Targets of Activity

Although ABD-B m and r proteins have distinct developmental functions, ectopic expression of either protein during embryogenesis induces the development of filzkörper and associated spiracular hairs (normally located in PS13) at ectopic sites in the larval thorax and abdomen. This suggests that other parasegmental differences contribute to the phenotype specified by ABD-B r activity in PS14. Both ABD-B m and r transcripts repress the expression of other homeotic genes, such as Ubx and abd-A, in PS10-14. Ectopic ABD-B m does not prevent transformations induced by ectopic UBX. Instead, ectopic UBX and ABD-B m proteins compete for the specification of segmental identities in a dose-dependent fashion. The evidence suggests a quantitative competition among the homeotic proteins rather than the existence of a strict functional hierarchy (Lamka, 1993).

The normal expression domain for abdominal-A extends from parasegments 7 to 13. However, while the anterior border of expression is precisely demarcated by a parasegmental boundary, the posterior border does not coincide with a lineage boundary. Within the normal domain, the expression of abd-A shows intrametameric modulation; the amount of product is relatively high in posterior compartments and in the most anterior cells of the anterior compartments and then gradually decreases. The abd-A gene is down-regulated in part of the Abdominal-B domain, precisely in those regions where the Abd-B gene is expressed at high levels (Macias, 1990).

Muscle diversification in the Drosophila embryo is manifest in a stereotyped array of myofibers that exhibit distinct segment-specific patterns. The homeotic genes of the bithorax complex control the identities of abdominal somatic muscles and their precursors by functioning directly in cells of the mesoderm. Whereas Ultrabithorax (Ubx) and abdominal-A (abd-A) have equivalent functions in promoting the formation of particular muscle precursors in the anterior abdominal segments, Abdominal-B (Abd-B) suppresses the development of these same myogenic cells in the posterior region of the abdomen. When expressed in the same mesodermal cells, however, either UBX or ABD-A can override the inhibitory influence of ABD-B, suggesting that these factors may compete in the regulation of common downstream genes. Furthermore, targeted ectopic expression of Ubx or abd-A indicates that these homeotic genes influence muscle cell fates (Michelson, 1994).

Genes that limit where the homeotic gene Sex combs reduced is expressed can affect cell fates in the Drosophila embryo. In the abdominal cuticle Scr is repressed by the three bithorax complex (BX-C) homeotic genes, thus prevented it from inducing prothoracic structures. However, two of the BX-C homeotic genes, Ultrabithorax and abdominal-A, have no effect on the ability of SCR to direct the formation of salivary glands. Instead, salivary gland induction by Scr is limited in the trunk by the homeotic gene teashirt and in the last abdominal segment by the third BX-C gene, Abdominal-B. Therefore, spatial restrictions on homeotic gene activity differ between tissues and result both from the regulation of homeotic gene transcription and from restraints on where homeotic proteins can function (Andrew, 1994).

Expression of Creb-A in salivary glands depends on Sex combs reduced, since Scr mutants do not express CrebA in salivary glands and embryos expressing Scr in new places also express CrebA in new places. Activation is blocked by the trunk gene, teashirt and the posterior homeotic gene Abdominal-B. As with two other salivary gland genes, forkhead and trachealess, activation of CrebA in the salivary gland by Scr is blocked by dpp (Andrew, 1997).

Segmental modulation of 18 wheeler expression later in the tracheal system is dependent upon the function of the homeotic genes of the bithorax complex. In Ultrabithorax mutant embryos, a larger, more intense patch of 18w extends to cells surrounding the tracheal pits in T2 and T3, indicative of a role for Ultrabithorax protein in repression of 18w in T2 and T3 tracheal pits and consistent with a homeotic transformation in Ubx mutants of posterior T2 and T3 towards a T1 identity. Expanded 18w similarly extends posteriorly to A6 in flies lacking both Ubx and abd-A functions and to A7 in a triple mutant also deficient in Abd-B, indicating a role for abd-A and Abd-B in the repression of 18w in the posterior abdominal segment. In the triple mutant, loss of intense posterior spiracle staining suggests that Abd-B may also be required in A8 for positive regulation of 18w. It is not known whether BX-C regulation of 18w is direct or indirect (Chiang, 1995a).

scabrous is a target for Ubx. Parts of the last intron and exon of the scabrous gene contain five ATTA sequences, the core sequence shared by most homeodomain binding sites. Mutation of Ubx results in the ectopic transcription of sca in the first abdominal segment. Transcript localization in several combinations of deficiencies in the bithorax complex indicates that sca is downregulated by Abdominal-A and Abdominal-B , and suggests that it is a common target of the three genes of BX-C (Graba, 1992).

The development of Drosophila larval filzkörper (structural specializations of the eighth abdominal segment) is dependent on the function of the homeotic selector gene Abdominal-B. The empty spiracles homeobox gene is also required for the development of the filzkörper. ems is a downstream gene, transcriptionally regulated by ABD-B proteins. This regulation is mediated by an ABD-B-dependent ems cis-regulatory element that in early- to mid-stage embryos is activated only in the eighth abdominal segment. Genetic epistasis tests suggest that both ems and Abd-B are required in combination for the specification of the filtzkörper primordia (Jones, 1993).

unplugged expression occurs in portions of the tracheal system that penetrate the CNS, including the cerebral branch specific to T1. To test the possibility that genes in the BX-C play a role in regulating unpg expression, the distribution of unpg transcript was examined in Ultrabithorax and abdominal-A mutants, and in Ubx, abd-A, Abd-B triple mutants. In Ubx mutant embryos additional unpg expression is observed in cells surrounding the tracheal pits of T2 and T3, indicative of a role for Ubx in repression of unpg in the posterior segments and consistent with homeotic transformation in Ubx mutants of posterior T2 and T3 toward a T1 identity. In abd-A mutants embryos extra patches of unpg-expressing cells around the tracheal pits extend posteriorly to A7, indicating a role for Abd-A in the repression of unpg expression in the abdominal segments. The homeotic gene abd-B probably contributes to the repression of unpg expression in A7, since slightly elevated expression in A7 is observed in the triple mutants (Chiang, 1995b).

Homeobox genes encode a class of highly evolutionarily conserved transcription factors that control embryonic development. The Drosophila melanogaster empty spiracles gene is the homolog of the two human homeobox genes EMX1 and EMX2. These genes are necessary for central nervous system development. A regulatory element of the empty spiracles gene was used to study the control of homeobox gene expression. The 1.2-kilobase (kb) cis-regulatory element located 3 kb 5' of the transcription start site of the empty spiracles gene was analyzed by evolutionary sequence comparisons, gel mobility shift assays, DNase footprinting, and the generation of transgenic flies. The corresponding element from a related species, Drosophila hydei, was cloned. Three discrete, approximately 100 base pair (bp) regions of sequence homology were identified. Each had two blocks of 10 to 40 bp of near perfect sequence identity. Fusion proteins were produced containing the Abdominal-B homeodomain or the Empty spiracles homeodomain, known regulators of empty spiracles gene expression. Gel mobility shift assays showed that each of the three regions is bound by both proteins. DNase footprinting revealed closely linked Empty spiracles and Abdominal-B binding sites. Transgenic flies containing a reporter linked to individual conserved regions of the enhancer were prepared. Reporter expression was evident only outside of the usual empty spiracles expression domain. These elements are not sufficient alone; a combinatorial model is proposed. Conserved discrete areas within a homeobox gene regulatory element, which function as homeodomain protein transcription factor binding sites, are used in a combinatorial fashion to regulate these developmentally important genes (Taylor, 1998).

Study of the posterior spiracles of Drosophila as a model to understand the genetic and cellular mechanisms controlling morphogenesis

The development of the posterior spiracles of Drosophila may serve as a model to link patterning genes and morphogenesis. A genetic cascade of transcription factors downstream of the Hox gene Abdominal-B subdivides the primordia of the posterior spiracles into two cell populations that develop using two different morphogenetic mechanisms. The inner cells that give rise to the spiracular chamber invaginate by elongating into 'bottle-shaped' cells. The surrounding cells give rise to a protruding stigmatophore by changing their relative positions in a process similar to convergent extension. In the larvae the spiracular chamber forms a very refractile filter, the filzkorper. The opening of the spiracular chamber, the stigma, is surrounded by four sensory organs; the spiracular hairs. Clones labeling the spiracular hairs show that each one is formed by four cells related by lineage, two neural and two support cells, the typical structure of a type I external sensory organ. When the larva is buried in the semi-liquid medium on which it feeds, the stigmatophore periscopes out of the medium allowing the larva to continue breathing. The genetic cascades regulating spiracular chamber, stigmatophore, and trachea morphogenesis are different but coordinated to form a functional tracheal system. In the posterior spiracle, this coordination involves the control of the initiation of cell invagination that starts in the cells closer to the trachea primordium and spreads posteriorly. As a result, the opening of the tracheal system shifts back from the spiracular branch of the trachea into the posterior spiracle cells (Hu, 1999).

Downstream of Abd-B the cascade can be subdivided into various levels. The activation of six genes -- cut, empty spiracles (ems), nubbin (nub), klumpfuss (klu), and spalt (sal) -- does not require the expression any of the other genes studied, suggesting that these six genes are at the top of the cascade under Abd-B regulation. The cut, ems, nub, and klu genes are expressed in the spiracular chamber in overlapping patterns. The sal gene is not expressed in the spiracular chamber but rather in the surrounding cells that will form the stigmatophore. The exclusion of sal from the spiracular chamber is partly due to repression by cut, because in cut mutants sal is expressed at low levels in the internal part of the spiracle. Downstream of these putative Abd-B targets other genes are activated. These include the transcription factors grainyhead (grh), trachealess (trh) and engrailed (en) (Hu, 1999).

The spiracle phenotypes in mutants for the early Abd-B downstream genes have been analyzed. In sal mutants the stigmatophore does not form, resulting in embryos with a normal spiraclular chamber that does not protrude. Conversely, mutations in ems and cut affect the spiracular chamber but not the stigmatophore. Mutations for ems result in a spiracular chamber that lacks a filzkorper and is not connected to the trachea. In cut mutants the filzkorper is almost completely missing, but the trachea is still connected to the spiracular chamber and the spiracular hairs are also missing. In trh mutants, where the tracheal pits do not form and there is no tracheal network, the spiracular chamber cells still invaginate, forming a filzkorper. However, this filzkorper is shorter than that of the wild type probably due to a secondary requirement of trh, which is also expressed in the spiracular chamber cells. These results show that the spiracular chamber, the stigmatophore, and the trachea develop independently of one another. No phenotypes for either klu or nub could be detected, indicting that although these genes are expressed in the spiracle, they are either redundant or their function is not required for spiracle morphogenesis (Hu, 1999).

The cut gene is the best marker for cells that will make the spiracular chamber because cut is expressed in these cells when they are still on the surface and continues being expressed after spiracle invagination. At stage 11 cut is expressed in a group of about 70 cells arranged as a two-dimensional sheet. Most of these cells are posterior to the A8 tracheal pit although a few coexpress both tracheal and spiracular markers. These cells are located in the dorsal half of the anterior compartment of A8; at this stage these cells have a shape similar to that of cells at homologous positions in more anterior segments (Hu, 1999).

To follow the movements of the spiracular chamber cells as they invaginate, constructs were examined that were made with particular enhancers of the cut, ems, and grh genes, each of which drive expression of beta-gal in a subset of cells that express the cut gene at stage 11. These enhancers do not drive the whole spiracular expression of their genes, but are good tools for studying cell specification and the morphogenetic movements of the posterior spiracle cells. The expression of cut in the posterior spiracle is controlled by at least three different enhancers, two of which have been used in this study. From stage 13, the ct-A4.2 enhancer marks the precursors of the four spiracular hairs. The grh-D4 enhancer of the grh gene is expressed in a single group of cells in this area. The expression of ems in the spiracle is driven by at least by one enhancer: ems-1.2. From stage 11 this enhancer marks a group of cells abutting the tracheal pit. Double stainings of the cut-D2.3, ems-1.2, and grh-D4 lacZ constructs show that they are expressed in non-overlapping subsets of cells. The correlation of the expression of these three constructs allows the fate mapping of the spiracular chamber primordium when it is a two dimensional sheet of cells. The different spatial expression of these enhancers at stage 11 shows that the two-dimensional sheet of cells is already patterned and that the cells invaginate to precise positions during development (Hu, 1999 and references therein).

