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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 |
EvoprintHD of scute
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).
Exons - one
Bases in 3' UTR - 290
Like Achaete, Scute has a central bHLH domain and a C-terminal acidic domain (Villares, 1987).
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).
date revised: 25 October 98
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