functional domain, amino acid sequence conservation is low (ranging from 20%-27% for the amino (N)-terminal portion to 25%-38% for the carboxy (C)-terminal part). A single stretch of 15 conserved amino acids, which appears to be restricted to insect ac-sc proteins, can be seen at the C terminus. The central tyrosine of this sequence has changed in the butterfly Precis coenia (Wülbeck, 2002).

The screening procedure used allowed the isolation of a single Anopheles ASC homolog, Ag-ASH, but examination of the recently published genome of this species reveals the existence of an asense gene. Ag-ASH is closest to Drosophila l'sc, but may be representative of an ancestral gene, which was present prior to the duplication events that gave rise to l'sc, sc and ac. This may have taken place after separation of the Nematocera (including the mosquitoes) and Brachycera (including Drosophila and Ceratitis), two lineages that diverged about 200 million years ago. A single ASC homolog has been described in the butterfly Precis coenia. When expressed in Drosophila, Ag-ASH has a conserved and strong, proneural function (Wülbeck, 2002).

The pnr gene of Drosophila comprises two zinc fingers characteristic of the GATA family of transcription proteins, and a C-terminal domain bearing two alpha helices. The protein contains two zinc fingers that are very strongly conserved. The proteins are, however, quite divergent in the C-terminal domain. The proteins of Ceratitis and Anopheles carry a single alpha helix, in contrast to the two in Drosophila (Wülbeck, 2002).

In Drosophila, pnr is expressed in a conserved broad medial domain but activates ac and sc in discrete proneural clusters within this domain. The ac-sc genes of Drosophila are organized into a complex containing multiple enhancer regions, each of which independently regulates expression in one or a small number of proneural clusters. In this species three proneural clusters arise in the domain of pnr expression and Pnr has been shown to directly activate ac-sc in the dorso-central cluster, through binding to a cis-regulatory sequence just upstream of ac. It is not entirely understood how the broad domain of Pnr is translated into the small clusters of ac-sc expression, but this is at least in part achieved through interaction of Pnr with regulatory co-factors. The spatially complex expression of sc in Calliphora and Ceratitis suggests that the ASC genes of these species may also have modular promoters. Furthermore, the expression domain of pnr in these species is conserved with that of Drosophila (Wülbeck, 2002).

In contrast, the regulatory interactions between the two genes appear to have diverged in Anopheles since Ag-ASH is expressed in all Ag-pnr-expressing cells. The common domains of expression suggest that Ag-Pnr may activate Ag-ASH in every cell in which it is expressed, in a simple straightforward fashion. This observation raises two possibilities. (1)for the regulation of Ag-ASH, Ag-Pnr may not associate with the various co-factors known to modulate its activity in Drosophila; (2) in order to be activated in all Ag-pnr-expressing cells, Ag-ASH would not need to have a modular promoter structure like that of the Drosophila locus, and could have a less complex organization. If so, the acquisition of position-specific enhancers may have occurred after the separation of Nematocera and Brachycera, at a time when further gene duplication events appear to have taken place. In addition, modulation of Pnr activity through the use of different co-factors may have accompanied the acquisition of cis-regulatory enhancer sequences in the lineage leading to Drosophila (Wülbeck, 2002).

Despite the inferred simple regulatory interaction between Ag-Pnr and Ag-ASH, it is remarkable that the effects of mis-expression of Ag-pnr in Drosophila are almost identical to those caused by mis-expression of Dm-pnr. For example, ectopic expression of either Dm-pnr or Ag-pnr on the lateral notum, causes the development of a tuft of ectopic dorso-central bristles. This is due to an expansion of the activity of the dorso-central enhancer element known to be regulated by Dm-Pnr. This result suggests that Ag-Pnr is able to recognise the relevant regulatory modules of the Drosophila ASC promoter; this may indicate that these enhancers are derived from an ancestral regulatory sequence also present in Anopheles. Alternatively, a number of regulatory modules may in fact be present in the Anopheles promoter and govern expression in the various domains on the notum. Further understanding of the structure and regulation of Ag-ASH will require investigation of regulatory sequences from this organism. The ectopic expression assay also indicates that Ag-Pnr is probably able to associate with Drosophila co-factors such as U-shaped and Chip. It has been shown that the N-terminal zinc finger of Dm-Pnr associates with U-shaped, while two C-terminal helical structures are components mediating association with Chip. The two zinc fingers are strongly conserved in Ag-Pnr, and there is a single alpha helix. Thus Ag-Pnr appears to contain the relevant binding regions for these two factors. This complexity of the Ag-pnr protein may indicate association with endogenous co-factors, perhaps in a different tissue (Wülbeck, 2002).