The connection of the posterior spiracle to the trachea is a regulated event. In mutants for the Drosophila FGF and FGF-receptor homologs branchless and breathless the tracheal pits do invaginate, but since they do not migrate toward one another, they do not form a continuous network. In contrast, in btl mutants, the posterior spiracle connects normally to the A8 spiracular branch of the trachea. In mutants for Abd-B the stigma of A8 does not slide posteriorly, but stays in the same position as in anterior abdominal segments, where the spiracular branch attaches to the outside epidermis. The contribution of the ems gene to coordination of morphogenetic movements has been examined. The spiracle-trachea connection occurs in cut and sal mutants but not in ems mutants. In ems mutants, invagination of the spiracle cells adjacent to the trachea does not occur, but more posterior cells of the spiracle invaginate normally. The elongation does not occur simultaneously in all cells, but starts in the more anterior ones and, in general, the invaginating cells keep contact with the external surface of the embryo. This results in the cells that have invaginated earlier being deeper in the spiracular chamber and more elongated. The defective invagination in ems mutants results in a spiracle without a lumen and with the tracheal opening located outside it. The results show that cell elongation and formation of a lumen are two independently controlled processes. The spiracles provide a good model for the study of cellular and molecular mechanisms controlling cell shape and cell rearrangements, two mechanisms which are used during the morphogenesis of a variety of organisms (Hu, 1999).

Genetic control and evolution of sexually dimorphic characters in Drosophila

Sexually dimorphic abdominal pigmentation and segment morphology evolved recently in the melanogaster species group of the Drosophila. These traits are controlled by the bric à brac gene, which integrates regulatory inputs from the homeotic and sex-determination pathways. bab expression is modulated segment- and sex-specifically in sexually dimorphic species, but is uniform in sexually monomorphic species. It is suggested that bab has an ancestral homeotic function, and that regulatory changes at the bab locus played a key role in the evolution of sexual dimorphism. Pigmentation patterns specified by bab affect mating preferences, suggesting that sexual selection has contributed to the evolution of bab regulation (Kopp, 2000).

An approach to bridging this gap between evolutionary genetics and comparative embryology is to analyze and compare the development of rapidly evolving morphological traits. In many animals, secondary sexual characteristics evolve rapidly, making them good candidates for analysis. One such character in Drosophila is the pigmentation of adult abdominal segments. In D. melanogaster, abdominal pigmentation is sexually dimorphic. Segments 1 to 6 in females and 1 to 4 in males carry only a posterior stripe of dark pigment. However, segments 5 and 6 (A5 and A6) in males are completely pigmented, giving the species its name. This pattern is of recent evolutionary origin; in most Drosophila species, male-specific pigmentation is absent, so that females and males are pigmented identically. To understand how this new pattern originated and evolved, the regulatory circuit that controls its development has been characterized, and its operation has been compared in sexually dimorphic and monomorphic species (Kopp, 2000).

The development of sexually dimorphic external characteristics is controlled by the doublesex (dsx) gene. Alternative splicing of the dsx transcript produces a male-specific product in males (dsxM), and a female-specific product in females (dsxF). Loss of dsx function in females results in the development of male-like pigmentation, which can be suppressed by heat-shock dsxF transgenes. Male-specific pigmentation is therefore expressed by default, and must be actively repressed by dsxF (Kopp, 2000).

Thus, the development of sexually dimorphic pigmentation requires integration of homeotic and sex determination gene inputs. In investigating how this integration is achieved, a newly evolved genetic circuit has been discovered that appears to be responsible for the origin of male-specific pigmentation (Kopp, 2000).

A gene near the left tip of the third chromosome contributes to the variation in female abdominal pigmentation. In investigating this genetic region, it was found that loss of one copy of the bab locus results in the development of male-specific pigmentation in females, but has no effect on the male abdomen. Ectopic pigmentation in heterozygous bab females is suppressed by reducing the dosage of Abd-B, but is not eliminated by loss of omb. This suggests that bab+ represses the development of male-specific pigmentation in females by opposing the function of Abd-B. The bab locus contains two closely related genes, bab1 and bab2, which encode putative transcription factors with multiple roles in development. Ectopic pigmentation in females increases in the order bab1/+ < bab1/bab1 bab1bab2/+ bab1bab2/bab1, indicating that both genes are involved in repressing male pigmentation. For simplicity, the entire locus has been treated as one gene, bab, unless noted otherwise (Kopp, 2000).

The expression pattern of bab at the pupal stage when the adult epidermis develops reflects its sex- and segment-specific function. In females, bab expression is strongest in segments A2 and A3, and progressively weaker in A4, A5 and A6. In males, bab expression is considerably weaker than in females in all segments. Most strikingly, it is completely absent from A5 and A6. This pattern of bab repression correlates with the presence of sex-specific pigmentation in males, and its absence in females (Kopp, 2000).

To test whether bab+ is sufficient to repress pigmentation, the bab genes were ectopically expressed in the pupal abdomen. Low-level expression of bab+ results in the loss of male-specific pigmentation, but has no other effects on external morphology, indicating that differential regulation of bab plays a central role in establishing sexual dimorphism. bab+ can also repress non-sex-specific pigment stripes when expressed at a higher level. This suggests that bab+ acts as a general repressor of pigmentation, but that its effects are overridden by omb in the posterior part of each segment. Consistent with this, complete loss of both bab genes results in ectopic pigmentation of A2 to A7 in both sexes. This phenotype is not caused by expansion of Abd-B expression, which appears normal in these mutants. In bab homozygotes, the intensity of pigmentation is higher in the more posterior segments than in those more anterior. This suggests that pigmentation does not develop by default in the absence of bab, but is actively promoted by Abd-B and abd-A (Kopp, 2000).

The sexually dimorphic repression of bab in the posterior abdomen suggests that bab integrates the homeotic and sex determination regulatory inputs. To test this, bab expression was examined in Abd-B and dsx mutant backgrounds. Ectopic expression of Abd-B in A3 and A4 eliminates bab expression from these segments in males, and downregulates it in females. Conversely, bab is derepressed in A5-A7 in the mutants that lack Abd-B function in these segments. Together, these results indicate that bab expression in A5 and A6 is normally repressed by Abd-B. The slight downregulation of bab in A4 suggests that it is also weakly repressed by abd-A (Kopp, 2000).

In dsx-intersexes, bab is expressed in a male-like pattern, suggesting that dsxF upregulates bab transcription in females. Abd-B and abd-A expression is identical in males, females and dsx -intersexes, indicating that bab is regulated independently by homeotic and sex-determination inputs. dsxDominant intersexes, which express both male- and female-specific dsx products, also show male-like expression of bab, indicating that dsxM can interfere with dsxF function. The two dsx isoforms encode transcription factors that bind the same DNA sequence, but have opposite effects on gene expression. dsx-intersexes differ from males in having a small unpigmented region at the anterior-lateral margin of A5, suggesting that dsxM may have a slight negative influence on bab expression (Kopp, 2000).

These results suggest that bab+ regulates sexually dimorphic pigmentation by integrating regulatory inputs from the homeotic genes and the sex determination pathway. In this regulatory circuit, bab+ acts as a general repressor of pigmentation, and Abd-B and abd-A promote pigmentation in both sexes. In addition, Abd-B, and to a lesser extent abd-A, repress bab transcription. In males, this results in the absence of bab from A5 and A6, allowing Abd-B and abd-A to promote pigmentation in these segments. However, in females, dsxF prevents bab transcription from being completely repressed by the homeotic genes. As a result, bab is present in A5 and A6 in females, where it blocks the ability of Abd-B and abd-A to promote pigmentation. In A2-A4, abd-A alone is not sufficient either to repress bab or to overcome its inhibitory effect on pigmentation; thus, only the omb-dependent striped pigmentation is generated. Because Abd-B, abd-A and dsx encode transcription factors, they may regulate bab expression directly (Kopp, 2000).

The central role of bab as an integrator of homeotic and sex-determination gene inputs suggests that changes in bab regulation may have been responsible for the evolution of sexually dimorphic pigmentation. In the subgenus Sophophora, male-specific pigmentation is present only in the melanogaster species group. Within this group, sexual dimorphism is seen in all species of the melanogaster subgroup and the closely related oriental subgroups, whereas the ananassae and montium subgroups contain both sexually dimorphic and sexually monomorphic species (Kopp, 2000).

In species with male-specific pigmentation of A5 and A6, bab expression is absent or strongly downregulated in these segments in males, but not in females. Moreover, in the sexually monomorphic species outside the melanogaster species group, bab expression is identical in both sexes and in all segments from A2 to A7. This correlation suggests that changes in the regulation of bab by Abd-B and dsx played an important role in the origin of sexually dimorphic pigmentation (Kopp, 2000).

bab+ regulates segment shape and bristle and trichome patterns in a manner reciprocal to Abd-B. Loss of bab+ function in females enhances posterior characteristics in A6, A7 and A8. No phenotype is seen in males, consistent with the absence of bab expression in posterior segments. Conversely, ectopic expression of bab transforms A6 and A7 to a more anterior identity in both males and females. These observations suggest that bab+ acts as an antagonist of Abd-B homeotic function, and that posterior abdominal characters are determined by the balance between Abd-B and bab activities (Kopp, 2000).

This model predicts that evolutionary changes in bab regulation should result in morphological transformation of Abd-B-expressing segments. Indeed, the entire suite of characteristics that distinguishes A5 and A6 from the more anterior segments in D. melanogaster is of recent evolutionary origin. In D. willistoni, bab is expressed strongly in A5 and A6 in males, whereas Abd-B is expressed in the same pattern as in D. melanogaster. As predicted, A5 and A6 are almost identical to the more anterior, non-Abd-B-expressing segments in the males of this species. In contrast, the melanogaster species group shows great diversity of bristle and trichome patterns in posterior abdominal segments. The two main lineages within this group show different patterns of evolution. In the clade composed of the melanogaster and oriental subgroups, male-specific pigmentation and bristle and trichome patterns have evolved in a concerted fashion. However, in the ananassae + montium lineage, these characteristics vary independently of each other, and sexually dimorphic bristle and trichome patterns are sometimes observed in species that do not show visible modulation of bab expression. This suggests that evolutionary changes have occurred not only in bab regulation, but also in the target genes of bab and in other genes regulated by Abd-B and dsx . Suppression of A7 development in males has occurred earlier in evolution than visible modulation of bab expression, despite the ability of bab to override this suppression (Kopp, 2000).

These findings indicate that changes in bab regulation have played an important part in the evolution of abdominal segment morphology. The presence of bab expression in all Drosophila species examined suggests that its roles in antagonizing the homeotic function of Abd-B and repressing pigmentation are ancestral. However, in the ancestral condition, bab expression was independent of Abd-B and dsx, resulting in sexually monomorphic pigmentation and segment morphology. In the melanogaster species group, bab evolved to be under the control of Abd-B and dsx. This eliminated bab from Abd-B-expressing segments in the male and resulted in a major transformation of male segment morphology. Subsequent diversification of pigmentation, bristle and trichome patterns was probably driven both by the fine-tuning of bab regulation and by changes in the downstream targets of bab and Abd-B (Kopp, 2000).

Two features of this genetic circuit make it highly plastic and evolvable: (1) the adult phenotype is sensitive to quantitative changes in bab expression; (2) the level of bab expression is determined by the balance between Abd-B and dsxF inputs. If bab is regulated directly by Abd-B and dsx, then the evolution of sexually dimorphic pigmentation and segment morphology may ultimately be traced to the acquisition and modification of binding sites for the Abd-B and Dsx proteins in the cis-regulatory region of bab. Thus, even a subtle molecular change could be expressed phenotypically and become subject to selection (Kopp, 2000).

Recurrent modification of a conserved cis-regulatory element underlies fruit fly pigmentation diversity

The development of morphological traits occurs through the collective action of networks of genes connected at the level of gene expression. As any node in a network may be a target of evolutionary change, the recurrent targeting of the same node would indicate that the path of evolution is biased for the relevant trait and network. Although examples of parallel evolution have implicated recurrent modification of the same gene and cis-regulatory element (CRE), little is known about the mutational and molecular paths of parallel CRE evolution. In fruit flies, the Bric-a-brac (Bab) transcription factors control the development of a suite of sexually dimorphic traits on the posterior abdomen. Female-specific Bab expression is regulated by the dimorphic element, a CRE that possesses direct inputs from body plan (Abd-B) and sex-determination (Dsx) transcription factors. This study finds that the recurrent evolutionary modification of this CRE underlies both intraspecific and interspecific variation in female pigmentation in the melanogaster species group. By reconstructing the sequence and regulatory activity of the ancestral Drosophila melanogaster dimorphic element, this study demonstrates that a handful of mutations were sufficient to create independent CRE alleles with differing activities. Moreover, intraspecific and interspecific dimorphic element evolution proceeds with little to no alterations to the known body plan and sex-determination regulatory linkages. Collectively, these findings represent an example where the paths of evolution appear biased to a specific CRE, and drastic changes in function are accompanied by deep conservation of key regulatory linkages (Rogers, 2013).

In the D. melanogaster pigmentation network, the bab genes function as an Input-Output node through the dimorphic element's integration of patterning inputs that include body plan (ABD-B) and sex determination (DSX) pathway inputs. These inputs are converted into a female-specific pattern of expression that culminates in the repression of the differentiation genes yellow and tan in females. In principle, changes in the expression or activity of a patterning gene, differentiation gene, or the Input-Output gene (bab) could alter pigmentation phenotypes. In application though, it is logical that bab expression and dimorphic element encodings were modified as those alterations minimize negative pleiotropic effects while being sufficient to alter the female pigmentation phenotype. For example, ectopic yellow expression fails to create additional melanic pigmentation, and changes in either DSX or ABD-B expression result in ectopic abdominal pigmentation in addition to several other trait phenotypes. Thus, sufficiency for pigmentation is counterbalanced by the negative pleiotropic affects for these genes. In contrast, increased Bab expression in the A5 and A6 segments is sufficient to suppress pigmentation, and ectopic abdomen pigmentation develops in bab heterozygous and homozygous null mutant females (Rogers, 2013).

Bab though is not dedicated to pigmentation. In the pupa, Bab expression includes the leg tarsal segments, abdomen epidermis, sensory organ precursor cells, oenocytes, and dorsal abdominal muscles, and each of these expression patterns are governed by a modular CRE (s). Thus, Bab itself is highly pleiotropic, however it's CREs are far less pleiotropic. For this reason, mutations altering female pigmentation would maximize sufficiency and minimize pleiotropy if they occurred in the dimorphic element, an expectation borne out in this study. Pigmentation of the A5 and A6 segments, though, is only one of many traits influenced by the regulatory activity of the dimorphic element. This CRE drives Bab expression in the female A7 and A8 segments, regulating numerous female-specific traits, including the size, shape, trichome density, and bristle morphologies of the resident dorsal tergites and ventral sternites. As expression in these more posterior segments require the ABD-B and DSX regulatory linkages, these regulatory linkages remain highly pleiotropic. For this reason, it seems logical that evolution would disfavor mutations that have deleterious consequences to these linkages and favor mutations that alter other CRE properties. This scenario reflects how dimorphic element function was modified in both the intraspecific and interspecific comparisons presented presented in this study as well as the long term conservation of the ABD-B and DSX linkages previously described (Rogers, 2013).

The current findings provide a unique contrast with previous investigations of the relationship between CRE conservation and CRE evolution. Although Drosophila non-coding DNA, including CRE sequences, evolves slower than synonymous sites, several well studied CREs were found to undergo substantial sequence evolution without matching regulatory activity evolution. During Drosophila embryonic development, the pair-rule gene even-skipped (eve) is expressed in seven stripes along the anteroposterior axis, with the second stripe of eve expression being specified by the stripe 2 element (S2E) CRE. In D. melanogaster, the S2E possesses binding sites for four transcription factors that collectively specify the eve expression output. The orthologous S2E from the species D. pseudoobscura differs in sequence for numerous binding sites, the overall content of binding sites, and spacing between conserved binding sites, yet the orthologous S2Es function equivalently in vivo. Hence, the S2E is an exemplar as to how selection acting at the level of the character (eve stripe expression) can accommodate a surprising amount of CRE evolution. Similarly, CRE sequence evolution without corresponding functional evolution was found between Drosophila species for the sparkling (spa) CRE that directs cone cell expression for the dPax2 gene. The content and spatial proximity of binding sites for neurogenic ectoderm enhancers (NEEs) evolved in order to conserve expression pattern outputs in response to changing regulatory inputs. These case studies, demonstrate how CRE sequence conservation is not a prerequisite for CRE functional conservation (Rogers, 2013).

In contrast, this study found little divergence in the content and sequence of known binding sites for the D. melanogaster dimorphic element alleles and orthologous sequences. At the sequence level, these CRE alleles and orthologs respectively posses identities of ~98% and ~80%. Indeed, the vast majority of binding sites in the dimorphic element have been conserved for over 30 million years, showing conservation to D. willistoni. At the functional level, these CREs exhibited striking differences in their regulatory activities. Thus, in contrast to S2E, spa, and the NEEs, the dimorphic element demonstrates how CREs can derive dramatic changes in function that drive phenotypic divergence, with little-to-no alteration to the characterized pre-existing regulatory linkages (Rogers, 2013).

While the regulatory activity of the Light and Dark dimorphic elements alleles correlated with female A5 and A6 pigmentation, some outcomes suggest that these variant sequences are affected by other features within or perhaps outside of the bab locus. For instance, the Light 2 and Dark 2 alleles exhibit the highest and lowest regulatory activities respectively. Surprisingly, the Light 1 and Dark 1 alleles and their intermediate regulatory activities are associated with the more extreme Light and Dark female pigmentation phenotypes. At the expression level, Bab1 and Bab2 showed similar patterns in females from the Light 1 (prominent expression in segments A5 and A6) and Dark 1 (reduced expression is A5 and A6) strains. In the Dark 2 strain, Bab1 but not Bab2 expression was reduced in females. Several possible explanations might explain the uncoupled expression of the Bab paralogs in Dark 2. For example, it is possible that a separate, as of yet unidentified CRE controls Bab2 expression. However, a screen of the entire ~160 kb locus failed to identify such a CRE. A second possibility is that a mutation(s) in the Dark 2 allele has paralog-specific regulatory effects, perhaps by modifying an interaction with the promoter for bab1 but not that of bab2 (Rogers, 2013).

Another possible explanation would involve the existence of CREs that coordinate communication between bab1 and bab2. In such a scenario, the Dark 2 allele could contain mutations that alter interaction with coordinating elements to result in paralog-specific expression patterns in the female A5 and A6 segments. This possibility is consistent with observations of bab locus evolution in another population where females differ in A6 segment pigmentation. For this population, fine-scale genetic mapping found that three disparate non-coding regions of the bab locus collaborate to compose a major effect QTL. One of these regions spans the dimorphic element, though no mutations reside with this CRE's core element. The other two regions include an intergenic sequence between bab1 and bab2 and a large sequence that includes the bab2 promoter. In the future, it will be important to understand what roles these other regions serve, and how they may interact with polymorphisms in the dimorphic element to produce paralog-specific effects on gene expression (Rogers, 2013).

With the centrality of CREs and their evolution to the diversification of phenotypic traits, a major obstacle to reaching this goal is understanding the processes by which CRE regulatory logics were modified to contemporary forms. Often studies of CRE evolution involve comparisons of two divergent derived regulatory states, where one sequence assumes the role of a surrogate for the ancestral function. This approach has been successful in making inferences about the ancestral states for regulatory linkages and identifying gains and losses of other key derived transcription factor binding sites. However, it is important to acknowledge a key limitation of this comparative approach; a CRE derived from an outgroup species that serves as a surrogate for the ancestor has also evolved along a unique lineage since divergence (Rogers, 2013).

Studies into the evolution of divergent protein activities encountered a similar problem when comparing extant proteins forms. For several cases, key amino acid residues necessary for a derived function were identified. When substituted into the surrogate ancestral protein, these changes were insufficient to impart the derived function and thereby indicating that the paths of evolution were more intricate. As a solution, the reconstruction of ancestral protein sequences, combined with functional testing of inferred ancestral proteins has allowed a more realistic simulation of evolutionary events. As a result, inferences about the paths of protein evolution were made that likely would not have been found from comparisons of extant proteins (Rogers, 2013).

A more ideal research program to study CRE evolution would include reconstruction of ancestral CREs as a starting point to trace the paths of evolutionarily relevant mutations. Few studies have used CRE reconstruction. For one study, a novel optic lobe expression pattern for the D. santomea Nep-1 gene occurred via the modification of a CRE that drove an eye field pattern of expression for an ancestor that existed ~0.5 million years ago. Importantly, by reconstructing and evaluating the ancestral CRE, the wrong conclusion - that this optic lobe activity evolved de novo – was avoided and the correct conclusion was found - a latent optic lobe CRE activity was augmented into a robust derived state. In the current study, had the Concestor element not been reconstructed, the Dark 1 and Dark 2 dimorphic element sequences would have been considered hypomorphic CRE alleles compared to the robust wild type-like activity of the Light 1 and Light 2 alleles. The Light alleles possessed activities more similar to a previously characterized dimorphic element allele and consistent with the narrative of D. melanogaster being a sexually dimorphic species where females lack posterior abdominal pigmentation. Reconstruction of the dimorphic element revealed a more complex reality, where neither alleles were good surrogates for the ancestral state. Using ancestral sequences as a starting point, this study found that the evolutionary paths for these alleles to be short in number of steps (one to two mutations) and in time frame (in the last ~60,000 years). Thus, demonstrating how simple and rapid an existing CRE regulatory logic can evolve (Rogers, 2013).

The cases of Nep1 optic lobe CRE and the bab dimorphic element evolution demonstrate the utility for reconstructing ancestral CRE states; though it must be pointed out that these cases involved comparisons of very closely-related species/populations. As a result of these short time frames for divergence, the extant CRE forms differ at fewer than two percent of the nucleotide sites. This made possible ancestral sequence reconstruction by the principle of parsimony. However, not all compelling instances of functional CRE evolution occur over similarly short time frames. Therefore, studies will need to reconstruct CREs that existed further in the past and for which the method of parsimony will need to be replaced by methods of maximum likelihood-based inference coupled with the testing of multiple alternate reconstructions (Rogers, 2013).

The functions of pannier during Drosophila embryogenesis; Abd-B targets pannier

Between stages 10 and 11 pannier loses expression in the A8 segment. Expectedly, it is under the control of Abd-B; in Abd-B mutants the gap in A8 does not appear. However, none of the known Abd-B target genes (sal, ems and grn) is involved in the regulation, since their mutations do not affect pnr expression. The finding that lin, which is considered as a co-factor of Abd-B, is involved, suggests that downregulation of pnr in the A8 segment is mediated either by an unknown Abd-B target or directly by interaction between the Abd-B and Lin products. It is not clear why pnr activity has to be eliminated precisely in the A8 segment. This segment gives rise to the spiracles, protruding structures that are very different from those differentiated by the other abdominal segments where pnr remains active. In fact, there are several Abd-B target genes specifically activated in the spiracles. It is possible that the formation of these structures demands that the pnr activity, which specifies larval epidermis of very different morphology, be turned off (Heranz, 2001).

Drosophila p27Dacapo expression during embryogenesis is controlled by a complex regulatory region independent of cell cycle progression

In the embryonic epidermis, dacapo expression starts during G2 of the final division cycle and is required for the arrest of cell cycle progression in G1 after the final mitosis. The onset of dacapo transcription is the earliest event known to be required for the epidermal cell proliferation arrest. To advance an understanding of the regulatory mechanisms that terminate cell proliferation at the appropriate stage, the control of dacapo transcription has been analyzed. dacapo transcription is not coupled to cell cycle progression. It is not affected in mutants where proliferation is arrested either too early or too late. Moreover, upregulation of dacapo expression is not an obligatory event of the cell cycle exit process. During early development of the central nervous system, Dacapo cannot be detected during the final division cycle of ganglion mother cells, while it is expressed at later stages. The control of dacapo expression therefore varies in different stages and tissues. The dacapo regulatory region includes many independent cis-regulatory elements. The elements that control epidermal expression integrate developmental cues that time the arrest of cell proliferation (Meyer, 2002).

To identify conserved sequence elements in the dap regulatory region, a dap homolog was isolated from Drosophila virilis. The gene product shares 65% amino acid sequence identity with p27DAP from Drosophila melanogaster. This comparison revealed several blocks of high sequence similarity not only within the coding region but also within the regulatory region. The most distal of these blocks (A8) is located within the region containing regulatory elements controlling expression in the epidermis. The significance of these blocks was examined, comparing the expression of transgenes with and without these blocks (dap-15l and dap-15l-A8). The results indicate that the A8 block is required for the early high level expression in a region of the epidermis within the abdominal segment A8. Wild-type dap expression occurs early and at high levels within this prospective posterior spiracle region. Interestingly, the A8 block contains binding sites for the homeodomain transcription factor encoded by Abdominal-B (Abd-B) which is expressed within the posterior abdominal region and required for the specification of posterior spiracles. prd-GAL4 driven expression of UAS-Abd-B induces expression of the endogenous dap gene (Meyer, 2002).

These results indicate that Abd-B is involved in the control of dap expression within the abdominal segment A8. Moreover, they indicate that dap expression is regulated by multiple control elements even within a tissue like the embryonic epidermis. While the regulatory sequences present in the dap-12l construct drive relatively uniform expression throughout the epidermis, the conserved block A8 adds an earlier onset within abdominal segment A8. Another element in dap-1l adds the early onset within the region of the tracheal pits. In summary, these analyses demonstrates that the dap regulatory region is composed of a complex array of stage- and tissue-specific enhancers (Meyer, 2002).