In Drosophila, it has been demonstrated, that pnr and the iro-C genes are selector genes involved in the subdivision of the dorsal component of segments of the head, thorax and abdomen of the adult into medial and lateral domains. While pnr regulates the pattern of the medial domain of the dorsal mesonotum, patterning of the lateral half is regulated by the iro-C genes. Thus, when either Dm-pnr or Ag-pnr is expressed from an early stage in the entire notum of Drosophila, only structures corresponding to the medial notum are formed: the lateral region fails to develop. Ubiquitous expression specifies a single medial domain thought to include cells originally destined to form the lateral region. In addition Ag-pnr is expressed in the medial, but not the lateral, mesonotum of Anopheles, consistent with a conserved function in the medial domain. Thus the selector gene function of pnr may have been conserved. The function of proteins of other selector genes of Anopheles, such as engrailed, has been shown to be conserved (Wülbeck, 2002).

The precursors of the sensory scales on the notum of Anopheles are distributed in a random fashion within the domains of expression of Ag-pnr/Ag-ASH. In some respects the sensory scales resemble the small bristles or microchaetes of cyclorraphous Diptera, which are often randomly distributed although sometimes lined up into rows. However, in the latter species they arise later than the large bristles or macrochaetes, from a second period of ac-sc expression, and are consequently positioned closer to one another than are the macrochaetes. In contrast, the precursors of scales and bristles appear to arise simultaneously in Anopheles, which is consistent with the fact that they are equidistant from each other in the imago. In cyclorraphous flies, the macrochaete pattern is the result of spatially complex sc (ac) expression: one (or a very small number) of bristle(s) develops from each small cluster (or stripe) of sc (ac) expression. In Anopheles, however, the patterning mechanism is different: remarkably, the precursors of the bristles are exclusively positioned along the borders of the expression domains. Thus the positions of the rows of AC and DC bristles, as well as the PST and SC bristles, are coincident with the borders of the four domains of Ag-pnr/Ag-ASH expression. This suggests that the boundaries of Ag-ASH/Ag-pnr expression convey specific positional information causing neural precursors to develop into bristles rather than sensory scales (Wülbeck, 2002).

Two observations in Drosophila may be relevant to this phenomenon. (1) Some of the macrochaete precursors arise from the edges of the corresponding proneural clusters of ac-sc expression, an observation that has been linked to distance from the source of the signaling molecules Wingless and Decapentaplegic. The expression pattern of these molecules in Anopheles is not yet known. (2) It has been demonstrated that the border between pnr-expressing and non-expressing cells does in fact display special properties. Cells of the medial domain manifest unique adhesive characteristics that prevent them from mixing with cells of the lateral domain. So, as for compartment boundaries, this interface between cells expressing pnr and those expressing iro may be an important patterning boundary. It has indeed been shown to be required for the growth and patterning of the Drosophila eye. Interestingly, the five macrochaetes on the medial notum of Drosophila are pnr-dependent, and they are all positioned on the lateral border of the domain of pnr expression. Experimentally contrived expression of ac-sc inside the pnr domain, however, results in the formation of ectopic macrochaetes, indicating that macrochaete formation in Drosophila, is not dependent on special properties at the border. Furthermore the prescutellar bristle of Ceratitis and the AC row of bristles in Calliphora, arise from sc-expressing cells situated inside the pnr expression domain (Wülbeck, 2002).