A green fluorescent protein reporter genetic screen that identifies modifiers of Hox gene function in the Drosophila embryo

Hox genes encode evolutionarily conserved transcription factors that play fundamental roles in the organization of the animal body plan. Molecular studies emphasize that unidentified genes contribute to the control of Hox activity. This study describes a genetic screen designed to identify functions required for the control of the wingless (wg) and empty spiracles (ems) target genes by the Hox Abdominal-A and Abdominal-B proteins. A collection of chromosomal deficiencies were screened for their ability to modify GFP fluorescence patterns driven by Hox response elements (HREs) from wg and ems. Fifteen deficiencies were found that modify the activity of the ems HRE and 18 that modify the activity of the wg HRE. Many deficiencies cause ectopic activity of the HREs, suggesting that spatial restriction of transcriptional activity is an important level in the control of Hox gene function. Further analysis identified eight loci involved in the homeotic regulation of wg or ems. A majority of these modifier genes correspond to previously characterized genes, although not for their roles in the regulation of Hox targets. Five of them encode products acting in or in connection with signal transduction pathways; this suggests an extensive use of signaling in the control of Hox gene function (Marabet, 2002).

This study surveyed 60% of the genome and 11 genomic regions were found acting as recessive activators of ems HRE; 4 were found acting as recessive repressors of ems HRE, and 18 were found acting as recessive repressors of wg HRE. So far, the only known gene in addition to AbdB required for ems activation is lines. Df(2R)H3E1, which uncovers lines, has been recovered from the screen for AbdB modifiers. A search for discrete mutations that reproduce the deficiency phenotypes allowed identification of four ems HRE modifier genes: dally, ds, scw, and ttk. Although ttk and scw have already been linked to filzkörper development, none of the four genes had previously been involved in the control of ems expression in posterior spiracles. The screen for AbdA modifiers was restricted to genomic regions leading to ectopic activation of the wg HRE; these response elements relate to functions that repress the enhancer. Accordingly, genomic regions or genes already known to play a role in wg activation, such as abdA, exd, hth, or genes coding for components of the Dpp signaling pathway, were not recovered. Five mutations at specific loci reproduce the phenotypes caused by original deficiencies. Four of these mutations identify tsl, ttk, and genes encoding a putative MPK and a putative CBP as candidate modifiers of wg HRE. None of these genes has so far been involved in the regulation of wg in the visceral mesoderm (Marabet, 2002).

Three of the four candidate genes identified from the ems screen encode molecules acting in or acting in connection with signal transduction pathways. The Scw protein is a secreted factor of the TGF-ß family. The loss of ems expression induced by Brk, a potent repressor of the Dpp/TGF-ß target gene, strongly supports this hypothesis. The involvement of additional signaling pathways in the regulation of ems is more indirectly suggested by the identification of ds and dally that act in connection with several signaling pathways. ds codes for a calcium-dependent cell adhesion molecule of the cadherin superfamily and genetically interacts with shotgun and rhomboid, two genes involved in epidermal growth factor (EGF) signaling, as well as with armadillo (arm), which produces a nuclear effector of the Wg transduction pathway. dally encodes a heparin sulfate proteoglycan involved in the reception of Wg. Although additional experiments are required to firmly establish the involvement of the Wg and EGF pathways, the integration of multiple signals seems to be required for accurate ems regulation by AbdB (Marabet, 2002).

Two modifier genes obtained from the wg screen are presumably involved in the signal transduction cascade. The first, tsl, encodes a ligand for the RTK Torso receptor and the second encodes a putative MKP. Signaling by Ras/MAPK could thus be part of the genetic network that controls wg expression in the midgut, which has been confirmed by showing that wg transcription is impaired by a constitutive active form of Ras. Interestingly, the Ras/MAPK pathway has been implicated in regulation of the Ubx and lab enhancer in the central midgut, and the ETS-domain-containing transcription factor Pointed, which acts as a nuclear effector of the Ras/MAPK pathway, is expressed in the third midgut chamber (Marabet, 2002).

Several modifiers of wg and ems HRE activities identified in this study encode molecules acting in signal transduction cascades. This indicates that signaling processes play important roles in the control of Hox gene function and extends previous observations from a screen for modifiers of a dominant Pb phenotype. Understanding how cell signaling and transcriptional control by Hox protein are mechanistically integrated requires further study (Marabet, 2002).

Segment-specific prevention of pioneer neuron apoptosis by cell-autonomous, postmitotic Hox gene activity

In vertebrates, neurons often undergo apoptosis after differentiating and extending their axons. By contrast, in the developing nervous system of invertebrate embryos apoptosis typically occurs soon after cells are generated. The Drosophila dMP2 and MP1 pioneer neurons undergo segment-specific apoptosis at late embryonic stages, long after they have extended their axons and have performed their pioneering role in guiding follower axons. This segmental specificity is achieved by differential expression of the Hox gene Abdominal B, which in posterior segments prevents pioneer neuron death postmitotically and cell-autonomously by repressing the RHG-motif cell death activators reaper and grim. These results identify the first clear case of a cell-autonomous and anti-apoptotic role for a Hox gene in vivo. In addition, they provide a novel mechanism linking Hox positional information to differences in neuronal architecture along the anteroposterior axis by the selective elimination of mature neurons (Miguel-Aliaga, 2004).

How does Abd-B prevent the function of RHG-motif genes? It is likely that Abd-B prevents pioneer neuron apoptosis by repressing the transcription of, at least, rpr and grim. This idea is supported by four facts: (1) the H99 deletion is epistatic to (functions downstream of) Abd-B; (2) Abd-B is a transcription factor; (3) rprGAL4 is activated posteriorly in Abd-Bm mutants; (4) when misexpressed postmitotically, Abd-B can fully rescue both types of pioneer neurons. Given that loss of rpr is critical for anterior dMP2 survival, whereas loss of grim is critical for anterior MP1s, Abd-B must prevent the expression of at least these two cell death activators (Miguel-Aliaga, 2004).

In the developing vertebrate neural tube, a number of studies have shown that Hox genes are critical for AP organization and for proper neuronal specification. Although their action may be largely confined to progenitor cells, recent studies have revealed that Hox genes can also act to control the identity of early postmitotic neurons. In the light of the current findings, it will be of interest to determine if selective, Hox-dependent elimination of mature neurons gives rise to differences in motor neuron numbers along the AP axis of the vertebrate spinal cord. Increased apoptosis of postmitotic motor neurons has been observed in mouse mutants lacking Hoxc-8, one of the vertebrate homologues of abd-A. This may be the result of the aberrant connectivity pattern of Hoxc-8-deficient motor neurons, which would restrict their access to target-derived neurotrophic factors. However, this increase in cell death is also consistent with the possibility that Hoxc-8 normally acts to prevent apoptosis of postmitotic neurons in its expression domain (Miguel-Aliaga, 2004).

The results contrast with the previous finding that Abd-B appears to activate rpr transcription to regulate segment boundary formation in the posterior region of early Drosophila embryos. Decreased apoptosis has also been observed in mouse mutants lacking Hoxb13, one of the vertebrate homologues of Abd-B. It has previously been shown that the target functions of Hox genes are highly dependent on cellular context, and the regulation of apoptosis appears to be no exception. This context dependence may not be unique to the Abd-B gene. abd-A has been previously reported to activate apoptosis in post-embryonic neuroblasts during normal development. When Antp and Ubx were misexpressed in these neuroblasts, they too were able to trigger apoptosis. In contrast, none of these genes acted in a pro-apoptotic manner in the current study. It is, therefore, conceivable that the pro-apoptotic function of Hox genes is confined to progenitors, at least in the nervous system. Alternatively, or additionally, availability of certain cofactors may determine whether a Hox gene activates or represses transcription of pro-apoptotic genes in a specific cell (Miguel-Aliaga, 2004).

In addition to their dependence on cellular context, specific Hox proteins may control pro-apoptotic genes differently. Abd-B and its vertebrate homologues share several properties that distinguish them from other Hox proteins, such as the absence of a hexapeptide motif and a preference for a different DNA core sequence. Together, these differences may confer unique transcriptional properties on proteins of the Abd-B family, and may explain why Abd-B is the only Hox protein capable of fully rescuing anterior pioneer neurons. The finding that Abd-B is the only Hox gene that was unable to rescue the embryonic brain phenotypes of Drosophila mutants for the Hox gene labial is consistent with this idea (Miguel-Aliaga, 2004).

Is the cellular control of Hox gene expression functionally relevant? The results show that while Hox genes are broadly expressed within their domains, they are largely absent from certain cell populations; at stage 16, few glial cells express Hox genes in the VNC. Since many Drosophila neuroblasts give rise to both neurons and glia, it is possible that Hox gene expression is actively suppressed by factors promoting glial fate. Alternatively, an initial wave of Hox expression in progenitors could be followed by a second, neuron-specific re-activation of Hox expression. In any case, it will be of interest to identify the molecular mechanism by which Hox gene expression is confined to specific populations of postmitotic cells in the nervous system (Miguel-Aliaga, 2004).

While cellular context may determine whether a Hox gene acts in a pro- or anti-apoptotic manner, apoptosis of specific cells within a Hox expression domain may also be achieved by differential Hox gene expression. For example, while Abd-A is broadly expressed in abdominal segments during larval stages, it is absent from post-embryonic neuroblasts. However, at the last larval instar, a neuroblast-specific pulse of abd-A results in the activation of the cell death program in these cells. Similarly, and given the novel role for Hox proteins in the apoptosis and differentiation of postmitotic neurons, the expression of Hox genes in specific postmitotic neurons is likely to be of functional significance. Together, these findings are not consistent with the view that Hox genes solely function as 'segment identity' factors specifying global properties of the segments in which they are active. Instead, they lend functional support to the proposal that Hox genes are required for a number of decisions taken at the cellular level (Miguel-Aliaga, 2004).

The combined activity of RHG-motif genes is critical to the initiation of all cell death in the Drosophila embryo. These genes act in an additive manner. However, not all cell death activators are simultaneously expressed in every cell fated to die, and their specific expression patterns do not always overlap. Therefore, it is likely that they are differentially regulated by specific developmental signals. While Abd-B acts to repress rpr and grim function in posterior pioneer neurons, the developmental stimulus activating their expression in these neurons throughout the cord is currently unknown. Three developmental signals are known to regulate the function of RHG-motif genes in the Drosophila nervous system. The insect hormone ecdysone appears to be important for blocking cell death of certain peptidergic neurons during metamorphosis. However, the ecdysone-receptor complex has also been shown to promote cell death by activating rpr transcription in other tissues during Drosophila metamorphosis. While an embryonic ecdysone pulse occurs around the time when pioneer neurons die, preliminary experiments have failed to lend any support to an ecdysone-dependent activation of apoptosis in these neurons. The EGF-receptor/Ras/MAPK pathway has been shown to phosphorylate Hid protein, thereby preventing apoptosis of midline glial cells. However, neither Rpr nor Grim appear to be regulated in this fashion, and this model would not address the specific transcriptional activation of these genes in pioneer neurons. Lastly, Notch signaling has been described as resulting in both activation and inhibition of apoptosis. In Drosophila, recent studies have revealed that Notch can act cell-autonomously to induce apoptosis during final mitotic divisions both in the central and peripheral nervous systems. Although this Notch-induced developmental apoptosis is prevented in H99 mutant embryos, the molecular mechanisms by which activated Notch signaling results in the activation of IAP inhibitors are still unknown. Nevertheless, Notch signaling is unlikely to be relevant to dMP2 death, since it is not active in dMP2 neurons. It is, therefore, likely that an as yet unidentified factor is responsible for the activation of the apoptotic machinery in pioneer neurons. This factor could be Odd-skipped, given its specific expression in dMP2 and MP1 neurons. Because of the early role of odd in embryonic patterning, its possible postmitotic function in these neurons cannot be addressed using the currently available odd mutants (Miguel-Aliaga, 2004).

Developmental apoptosis in invertebrate embryos typically occurs shortly after cells are generated. In Drosophila, this has often precluded the identification of dying cells until apoptosis has been genetically prevented. Consequently, progress in identification of the mechanisms controlling apoptosis has been relatively slow, and little is known about the upstream pathways that initiate cell death in specific tissues or lineages. Furthermore, in the Drosophila VNC, studies have shown that apoptotic corpses are engulfed by glia, transported to the dorsal surface of the VNC and transferred to macrophages for final destruction. The molecular genetic mechanisms underlying this intriguing series of events are only just beginning to be unraveled. The identification of a late apoptotic event in two of the best-studied and least complex lineages in the Drosophila CNS, as well as the characterization of the dMP2-GAL4 line, should contribute to the elucidation of the mechanisms involved in both the developmental initiation and execution of apoptosis (Miguel-Aliaga, 2004).

A critical role for cyclin E in cell fate determination in the central nervous system of Drosophila; cycE is targeted by abd-A and Abd-B

This study examined the process by which cell diversity is generated in neuroblast (NB) lineages in the central nervous system of Drosophila. Thoracic NB6-4 (NB6-4t) generates both neurons and glial cells, whereas NB6-4a generates only glial cells in abdominal segments. This is attributed to an asymmetric first division of NB6-4t, localizing prospero (pros) and glial cell missing (gcm) only to the glial precursor cell, and a symmetric division of NB6-4a, where both daughter cells express pros and gcm. This study shows that the NB6-4t lineage represents the ground state, which does not require the input of any homeotic gene, whereas the NB6-4a lineage is specified by the homeotic genes abd-A and Abd-B. They specify the NB6-4a lineage by down-regulating levels of the G1 cyclin, DmCycE (CycE). CycE, which is asymmetrically expressed after the first division of NB6-4t, functions upstream of pros and gcm to specify the neuronal sublineage. Loss of CycE function causes homeotic transformation of NB6-4t to NB6-4a, whereas ectopic CycE induces reverse transformations. However, other components of the cell cycle seem to have a minor role in this process, suggesting a critical role for CycE in regulating cell fate in segment-specific neural lineages (Berger, 2005).

In Drosophila, individual neuroblasts deriving from corresponding neuroectodermal positions among thoracic and abdominal segments generally acquire similar fates. However, some of these serially homologous neuroblasts produce lineages with segment-specific differences that contribute to structural and functional diversity within the CNS. The NB6-4 lineage was selected as a model to determine how this diversity evolves from a basic developmental ground state. As an experimental system, NB6-4 has an additional advantage, since Eagle (Eg) is expressed in all the cells of both thoracic and abdominal lineages and can thus be used as a lineage marker (Berger, 2005).

First the expression patterns of different homeotic genes were examined in thoracic and abdominal lineages of NB6-4. Antennapedia (Antp) is expressed in NB6-4t lineages of thoracic segments T1-T3. Abdominal A (Abd-A) is expressed in the NB6-4a lineage of abdominal segments A1-A6, whereas Abdominal B (Abd-B) is expressed in the NB6-4a lineage of segments A7-A8. Whereas loss of Antp function does not affect the NB6-4t lineage in any of the thoracic segments, loss-of-function mutations in abd-A and Abd-B cause NB6-4a-to-NB6-4t homeotic transformations in their corresponding segments. Interestingly, Ultrabithorax (Ubx), which is expressed in most of the cells of T3, is specifically absent in the NB6-4t lineage of that segment, and its loss-of-function alleles do not show any thoracic phenotypes. However, overexpression of Ubx as well as abd-A causes NB6-4t-to-NB6-4a transformations. Thus, it seems that the NB6-4t fate is the ground state and the NB6-4a state is imposed by the function of homeotic genes of the bithorax-complex (BX-C). This is consistent with previous reports that the T2 state is the ground state (for epidermis, including adult appendages) and other segmental identities are conferred by the function of homeotic genes (Berger, 2005).

The mechanism was examined by which abd-A or Abd-B specify the NB6-4a lineage compared with the NB6-4t lineage. As the mode and number of mitoses is the most obvious characteristic by which the NB6-4a lineage differs from NB6-4t, it was wondered whether factors regulating the cell cycle might be involved in controlling NB6-4 cell fate. One major factor that regulates the cell cycle is the G1 Cyclin CycE, which is needed for various aspects of the G1-to-S-phase transition (Berger, 2005).

To examine possible effects on cell fate decisions in the NB6-4t lineage, CycEAR95-mutant embryos were stained for gcm transcripts and Pros and Repo proteins. In wild-type embryos, gcm is initially distributed to both daughter cells during the first division of NB6-4t, but subsequently gets rapidly removed in the cell that functions as a neuronal precursor. Pros is transferred asymmetrically into only one cell, where it is needed to maintain and enhance the expression of gcm, thereby promoting glial cell fate. In CycEAR95 embryos, even at late stages (up to stage 14), gcm mRNA is strongly expressed in both daughter cells after the first division of NB6-4t. Even distribution of Pros was observed in both daughter cells, which could be the cause of continued expression of gcm. Furthermore, the glial marker Repo revealed that both cells differentiate as glial cells. The NB6-4a lineage is not affected in CycEAR95-mutant embryos, suggesting that the requirement for zygotic CycE is specific to NB6-4t (Berger, 2005).

Whether ectopic expression of CycE in abdominal lineages causes the opposite effect was tested. The sca-GAL4 line was used to drive UAS-CycE to achieve early expression in the neuroectoderm. An asymmetric distribution of Pros to one of the two progeny cells was observed just after the first division of NB6-4a. At later stages an increase was observed in the number of cells in the NB6-4a lineage (up to 5 cells). Some of these cells migrated medially, as NB6-4 glial cells normally do, maintaining Pros expression at a lower level. They also expressed Repo, which confirmed their glial identity. Other cells stayed in a dorso-lateral position and did not stain for Repo, suggesting neuronal identity (Berger, 2005).

To further investigate if ectopic CycE had indeed induced a neuronal sublineage in NB6-4a, and to test whether CycE can function cell-autonomously, a cell transplantation technique was employed. Single progenitor cells (stage 7) from the abdominal neuroectoderm of horseradish peroxidase (HRP)-labelled donor embryos overexpressing CycE were transplanted into the abdominal neuroectoderm of unlabelled wild-type hosts (at the same stage). The lineages produced by the transplanted cells were identified by morphological criteria. In all six cases, where cell clones were derived from NB6-4a, they were composed of both glial cells and neurons exhibiting their respective characteristic structures and positions. Because the clones are located in a wild-type abdominal environment, this experiment provides evidence that ectopic expression of CycE causes asymmetric division of NB6-4a and confers neuronal identity to one part of the lineage in a cell-autonomous manner. In these single-cell transplantation experiments, similar observations were made for NB1-1 and NB5-4, which also generate segment-specific lineages. Thus, CycE seems to have a general role in establishing segment-specific differences in neuroblast lineages (Berger, 2005).

Next whether the requirement for CycE to specify the neuronal lineage in NB6-4t is due to altered cell-cycle phases was examined. In string mutants, NB6-4t (whose proliferation is blocked before its first division) expresses gcm mRNA, as well as Pros and hunchback protein, although it does not differentiate as a glial cell. The composition of NB6-4t and NB6-4a lineages were further analysed in embryos mutant for other factors that interact with CycE in cell-cycle regulation. dacapo (dap) is the Drosophila homologue of members of the p21/p27Cip/Kip inhibitor family, which specifically block CycE-cdk complexes. Interestingly, in dap-null-mutant embryos an additional glial cell was observed in the NB6-4a lineage, but the appearance of any neuron-like cells was not observed. Consistent with these results, overexpression of Dap resulted in a reduction in the number of neurons in the NB6-4t lineage (from the normal number of 6 to 2-4), but homeotic transformation of the lineage did not occur. The number of glial cells was never affected, neither in the thorax nor in the abdomen. Ectopic expression of p21, the human homologue of the Drosophila dacapo gene, generated similar phenotypes (Berger, 2005).

The influence of the transcription factor dE2F, which mediates the activation of several genes needed for the initiation of S phase, was tested. In dE2F-mutant embryos, unlike in CycE mutants, no homeotic transformation of NB6-4t to NB6-4a was observed, although the number of neurons was reduced from 5-6 to 2-4. Ectopic expression of dE2F resulted in an increase in cell number in some abdominal hemisegments. In only a small percentage of those embryos, cells at lateral positions in abdominal segments did not show expression of gcm or Repo, suggesting their neuronal identity. Thus, although dE2F activation in the CNS depends on CycE, ectopic expression of dE2F cannot fully bypass the requirement for CycE in a NB6-4a-to-NB6-4t transformation. Similarly, ectopic expression of Rbf, a potent inhibitor of E2F target genes, did not cause any changes in the segregation of gcm mRNA, Pros or Repo in the NB6-4t lineage (Berger, 2005).

Whether interfering with another checkpoint of the cell cycle, the transition from G2 to M phase, affects NB6-4 cell fate was tested. Previous studies show that loss of CycA function prevents further mitosis after the first division of NB6-4t. However, the first division of NB6-4t follows the normal pattern; it gives rise to one glial and one neuronal cell. Similar effects were observed in CycA mutants. The cyclin-dependent kinase cdc2 heterodimerizes with CycA and CycB, and high levels of cdc2 expression have been shown to be required for maintaining the asymmetry of neuroblast divisions. In a cdc2 loss-of-function background, NB6-4t generated a normal lineage consisting of two glial cells and five to six dorso-lateral neurons. These observations show that NB6-4 cell fates do not change after manipulation of the transition from G2 to M phase (Berger, 2005).

These results suggest a critical role for CycE per se in regulating the NB6-4t lineage. Therefore whether CycE itself is differentially expressed between thoracic and abdominal NB6-4 lineages was tested. In situ hybridization with CycE RNA on wild-type embryos revealed that CycE is expressed just before the first division in NB6-4t. After the first division, CycE mRNA was detected in the neuronal precursor only and not in the glial precursor. In abdominal segments, no CycE expression was detected in NB6-4a before or after the division. Consistent with the role of CycE in specifying the NB6-4t lineage, notable levels of CycE transcripts were detected in the homeotically transformed NB6-4a lineages in abd-A-mutant embryos. Conversely, overexpression of abd-A caused down-regulation of CycE levels in thoracic segments and homeotic transformation of NB6-4t to NB6-4a. The importance of CycE in generating neuronal cells in the NB6-4t lineage was confirmed in an epistasis experiment involving abd-A and CycE mutants. As described above, loss of abd-A leads to transformation of NB6-4a to NB6-4t. Such homeotic transformation was suppressed by mutations in CycE, suggesting an absolute requirement for CycE in specifying the NB6-4t lineage. Finally, nine potential AbdA-binding sites (five of which are evolutionarily conserved in Drosophila pseudoobscura) were identified in a 5.0-kb enhancer fragment of CycE that is known to harbour cis-acting sequences for driving CycE expression in the CNS (Berger, 2005).

It is concluded that, in addition to its role in cell proliferation, CycE is necessary and sufficient for the specification of cell fate in the NB6-4 lineage. These results suggest that the function of CycE in regulating cell fate in NB6-4 lineages is independent, albeit partially, of its role in cell proliferation. The absence of any cell fate changes in the loss-of-function mutants of string, dap, cdc2 or CycA and in the Dap or Rbf gain-of-function genetic background may be attributed to the presence of CycE, which is strongly expressed in the NB6-4t, but not the NB6-4a, lineage. However, CycE may still function by controlling cell-cycle progression. NB6-4a, which does not express CycE, divides once followed by a cell-cycle arrest, presumably in G1. After the first division in the thorax, one daughter cell expresses high levels of CycE and divides roughly three times to generate neuronal cells. Therefore, this daughter cell presumably progresses through S phase. Chromatin reorganization during S phase might allow cell fate regulators to access their target genes, driving neuronal differentiation. Contrary to this interpretation, the other daughter cell of NB6-4t, which does not express CycE, divides twice but still generates glial cells. Thus, it remains to be investigated whether the role of CycE in neuronal cell fate determination is entirely independent of its role in cell proliferation. The results on the role of CycE in specifying neuronal compared with glial cell fate in the CNS are consistent with data from Xenopus on the role of cyclin-cdk complexes in specifying neuronal cell fate, inhibition of which promotes glial cell fate. In addition, this study shows that homeotic genes contribute to regional diversification of cell types in the CNS through the regulation of CycE levels (Berger, 2005).

Regulation of body pigmentation by the Abdominal-B Hox protein and its gain and loss in Drosophila evolution

Hox genes have been implicated in the evolution of many animal body patterns, but the molecular events underlying trait modification have not been elucidated. Pigmentation of the posterior male abdomen is a recently acquired trait in the Drosophila melanogaster lineage. This study shows that the Abdominal-B (Abd-B) Hox protein directly activates expression of the yellow pigmentation gene in posterior segments. Abd-B regulation of pigmentation evolved through the gain of Abd-B binding sites in a specific cis-regulatory element of the yellow gene of a common ancestor of sexually dimorphic species. Within the melanogaster species group, male-specific pigmentation has subsequently been lost by at least three different mechanisms, including the mutational inactivation of a key Abd-B binding site in one lineage. These results demonstrate how Hox regulation of traits and target genes is gained and lost at the species level and have general implications for the evolution of body form at higher taxonomic levels (Jeong, 2006).

The functions of homeotic genes in the development of body parts have long been viewed as operating at the top tier of a hierarchical process. In the conceptual framework first formulated by Garcia-Bellido, individual Hox genes act as “selector” genes that direct the differentiation of initially similar developmental fields along one of several alternative paths. They were envisioned to operate by regulating the activity of teams of “realizator” genes that determine the size and shape of individual structures. The reaper gene, which encodes a cell-death-promoting protein, is one such realizator that affects segment shape. It has not been shown previously whether realizator genes also include structural genes involved in terminal cell differentiation. Most direct Hox-regulated target genes identified thus far encode transcription factors and signaling proteins that act by controlling the expression of other genes. The current results show that Hox regulation of morphological traits is not strictly hierarchical in that direct Hox regulation of genetic circuits extends to the level of terminal differentiation genes (Jeong, 2006).

It was not anticipated that Abd-A regulation of pigmentation patterns would be mediated by the direct control of pigmentation genes. Throughout the melanogaster species group, sexually dimorphic pigmentation patterns correlate with dimorphic regulation of the bab locus, which depends on Abd-B function. Therefore, it was initially inferred that bab and perhaps other regulatory loci were interposed between Abd-B and pigmentation genes. The available evidence suggests that Bab regulates yellow indirectly, through the regulation of other transcription factors (Jeong, 2006).