Although the bristles on the notum of Anopheles are aligned into rows, the number and position of bristles within a row varies greatly between individuals, a feature that is thought to be ancestral. In contrast, species of cyclorraphous Schizophora have very defined rows in which the number and position of bristles varies little if at all. The stereotyped notal bristle patterns of species such as Drosophila are thought to be derived from an ancestral pattern of four longitudinal rows of bristles, still present in many extant species of Schizophora. These include the AC and DC bristle rows that are in the medial domain of the notum. So, for example, the two DC bristles of Drosophila would be vestiges of the DC row. Whether the rows of bristles seen in some families of Nematocera such as the Culicidae, are in any way related by ancestry to the four rows of Schizophoran flies, is more difficult to assess. Nevertheless the DC row of Anopheles is positioned on the lateral border of the Ag-pnr expression domain, as in Ceratitis, Calliphora and Drosophila, which may indicate a common origin for this row. If so, this would mean that an ancestral pattern of bristle rows was already present in a common ancestor of the Brachycera and at least some families of Nematocera (Wülbeck, 2002).

These results indicate a conserved function for pnr in the regulation of the bristle pattern on the medial notum. This argues in favour of an ancient role for pnr as a selector gene specifying the dorsal medial pattern. The nature of the regulatory interactions between Pnr and its target genes ac-sc appears to have changed, however, over evolutionary time. It is hypothesized that in Culicid mosquitoes, which have fewer ac-sc genes, the regulatory regions of this locus may not be organized in a modular fashion. Evolution of the stereotyped bristle patterns characteristic of species such as Drosophila and Ceratitis may have entailed the acquisition of a number of additional factors. These would include gene duplication within the ASC and the co-option of cis-regulatory sequences. Co-factors for Pnr, such as Ush and Chip, are also likely to have been co-opted for use in constructing the notal pattern at a later evolutionary stage, although the current results suggest that Ag-Pnr has the requisite domains for association with these proteins. In the lineage leading to Drosophila, these different levels of regulation might have been superimposed onto an ancestral patterning mechanism, similar to that of Anopheles, at different times in the 200 million years separating Drosophila from the Nematocera (Wülbeck, 2002).

Temporal shifts in the expression of regulatory genes, relative to other events taking place during development, can result in changes in morphology. Such transcriptional heterochrony can introduce dramatic morphological changes that involve rather few genetic events and so has the potential to cause rapid changes during evolution. Stereotyped species-specific bristle patterns on the notum of higher Diptera correlate with changes in the spatial regulation of scute expression. scute encodes a proneural gene required for the development of sensory bristle precursors and is expressed before pupation in discrete domains on the presumptive notum at sites where the macrochaete precursors arise. Thus, for Ceratitis capitata and Calliphora vicina, species separated from Drosophila melanogaster by about 80 and 100 million years respectively, the domains of sc expression differ. In all three species, a second phase of ubiquitous sc expression, after pupation, precedes formation of the microchaete precursors (Skaer, 2002).

Higher Diptera of the Brachycera suborder often display two distinct categories of bristles of very different size: macrochaetes and microchaetes (taxonomists refer to bristles and hairs, respectively). Macrochaetes are generally absent in the more basal suborder Nematocera. Many families of Brachycera bear macrochaetes but, interestingly, most families include species devoid of them. This means that either the macrochaetes have appeared independently several times in different lineages during the history of the Brachycera or they arose once and have been lost many times since. On the notum, macrochaetes are invariably arranged in longitudinal rows or in stereotyped patterns. Comparison of species within the derived Schizophora taxon suggests that changes in the positions of macrochaetes have taken place only gradually. Closely related species tend to have closely related patterns, whereas phylogenetically more distant species may display greater differences. There has been a gradual tendency towards the evolution of stereotyped patterns. Neuronal specificity of macrochaetes is dependent on their position, suggesting that bristle position is important for behaviour and that the genetic regulation underlying patterning is under strong selective pressure. In contrast, the microchaetes do not display conserved patterns and are frequently randomly arranged, the number and position of each bristle varying between individuals of a species (Skaer, 2002 and references therein).

In those species examined to date, D. melanogaster, C. capitata, and C. vicina, precursors for the macrochaetes arise earlier in development than those for the microchaetes. This is probably true of most Brachycera, since the macrochaetes are spaced farther apart from one another than are the microchaetes, suggesting that there has been a longer interval for division of the intervening epidermal cells. Two temporally separate waves of precursor segregation have been described, one largely before, and one after the pupal moult. The macro- and micro-chaete precursors thus arise at different times during early pupal development (Skaer, 2002).

This study describes sc expression in Phormia terranovae, a species belonging to the family Calliphoridae that is closely related to C. vicina. Spatial regulation is almost identical between P. terranovae and C. vicina, in spite of their different bristle patterns. The timing of sc expression differs, however, between the two. The first spatially restricted phase of expression is slightly delayed and the second ubiquitous phase remarkably accelerated, such that there is a period of overlap. As a result, the last precursors from the first phase of expression arise at the same time as the first precursors from the second phase of expression and are morphologically indistinguishable from the late-arising microchaetes. These observations illustrate the power of developmental heterochrony in bringing about rapid morphological change (Skaer, 2002).

Achaete-Scute complex of Drosophilids derived from simple ur-complexes preserved in mosquito and honeybee

In Drosophila melanogaster the Enhancer of split-Complex [E(spl)-C] consists of seven highly related genes encoding basic helix-loop-helix (bHLH) repressors, intermingled with four genes that belong to the Bearded (Brd) family. Both gene classes are targets of the Notch signalling pathway. The Achaete-Scute-Complex [AS-C] comprises four genes encoding bHLH activators. Focussing on Diptera and the Hymenoptera Apis mellifera, the question arose how these complexes evolved with regard to gene number in the evolution of insects. In Drosophilids, both gene complexes are highly conserved, spanning roughly 40 million years of evolution. However, in species more diverged, like Anopheles or Apis, dramatic differences are found. Here, the E(spl)-C consists of one bHLH () and one Brd family member (malpha) in a head to head arrangement. Interestingly in Apis but not in Anopheles, there are two more E(spl) bHLH like genes within 250 kb, which may reflect duplication events in the honeybee that occurred independently of those in Diptera. The AS-C may have arisen from a single sc/l'sc like gene which is well conserved in Apis and Anopheles and a second ase like gene that is highly diverged, however, located within 50 kb. Thus, E(spl)-C and AS-C presumably evolved by gene duplication to the current complex composition in Drosophilids in order to govern the accurate expression patterns typical for these highly evolved insects. The ancestral ur-complexes, however, consisted most likely of just two genes: (1) E(spl)-C contains one bHLH member of type and one Brd family member of malpha type, and (2) AS-C contains one sc/l'sc and a highly diverged ase like gene (Schlatter, 2005).

The Achaete-Scute complex (AS-C) is well conserved in D. virilis: all four genes, achaete (ac), lethal of scute (l'sc), scute (sc) and asense (ase) are found in the same order and orientation on the X-chromosome. As in D. melanogaster, the genes are without introns. All proteins share the typical bHLH motif of the AS-C proteins and this domain reveals the lowest evolutionary rate. However, compared with the bHLH proteins of the E(spl)-C the bHLH proteins of the AS-C evolve faster. The complex can be separated into two clusters that are distinguished by their rates of conservation. L'sc and Sc are well conserved with an identity between D. melanogaster and D. virilis of more than 75% ; in contrast, Ac and Ase are conserved with an identity of less than 69%. Note that the highest divergence that was found between these two species in the E(spl)-C was for M8 with still almost 81% identity (Schlatter, 2005).

Of the four AS-C gene members in D. melanogaster, ase stands out because it is much larger than the other three. In D. virilis, the size increase is even more striking: D.v.Ase is predicted to comprise 619 residues, whereas D.m.Ase is only 486 residues in length. This extension of more than 20% additional residues is caused by multiple insertions of repetitive sequences that code for poly-glutamine (Q), poly-alanine (A) and poly-asparagine (N) stretches. Like in D. melanogaster the unrelated gene pepsinogen-like (pcl) is located between l'sc and ase (Schlatter, 2005).