At least three regulatory interactions have evolved for the expression of male-specific pigmentation of the A5 and A6 segments: the direct control of pigmentation genes by Abd-A, the sexually dimorphic regulation of bab, and the repressive function of bab on abdominal pigmentation. Analysis of the evolution of the body cis-regulatory element (CRE) sequence and function and previous studies of bab expression enable mapping of the evolutionary gain and loss of these interactions onto the history of the species considered in this study. To do so, the state of regulatory interactions in common ancestors of these species must first be inferred (Jeong, 2006).

Based upon the distribution of Abd-A binding sites, Abd-A-responsive body CREs, and dimorphic BAB regulation, it is inferred that all three regulatory interactions existed in the common ancestor of the melanogaster group, and perhaps in a common ancestor of the melanogaster and obscura groups. The reason for the uncertainty between these two nodes is the surprising evidence for Abd-A regulation of the body CRE in D. subobscura (and D. pseudoobscura). From mature adult phenotypes alone, one would not infer any sexually dimorphic abdominal pigmentation or regulation in the obscura group. However, in one member of the obscura species group (D. guanche), more intense pigmentation of the posterior segments was observed. Coupled with the discovery of a weak but functional Abd-A BS7 binding site and Abd-A responsiveness of the D. subobscura body CRE, these observations suggest that Abd-A regulated yellow expression and pigmentation in an ancestor of the obscura group. These results underscore the potential for knowledge of the underlying regulatory architecture of trait formation to inform evolutionary reconstructions beyond what is possible with phenotypes alone (Jeong, 2006).

The absence of sexually dimorphic posterior pigmentation and the monomorphic expression of body CREs in groups outside of the melanogaster and obscura groups place the origin of male-specific pigmentation to within the clade consisting of the melanogaster and obscura species groups. The mechanism for the origin of elevated posterior pigmentation is evident from the functional dissection of the body CRE here—namely, via the gain of Abd-A binding sites and sites for any obligatory cofactors in a CRE that controlled expression of the yellow gene in the abdomen. This scenario for the gain of Abd-A regulation is conceptually identical to that which has been put forth for the co-option of extant transcription factors and CREs in the evolution of novel patterns of gene expression in the wing (Jeong, 2006).

Evidence was found for three different genetic mechanisms for the loss of male-specific pigmentation. First, in D. kikkawai, the mutational inactivation of the key Abd-A binding site BS7 is sufficient to inactivate an otherwise functional element. Second, in D. bipectinata, a change in the regulation of the bab locus from dimorphic to monomorphic expression is sufficient to account for the evolutionary loss of male-specific pigmentation (while the body CRE has retained responsiveness to Abd-B). And third, in D. santomea, the sexually dimorphic activity of the D. santomea body CRE in D. melanogaster, the perfect conservation of Abd-A sites, and the conserved dimorphic bab expression pattern in D. santomea indicate that none of these three regulatory interactions have changed in D. santomea; rather, evolutionary changes at other loci have been involved in the loss of pigmentation. The frequent loss of pigmentation patterns suggests that evolutionary loss of parts of regulatory circuits may be fairly common (Jeong, 2006).

One of the longstanding goals of evolutionary biology has been to identify genetic events responsible for morphological change and to elucidate how changes at the molecular level translate into phenotypic diversity. In recent years, two distinct approaches have been taken to meet this goal. The first has been comparative studies of gene expression during development, which have identified correlations between the deployment of regulatory genes, particularly Hox genes, and differences in morphology. The second approach has been direct genetic analysis of intraspecific variation and interspecific differences. For many years, these two approaches have been far apart because of their different scales of analysis. Most comparative studies have focused on slowly evolving traits among higher taxa, and genetic analyses have been necessarily restricted to species that produce fertile hybrids, among which the type and extent of morphological variation is much more limited. The question has remained open in some minds as to whether the mechanisms that produce morphological variation and divergence at the species level are sufficient to explain the larger-scale divergences at higher taxonomic levels (Jeong, 2006).

The approach to bridging this gap between comparative embryology and evolutionary genetics has been to analyze more rapidly evolving characters. Because of the major influence that the comparative study of Hox genes and Hox-regulated structures has had to date in evolutionary developmental biology, the demonstration of the evolution of a direct Hox-regulated trait and target gene among closely related Drosophila species should help address concerns about the extrapolation of microevolutionary processes to macroevolution. It is seen here, in the evolution of segmental pigmentation and the molecular mechanisms of the evolutionary gain and loss of Hox target regulation among closely related species, the very sort of process that has been proposed to explain the large-scale divergence of homologous structures over much greater evolutionary distances (Jeong, 2006).

Co-ordinated control of cell adhesión, cell polarity and cytoskeleton underlies Hox-induced organogenesis in Drosophila

Hox genes control animal body plans by directing the morphogenesis of segment-specific structures. As transcription factors, HOX proteins achieve this through the activation of downstream target genes. Much research has been devoted to the search for these targets and the characterization of their roles in organogenesis. This has shown that the direct targets of Hox activation are often transcription factors or signaling molecules, which form hierarchical genetic networks directing the morphogenesis of particular organs. Importantly, very few of the direct Hox targets known are 'realizator' genes involved directly in the cellular processes of organogenesis. This study describes a complete network linking the Hox gene Abdominal-B to the realizator genes it controls during the organogenesis of the external respiratory organ of the larva. In this process, Abdominal-B induces the expression of four intermediate signaling molecules and transcription factors, and this expression results in the mosaic activation of several realizator genes. The ABD-B spiracle realizators include at least five cell-adhesion proteins, cell-polarity proteins, and GAP and GEF cytoskeleton regulators. Simultaneous ectopic expression of the Abd-B downstream targets can induce spiracle-like structure formation in the absence of ABD-B protein. It is concluded that Hox realizators include cytoskeletal regulators and molecules required for the apico-basal cell organization. HOX-coordinated activation of these realizators in mosaic patterns confers to the organ primordium its assembling properties. It is proposed that during animal development, Hox-controlled genetic cascades coordinate the local cell-specific behaviors that result in organogenesis of segment-specific structures (Lovegrove, 2006).

To initiate spiracle organogenesis, ABD-B, in combination with local signaling molecules, activates a set of targets within the dorsal area of A8. This study shows that there may be as few as four direct targets for the posterior spiracle. The expression of the primary targets, with their corresponding cofactors, subdivides the organ into specific regions. After this patterning stage, specific cell behaviors are controlled by another set of transcription factors that include the GATA transcription factor Grn to bring about cell rearrangements, and the JAK/STAT signaling pathway, which induces posterior spiracle-cell elongation. The partially overlapping expression of these transcription factors has the potential to activate in particular subsets of spiracle cells different sets of realizator genes. In the spiracles, these realizators include cell-adhesion molecules, apico-basal polarity proteins, and cytoskeletal regulators. Thus, in this way, ABD-B activates a genetic cascade coordinating the local cell-specific behaviors that result in organogenesis (Lovegrove, 2006).

Two main issues may explain why identification of the realizator genes has been so difficult. Primarily, by nature, many of these molecules are required for general functions in all cells. A screen for Hox realizators based on finding segment-specific defects would miss molecules like E-Cad or the Rho GTPases because of generalized embryonic malformations. Thus, their realizator nature can only be uncovered when, through intermediate regulators, a link to the HOX protein is found. This is demonstrated in the case of crb where a specific spiracle enhancer was found, that directs its increased transcription. In the case of the cytoskeleton, the link is made through the use of specific regulatory GEF and GAP proteins that modulate the activity of the GTPases. A second problem has been that some of the realizator molecules function redundantly and therefore a mutational approach yields no result. This is the case with the nonclassic cad88C and cad96C, which only show a mutant phenotype if E-cad is also mutated. Although cell-adhesion molecules had been originally proposed to be realizators, it is surprising to find that there are four nonclassical cadherins with restricted expression in the spiracle (Lovegrove, 2006).

Another unexpected finding has been the observation that the expression of apical- and basolateral-membrane proteins is modulated in the spiracle during the elongation stages. This study has established a link between ABD-B and the apical determinant crb through the JAK/STAT pathway. During invagination, spiracle cells are going through major membrane reorganization, including apical constriction and basal elongation. Thus, Crb, which is required in many epithelia for maintenance of a proper zonula adherens, may be playing an important role for the polarized remodeling along the apico-basal cell axis. Crb upregulation is functionally important for cell elongation, but it is not the only function controlled by STAT. In this respect, it is important to note that spiracle-cell elongation occurs mainly through the increase of basolateral membranes. It is thus likely that the spiracle-gene network will also be controlling basal polarity determinants (Lovegrove, 2006).

A role for ABD-B in regulation of the cytoskeleton in the posterior spiracles was expected because of the initial observations on cell elongation taking place in the spiracular chamber. The observed effects of the dominant-negative and constitutively active forms of Rho GTPases on spiracle development support this hypothesis. The finding of Gef64C regulation by ABD-B in the spiracle cascade and the finding of spiracle invagination defects in RhoGAP cv-c mutants confirms that specific control of the Rho GTPases is an important feature of spiracle development (Lovegrove, 2006).

Although all the realizators analyzed in this study are activated indirectly by ABD-B, the possibility cannot be excluded that ABD-B can also activate some others directly. Direct regulation of realizator genes by HOX may be important for differentiation of specific cell types (Lovegrove, 2006).

This study has linked the activity of a HOX protein, through the regulation of a small number of intermediate regulators, to a battery of realizator genes. The local-specific modulation of these genes that in other contexts control cell adhesion, polarity, and organization of the cytoskeleton, would be sufficient to confer unique morphogenetic properties to the cells leading to the formation of a segment specific organ. Other examples in Drosophila include salivary-gland organogenesis, where SCR initially activates a cascade of downstream genes, and head formation where DFD activates Dll but similar processes must be occurring in Hox-controlled organogenesis in vertebrates (Lovegrove, 2006).

Drosophila castor is regulated negatively by the Ubx and abdA genes, but positively by the AbdB gene

The ventral nerve cord (VNC) of Drosophila exhibits significant segmental-specific characteristics during embryonic development. Homeotic genes are expressed over long periods of time and confer identity to the different segments. castor (cas) is one of the genes which are expressed in neuroblasts along the VNC. However, at late embryonic stages, cas transcripts are found only in head and thoracic segments and terminal abdominal segments, while Cas protein lasts longer in all segments. This study investigated the regulation of temporal and spatial expression of cas by the bithorax complex genes. In the loss-of-function mutants of Ultrabithorax (Ubx) and abdominal-A (abdA), cas transcripts were ectopically expressed in abdominal segments at late embryonic stage. However, unlike in Ubx and abdA mutants, in Abdominal-B (AbdB) loss-of-function mutant embryos, cas disappeared in the terminal region. Ectopic Ubx and abdA suppressed cas expression, but ectopic AbdB activated cas expression in most abdominal segments. Moreover, cas was co-expressed in the cells in which AbdB was normally expressed, and overexpressed in the ectopically expressed AbdB embryos. These results suggest that the expression of cas is segment-specifically regulated negatively by Ubx and abdA genes, but positively by the AbdB gene (Ahn, 2010).

cas is transiently expressed in a subset of neuroblasts in their cell lineage. Its transcripts are present with homologous patterns in thorax and abdomen at early embryonic stages. At late embryonic stages, cas transcripts are found in only a few cells per hemisegment in thoracic and posterior abdominal segments, but not in other abdominal segments. This indicates that cas is expressed in segment-specific mode during late embryonic stages. This study investigated how this regional diversity was produced (Ahn, 2010).

The segment-specific expression of cas was regulated by the homeotic genes. Ubx or abdA mutation caused the homeotic transformation of abdominal cuticle belts to the more anterior ones. These transformation patterns were also observed in cas expression. Mutations in Ubx or abdA genes caused ectopic cas expression in abdominal segments, which was normally observed in the thoracic segments at stage 15 of wild-type embryo, suggesting transformation of the thoracic pattern to an abdominal pattern at that stage. This transformation was synergistically enhanced in the Ubx and abdA double mutants (Ahn, 2010).

cas was ectopically expressed in A1 segment in Ubx mutant embryos. This result is coincident with the function of Ubx to specify the posterior thorax and a portion of A1 segment. cas was also ectopically expressed in A1 to A4 segments in abdA mutant embryos, which coincide with the function of abdA. In Ubx abdA double mutant embryos, cas was expressed in virtually all abdominal segments and in more cells than in each single mutant embryo. The roles of Ubx and abdA on cas expression in the abdominal segments were confirmed in the ectopically expressed Ubx and abdA mutant embryos. For this experiment, proper embryonic stages were very important because cas expression changed dramatically in the abdominal segments between stages 14 and 15. Whether ectopic Ubx or abdA repressed cas expression in the abdominal segments was tested at this stage. The GAL4-mediated induction of Ubx or abdA suppressed cas expression in the abdominal segments (Ahn, 2010).

However, in contrast to the Ubx and abdA mutations, AbdB mutation caused reverse effects on cas expression in the abdominal segments, which have never been reported. Loss-of AbdB function caused lack of cas expression, while ectopic ABDB activated cas expression in the abdominal segments. This phenotype was also observed in Polycomb mutant embryos. Although Polycomb mutation induced ectopic expression of Ubx, abdA and AbdB at the same time, cas was ectopically expressed in the abdominal segments of stage15 embryos, suggesting that ABDB dominated the effects of ectopic UBX or ABDA. The co-localization of cas and ABDB is found in a few cells in the posterior abdominal segments, supporting the positive regulation of cas by ABDB (Ahn, 2010).

This idea was further intensified by the appearance of the ectopic cas mRNA in the numerous abdominal cells with the ectopic AbdB expression. Real-time PCR experiment showed the overexpession of cas mRNA in the ectopically expressed AbdB embryos, also supporting the positive regulation of cas by ABDB. Furthermore, seven AbdB DNA binding sites were found within 5kb upstream from the cas transcription start site enhancing the possibility that ABDB directly regulates the cas expression. ABDB binds preferentially to a sequence with an unusual 5'-TTAT-3' core (Ahn, 2010).

One of questions was why all the cells with the AbdB expression does not show cas mRNA expression. In wild-type embryos, all AbdB-expressing cells does not show cas mRNA. Only a few cells among AbdB-expressing cells could maintain the expression of cas and the other cells lost it. This might be that the homoetic proteins carry out their function by interacting with other cofactors to regulate distinct sets of downstream genes (Ahn, 2010).

Accumulating evidence shows that the bithorax complex genes are involved at different steps in the segment-specific divergence of the CNS. Ubx or abdA activity is required for the abdominal pathway of the NB1-1 lineage. Both ectopic induction of Ubx- or abdA expression until several hours after gastrulation and homeotic de-repression in Polycomb mutants, override thoracic determination of NB1-1. The abdominal NB6-4 lineage is also specified by the abdA and AbdB. abdA is expressed in the NB6-4 lineage of abdominal segments A1-A6, whereas AbdB is expressed in the NB6-4 lineage of segments A7-A8. They specify the abdominal NB6-4 lineage by down-regulating levels of G1 Cyclin (CycE) (Ahn, 2010).

In summary, UBX and ABDA suppress cas expression in abdominal segments, so that mutation in both genes causes ectopic expression of cas in abdominal segments at late embryonic stage. However, ABDB activates cas expression, which is supported by co-localization of cas and ABDB in cells ectopically expressing AbdB, and real-time PCR in ectopically expressed AbdB embryos (Ahn, 2010).

Drosophila Hox and sex-determination genes control segment elimination through EGFR and extramacrochetae activity

The formation or suppression of particular structures is a major change occurring in development and evolution. One example of such change is the absence of the seventh abdominal segment (A7) in Drosophila males. This study shows that there is a down-regulation of EGFR activity and fewer histoblasts in the male A7 in early pupae. If this activity is elevated, cell number increases and a small segment develops in the adult. At later pupal stages, the remaining precursors of the A7 are extruded under the epithelium. This extrusion requires the up-regulation of the HLH protein Extramacrochetae and correlates with high levels of spaghetti-squash, the gene encoding the regulatory light chain of the non-muscle myosin II. The Hox gene Abdominal-B controls both the down-regulation of spitz, a ligand of the EGFR pathway, and the up-regulation of extramacrochetae, and also regulates the transcription of the sex-determining gene doublesex. The male Doublesex protein, in turn, controls extramacrochetae and spaghetti-squash expression. In females, the EGFR pathway is also down-regulated in the A7 but extramacrochetae and spaghetti-squash are not up-regulated and extrusion of precursor cells is almost absent. These results show the complex orchestration of cellular and genetic events that lead to this important sexually dimorphic character change (Foronda, 2012).

The elimination of a part of an animal body is a major change occurring during morphogenesis and evolution. This study has analyzed the mechanisms required for one such change, the absence of the male seventh abdominal segment. The study shows that the suppression of this segment involves the interplay between Hox and the sex determining genes, which regulate targets implementing the morphological change. The reduction or suppression of this segment is also a sexually dimorphic feature characteristic of higher Diptera, so the mechanisms shown here may be relevant for the evolution of morphology (Foronda, 2012).

In early pupa, during the second phase of cell division, there is a reduction in the number of A7 histoblasts, both in males and females, but stronger in males perhaps because wg is not expressed in the male A7 histoblasts. It has been shown that fewer histoblasts result in a smaller adult segment. Therefore, the reduced number of A7 histoblasts may account in part for the reduced size of the A7 segment in females. The control of the second phase of cell division involves the EGFR pathway, and Abd-B was found to reduce the number of histoblasts in the A7 through down-regulation of EGFR activity. If this activity is eliminated in the male A7, an increase is observed the number of histoblasts, that many of these cells remain at the surface at the time of extrusion and that a small A7 forms in the adult. It was also previously reported that a small A7 is observed in the male adult when expressing vein, an EGFR ligand. It is possible that the high number of histoblasts obtained when over-expressing elements of the EGFR pathway makes many of them unable to be extruded by a 'titration' effect, that is, there may be 'too many' histoblasts for the invagination mechanism to extrude them at the correct time. However, the EGFR pathway may also hinder extrusion since lower levels are seen of emc-GFP and also many histoblasts remain at the surface after high EGFR activation (Foronda, 2012).

At later pupal stages (around 35-40 h APF) there is the extrusion of the male A7 histoblasts. It was observed, however, that a few histoblasts also invaginate in the female A7, suggesting the male intensifies a mechanism present in both sexes. The extrusion requires the activity of emc, and correlates with higher emc expression in the male A7 histoblasts at about the time of extrusion. The invagination of histoblasts superficially resembles that of larval cells, and it also requires myosin activity. This would suggest that, due to the higher levels of Abd-B and DsxM, male A7 histoblasts may have adopted a mechanism similar to that used by cuticular larval epidermal cells (LECs) for their elimination. Recent reports, however, suggest an alternative mechanism. It has been demonstrated that an excess of proliferation in the epithelium leads to cell death-independent cell extrusion. Since this study has observed that prevention of cell death in the male A7 does not cause the development of an A7 (although delamination is delayed), the mechanism driving extrusion may be more similar to that of an overproliferating epithelium than to that taking place in larval cells (Foronda, 2012).

The data are consistent with emc increasing the expression of spaghetti-squash to accomplish apical constriction and extrusion. However, high expression of emc may not be sufficient to effectively induce histoblast extrusion, suggesting other genes are required. Besides, a strong reduction of emc leads to a very small and poor differentiated male A7 segment, reflecting that this gene is required for several cellular functions, among them cell survival. Perhaps significantly, emc is also expressed in embryonic tissues preceding invagination of different structures in the embryo, suggesting a common requirement for invagination at different developmental stages. It is thought that emc forms part of complex networks that have, among other cellular functions, that of contributing to the extrusion of A7 histoblasts (Foronda, 2012).

Although regulation of the EGFR pathway and emc are two key events in controlling male A7 development, previous experiments have also shown the contribution of the wingless gene, absent in male A7 but present in male A6 and female A7, in the development of this segment. These results have been confirmed and it was also shown that a reduction in wg expression can partially suppress the Abd-B mutant phenotype. Absence of wg is probably required to reduce cell proliferation in the male A7 but the data suggest wg may also be needed to maintain high emc levels. Apart from the role of wg, it was also shown that some A7a cells are transformed into A6p cells, thus reducing the number of A7 cells that might contribute to the adult segment. Finally, the expression of bric-a-brac must also be down-regulated in male A7 histoblasts to eliminate this metamere. Thus, this suppression is a complex process using different genes and mechanisms (Foronda, 2012).

The suppression of the male A7 depends ultimately on the levels of Abd-B expression. The role of this Hox gene is probably mediated in part by dsx, since Abd-B regulates dsx transcription and dsx governs, in turn, the expression of genes required for cell proliferation and extrusion. That Hox genes regulate dsx expression has also been demonstrated in the male foreleg, suggesting that Hox genes specify the different parts of the body where sexual dimorphism may evolve. The different dsx isoforms (DsxF and DsxM) determine the outcome of this regulation. A significant difference between the activities of these two proteins in the A7 is the regulation of emc levels. In the female, emc expression is similar in the A7 and the A6 and, accordingly, histoblast extrusion in females is small and confined to the central dorsal region, a domain virtually absent in the adult tergite. By contrast, the DsxM isoform increases Emc expression to drive large extrusion of A7 cells and elimination of the segment (Foronda, 2012).

Only the male A7, but not anterior abdominal segments, is eliminated. Therefore, the increase in emc expression, and subsequent events observed in the A7, depends on the higher Abd-B expression in the A7 in relation to the A6. Several Hox loci, like Sex combs reduced, Ultrabithorax or Abd-B are haplo-insufficient, and relatively small differences in the amount of some of these Hox proteins can drive major phenotypic changes, suggesting some downstream genes can sense these slight differences and implement major changes in morphology (Foronda, 2012).

Previous studies have shown the cooperation of Abd-B and the sex determination pathway in controlling the pigmentation of the posterior abdomen. It is thought that Abd-B plays a dual role in regulating the morphology of the posterior abdomen. First, it regulates dsx expression, thus allowing the possibility to develop sexually dimorphic characters; second, it cooperates with Dsx proteins in establishing pattern. Part of the effect implemented by Abd-B may be mediated by the levels of expression of dsx (distinguishing male A6 from male A7), and from the nature of the Dsx proteins (male and female ones). Although there is no conclusive evidence that the different levels of dsx in the A6 and A7 play a role in development, it is noted that this difference correlates with that of Abd-B (and depends on it), that high levels of DsxM are sufficient to increase emc-GFP in the A7 of females and eliminate this segment, and that these same high levels similarly increase emc-GFP and partially rescue the Abd-B mutant phenotype in males. Hox genes, therefore, may provide a spatial cue along the anteroposterior axis to activate dsx transcription and allow the formation of sexually dimorphic characters, but they may also cooperate with Dsx proteins to determine different morphologies. This double control by Hox genes may apply to all the sexually dimorphic characters and be also a major force in evolution (Foronda, 2012).

Hox-mediated regulation of doublesex sculpts sex-specific abdomen morphology in Drosophila

Hox transcription factors are deeply conserved proteins that guide development through regulation of diverse target genes. Furthermore, alteration in Hox target cis-regulation has been proposed as a major mechanism of animal morphological evolution. Crucial to understanding how homeotic genes sculpt the developing body and contribute to the evolution of form is identification and characterization of regulatory targets. Because target specificity is achieved through physical or genetic interactions with cofactors or co-regulators, characterizing interactions between homeotic genes and regulatory partners is also critical. In Drosophila, sexually dimorphic abdominal morphology results from sex-specific gene regulation mediated by the Hox protein Abdominal-B (Abd-B) and products of the sex-determination gene doublesex (dsx). Together these transcription factors regulate numerous sex-specific characters, including pigmentation, cuticle morphology, and abdominal segment number. This study shows that Dsx expression in the developing D. melanogaster pupal abdomen is spatiotemporally dynamic, correlating with segments that undergo sexually dimorphic morphogenesis. Furthermore, genetic analyses show Dsx expression is Abd-B dependent. It is concluded that Doublesex and Abd-B are not only requisite co-regulators of sexually dimorphic abdominal morphology, but also that dsx is itself a transcriptional target of Abd-B. These data present a testable hypothesis about the evolution of sexually dimorphic segment number in Diptera (Wang, 2012).

That dsx is transcriptionally regulated downstream of Abd-B presents testable hypotheses about the evolution of posterior abdominal morphology in the Cyclorrhapha. One possibility is that dsx was an Abd-B target before the Cyclorrhaphan radiation. Ancestral to Cyclorrhapha, this spatial regulation of dsx may have contributed to dimorphic traits more subtle than segment number. Although evidence is still needed, it is proposed that, by analogy with bab regulation, male-specific wg repression is mediated directly by Abd-B and DsxM acting on a wg abdominal CRE. The gain of Abd-B and Dsx binding sites in a wg abdominal CRE may have been a critical step in the evolution of sexually dimorphic segment number in the Cyclorrhapha (Wang, 2012).

Alternatively, evolution of the Cyclorrhaphan body plan may first have required changes to the abdominal trans-regulatory landscape: a critical step being novel dsx abdominal expression under control of Abd-B. This evolution of overlapping positional and sex-specific regulatory proteins may have co-opted target genes of this pair of transcription factors, necessary for sexually dimorphic development of the genital, into processes that pattern development of the posterior abdomen. Novel abdominal specific targets of this regulatory partnership would likewise have been acquired through independent cis-regulatory evolution (Wang, 2012).