Genes related to achaete or scute have been identified in a large number of species, from hydra to mouse, and so these are also to be expected in the different insects. The AS-C was most intensely studied in various species of Schizophora flies, apart from Drosophila. The number of genes varies between one and four, however, is not strictly correlated with the position in the phylogenetic tree. For example, AS-C of Calliphora vicina contains three genes, whereas other dipteran flies like Drosophila contain four. Two genes are found in the branchiopod crustacean Triops longicaudatus. In Dipteran flies the expression patterns of the proneural genes are largely varied. This is regulated by positional information through the Iroquois Complex and pannier and in addition by a transcriptional feed-back loop involving AS-C proteins. Eventually, neural precursors are selected by the repressive activity of E(spl) bHLH proteins. In this way, location and number of the large bristles on the notum is precisely controlled. The mosquito is covered with rows of large sensory bristle, where number and position varies between individuals. This is in accordance with the fact that there is only one scute-like gene, A.g.ash that is expressed all over the presumptive notum in a modular pattern. Recently it was shown that the Anopheles A.g.ash gene can mimic the endogenous Drosophila genes and that overexpression leads to many ectopic bristles (Schlatter, 2005).

Although the bristle pattern on the notum of different Drosophilids varies slightly, bristle number and position is highly stereotyped. Therefore, it is not surprising to find the AS-C highly conserved within Drosophilids. Yet, the rate of change came unexpectedly and is quite remarkable outside of the bHLH domain. Compared to E(spl) bHLH proteins, those encoded by AS-C have a rather low degree of similarity, most notably Ac. In fact, the big flesh fly Calliphora vicina, which like Drosophila belongs to the Schizophora, is totally lacking the ac gene and is covered with bristles. In agreement, no ac was found in Anopheles or Apis, arguing for rapid evolution. The best conservation rate is found in Sc and L'sc suggesting high evolutionary pressure and maybe common ancestry. Not only the bHLH domain, but also two small stretches outside (aa 203; SPTPS in D. melanogaster L'sc) and also the C-terminus are of high similarity, the latter found identical in Calliphora. Presumably these protein domains are of functional importance. Indeed, the C-terminus acts as a transcriptional activation domain and is also used to recruit E(spl) bHLH proteins. Although the alignments of the respective genes of honeybee and mosquito to sc and l'sc are very similar, the tendency is toward a closer relationship to l'sc. However, it is proposed that this gene pair arose by duplication in the course of Drosophilid evolution, such that a common ancestor may be present in the other two species (Schlatter, 2005).

The rate of conservation is very limited for the Ase homologs. Decent conservation is found within the bHLH domain, and moreover, a further well-conserved box is present (NGxQYxRIPGTNTxQxL; x are differences between A. gambiae and D. melanogaster). This sequence is likewise detected in the Ase protein of C. vicina, which shares many more similarities with D.m.Ase. In Apis, there is no such conservation outside of the bHLH domain, which itself is highly diverged. The overall degree of conservation is so poor that further statements about the relationship are difficult. It is argued that this gene represents A.m.ase by its close proximity to A.m.ash, although other interpretations are similarly possible. An analysis of its expression pattern in honeybee may help to solve these questions (Schlatter, 2005).

In conclusion this study found that both E(spl)-C and AS-C expanded rather recently because they are only present in their current complex structures in Drosophilids. In Apis and in Anopheles, very similar arrangements are found indicative of an ancient ur-complex. The E(spl)-C seems to have evolved from two genes, one HES-like and one Brd-like that are arranged in a head to head orientation. Both types of genes are responsive to Notch signalling in Drosophila. The data suggest that the most ancient genes are E(spl) bHLH and E(spl) malpha from which the other E(spl)-C genes derived by duplication and subsequent change. Moreover, an E(spl) ur-complex is likewise detected in Tribolium castaneum that belongs to the order Coleoptera. In Drosophila the complex also gained unrelated genes like m1 and gro. The latter is highly conserved, however, located at different genomic positions. Whereas in Anopheles the ur-complex seems to exist in its original form, two additional -like bHLH genes are found in the Apis genome that possess introns. These introns are at similar positions as the introns of two other HES-like genes, dpn and h which themselves are highly conserved in the three insect species, arguing for a common evolutionary history. Presumably, the introns are evolutionarily ancient because they are also found in the C. elegans E(spl)/h like gene lin-22. The AS-C seems to originate from a single sc/l'sc like bHLH gene and a second largely diverged bHLH gene that shares similarity with Drosophila ase. The high degree of variation in the latter makes it difficult to conclusively decide on the original arrangement of this gene complex (Schlatter, 2005).


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

date revised: 25 October 98

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