Sequence-specific interaction between ABD-B homeodomain and castor gene in Drosophila

The effect were analyzed of bithorax complex genes on the expression of castor gene. During the embryonic stages 12-15, both Ultrabithorax and abdominal-A regulated the castor expression negatively, whereas Abdominal-B showed a positive correlation with the castor gene expression according to real-time PCR. To investigate whether ABD-B protein directly interacts with the castor gene, electrophoretic mobility shift assays were performed using the recombinant ABD-B homeodomain and oligonucleotides, which are located within the region 10 kb upstream of the castor gene. The results show that ABD-B protein directly binds to the castor gene specifically. ABD-B binds more strongly to oligonucleotides containing two 5'-TTAT-3' canonical core motifs than the probe containing the 5'-TTAC-3' motif. In addition, the sequences flanking the core motif are also involved in the protein-DNA interaction. The results demonstrate the importance of HD for direct binding to target sequences to regulate the expression level of the target genes (Kim 2013).

Hox proteins mediate developmental and environmental control of autophagy

Hox genes encode evolutionarily conserved transcription factors, providing positional information used for differential morphogenesis along the anteroposterior axis. This study shows that Drosophila Hox proteins are potent repressors of the autophagic process. In inhibiting autophagy, Hox proteins display no apparent paralog specificity and do not provide positional information. Instead, they impose temporality on developmental autophagy and act as effectors of environmental signals in starvation-induced autophagy. Further characterization establishes that temporality is controlled by Pontin, a facultative component of the Brahma chromatin remodeling complex, and that Hox proteins impact on autophagy by repressing the expression of core components of the autophagy machinery. Finally, the potential of central and posterior mouse Hox proteins to inhibit autophagy in Drosophila and in vertebrate COS-7 cells indicates that regulation of autophagy is an evolutionary conserved feature of Hox proteins (Banreti, 2013).

Autophagy is a cellular process whose induction or inhibition involves multiple levels of regulation, including developmental signals conveyed by the steroid hormone ecdysone, and environmental signals, sensed in the case of amino acid starvation by the InR/dTOR pathways. These regulatory paths do not act independently but seem rather to be interconnected as illustrated by developmentally induced ecdysone-mediated autophagy that acts by repressing the inhibitory function of the InR pathway. This indicates that whereas upstream control is distinct, downstream control may be common (Banreti, 2013).

This study shows that Drosophila Hox proteins are potent inhibitors of autophagy, with a potent and equivalent impact on both developmental and starvation-induced autophagy, and establish that both converge in the regulation of Hox gene expression. This highlights Hox genes as central regulators of autophagy, acting as a node for mediating autophagy inhibition. In regulating autophagy, Hox proteins act at least through regulation of Atg genes and other autophagy genes. Consistent with a direct transcriptional effect of Hox proteins in controlling Atg genes, Ubx DNA binding was found to be essential for autophagy inhibition, whereas have previously shown that Ubx associates to genomic regions immediately adjacent to Atg5 and Atg7 genes (Banreti, 2013).

A key aspect underlying Hox-mediated autophagy control is the regulation of Hox gene expression, where Hox downregulation induces autophagy. This aspect is true for both developmental- and starvation-induced autophagy, where the dynamics of Hox proteins respond to ecdysone (developmental autophagy) and to InR/dTOR (starvation) signaling. Signals mediating changes in Hox gene expression result from changes in the expression of Pont, a facultative component of the Brm complex known to act as a global and positive regulator of Hox genes. Although not establishing changes in Brm complex composition at the L3 feeding/L3 wandering transition, the dynamics of Pont expression suggest that a Pont-depleted Brm complex loses its ability to maintain the expression of Hox genes, resulting in the release of Hox-mediated inhibition of autophagy (Banreti, 2013).

Hox proteins are widely described as providing spatial information required for differential morphogenesis along the A-P axis, within which they largely display paralog-specific activities. However, in regulating autophagy, Hox function is distinct. First, it appears to be generic, with all Hox proteins tested providing inhibitory activity. The need to alleviate global Hox gene function (achieved in this study by impairing the activity of the Brm complex) in order to induce autophagy, further supports their redundant function in inhibiting autophagy. Second, they provide temporal, instead of spatial, information, mediating the temporality of developmental autophagy downstream of ecdysone signaling. Third, in the case of starvation-induced autophagy, Hox genes respond to the InR/dTOR pathways, acting as environmental effectors (Banreti, 2013).

Investigating the evolutionary conservation of Hox-mediated inhibition of autophagy by exploring the activity of mouse Hox proteins in Drosophila fat body cells as well as in vertebrate COS-7 cells indicates that vertebrate Hox proteins also act as potent autophagy inhibitors. Further studies in vertebrate cells should frame their activity to the multiple physiological and pathological situations that involve autophagy and allow for deciphering the molecular modalities of their regulatory roles (Banreti, 2013).

In summary, these findings broaden the framework of Hox protein functions, showing that besides providing spatial information during development, they also coordinate temporal processes and, more surprisingly, act as mediators of environmental signals for autophagy regulation (Banreti, 2013).

Transvection-based gene regulation in Drosophila is a complex and plastic trait

Transvection, a chromosome pairing-dependent form of trans-based gene regulation, is potentially widespread in the Drosophila melanogaster genome, and varies across cell types and within tissues in D. melanogaster, characteristics of a complex trait. This study demonstrate that the trans-interactions at the Malic enzyme (Men) locus are, in fact, transvection as classically defined, and are plastic with respect to both genetic background and environment. Using chromosomal inversions, trans-interactions at the Men locus were eliminated by changes in chromosomal architecture that presumably disrupt somatic pairing. It was further shown that the magnitude of transvection at the Men locus is modified by both genetic background and environment (temperature), demonstrating that transvection is a plastic phenotype. These results suggest that transvection effects in Drosophila are shaped by a dynamic interplay between environment and genetic background. Interestingly, cis-based regulation of the Men gene was found to be more robust to genetic background and environment than trans-based. Finally, this study begins to uncover the non-local factors that may contribute to variation in transvection overall, implicating Abd-B in the regulation of Men in cis and in trans in an allele-specific and tissue-specific manner, driven by differences in expression of the two genes across genetic backgrounds and environmental conditions (Bing, 2014).

The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans

The origination and diversification of morphological characteristics represents a key problem in understanding the evolution of development. Morphological traits result from gene regulatory networks (GRNs) that form a web of transcription factors, which regulate multiple cis-regulatory element (CRE) sequences to control the coordinated expression of differentiation genes. The formation and modification of GRNs must ultimately be understood at the level of individual regulatory linkages (i.e., transcription factor binding sites within CREs) that constitute the network. This study investigated how elements within a network originated and diversified to generate a broad range of abdominal pigmentation phenotypes among Sophophora fruit flies. The data indicates that the coordinated expression of two melanin synthesis enzymes, Yellow and Tan, recently evolved through novel CRE activities that respond to the spatial patterning inputs of Hox proteins and the sex-specific input of Bric-a-brac transcription factors. Once established, it seems that these newly evolved activities were repeatedly modified by evolutionary changes in the network's trans-regulators to generate large-scale changes in pigment pattern. By elucidating how yellow and tan are connected to the web of abdominal trans-regulators, this study discovered that the yellow and tan abdominal CREs are composed of distinct regulatory inputs that exhibit contrasting responses to the same Hox proteins and Hox cofactors. These results provide an example in which CRE origination underlies a recently evolved novel trait, and highlights how coordinated expression patterns can evolve in parallel through the generation of unique regulatory linkages (Camino, 2015).

This study has traced the evolutionary history of two CREs required for a novel trait, and show that they have recently evolved similar expression patterns through remarkably different architectures in a common trans-regulatory landscape. The data indicates that the tergite-wide activities of the yBE and t_MSE did not exist in the monomorphic ancestor for Sophophora, but evolved in the lineage leading to the common ancestor of the melanogaster species group. The results support a scenario where the subsequent expansion and contraction of male pigmentation pattern was driven primarily by alteration of the trans-regulators, whereas repeated losses involved both cis- and trans-evolution with respect to these CREs. Though the t_MSE and yBE drive coordinated patterns of gene expression, striking differences were found in their upstream regulators and direct regulatory linkages. These results bear on the understanding of how new gene regulatory networks form, diversify, and how coordinated regulatory activities can arise through the independent evolution of unique regulatory codes (Camino, 2015).

Hox transcription factors play a prominent role in generating the differences in serially homologous animal body parts, and the origin of novelties. The diversification of homologous parts can be driven by changes in the spatial domains of Hox protein expression, as has been shown for crustacean appendage morphology, snake limblessness, and for the water strider appendage ground plan. Changes in the downstream Hox targets are evident in cases such as the hindwings of insects, and for fruit fly tergite pigmentation. The origin of novel structures can also be traced to the co-option of Hox proteins, as exemplified by cases such as the Photuris firefly lantern and the sex combs residing on the forelegs of certain Drosophila species. For many of these evolved traits, the molecular mechanisms by which Hox expression patterns and target genes evolve remain unknown (Camino, 2015).

While mechanistic studies on the evolution of Hox-regulated CREs remain limited, several target gene CREs have been thoroughly characterized and serve as exemplars of Hox-regulation during development. Hox proteins can interact with CRE binding sites as monomers or through cooperative interactions with Hox-cofactors. The activity of these bound complexes can be further modulated through interactions with collaborating transcription factors. However, to date, few direct Hox target linkages have been traced to their evolutionary beginnings. Expression of yellow in the male A5 and A6 segments required the gain of two binding sites for Abd-B, but it remains uncertain whether these binding events require cooperative interactions with Hox cofactors and which transcription factors are acting as collaborators (Camino, 2015).

The t_MSE presented an opportunity to study how a second Hox-responsive CRE evolved in parallel to the activity at yellow. This study shows that Abd-A and Abd-B respectively are necessary and sufficient for t_MSE regulatory activity. However, the ablation of the resident Hox sites had little effect on this CRE's activity in the A5 and A6 segments, though mutations to nearby CRE sequences resulted in dramatically reduced activity. This result strongly implies that both Abd-A and Abd-B indirectly activate the t_MSE through a downstream factor or factors. While it can't be entirely ruled out that these factors are operating directly through other non-canonical Hox sites, the gel shift assays did not provide convincing evidence that such sites exist. While the Hox sites were not necessary for activation in the A5 and A6 segments, their ablation resulted in a drastic gain of regulatory activity in the A4 and A3 segments, a setting in which Abd-A is the only Hox protein present. This indicates that Abd-A is a direct repressor of t_MSE function in these anterior abdomen segments. The observed dichotomy in Abd-A function can be explained by at least two-not necessarily mutually exclusive-scenarios. First, in the A5 and A6 segments Abd-B may not act as a direct activator of the t_MSE but its occupancy of Hox sites might preclude the direct repressive effects of Abd-A. Secondly, Abd-A may interact cooperatively or collaboratively with other transcription factors in the more anterior segments to impart repression. The results with Hth support this second scenario (Camino, 2015).

The Hox co-factors Hth and Exd were prime candidates to mediate the context-dependent modulation of Abd-A activity. First, RNAi suppression of hth and exd expression each resulted in ectopic pigmentation (Rogers, 2014) and t_MSE activity in the male A4 and A3 segments. Furthermore, inspection of the t_MSE sequence revealed sites characteristic of Hth (AGACAG) and Exd (GATCAT) binding that reside in close proximity to Hox sites. This site content and arrangement is strikingly similar to that found in an abdominal-repressive module for the CRE controlling thoracic Distalless expression. Along a similar vein, this study shows that the ablation of the Hth-like site led to an anterior expansion in t_MSE activity similar to that induced by the Hox site mutations. This outcome supports the interpretation that the more recent origin of the t_MSE involved the formation of novel regulatory linkages with Hox proteins and Hox cofactors (Camino, 2015).

Morphological traits result from the activities of gene regulatory networks, in which each network is governed by a trans-regulatory tier of transcription factors and cell signaling components that ultimately regulate the expression of a set of differentiation genes. For animals, the trans-regulatory genes are remarkably conserved. It is plausible that the origin of new morphologies occurs through the formulation of new gene regulatory networks, while diversification and losses in traits would likely occur through the modification and dismantling of extant networks. The empirical evaluation of such trends of network evolution necessitates the study of trait evolution at the level of networks, CREs, and their encoded binding sites for multiple animal lineages, traits, and evolutionary time frames. The Drosophila pigmentation system is particularly well poised to make pioneering contributions to this growing body of knowledge (Camino, 2015).

The most recent common ancestor of monomorphic and dimorphic Sophophora lineages was inferred to have possessed monomorphic tergite pigmentation, in the context of an otherwise invariant morphological landscape, in which segment number and form has remained conserved at the genus level. Hence, the origin of this novel pigmentation trait may be expected to have co-opted spatial and sex-specific patterning mechanisms that shape the conserved abdomen features. Comparative analysis of orthologous yellow and tan non-coding sequences indicate that these co-option events involved the origination of novel CRE activities that connected a trans-regulatory tier of Hox, Hox-cofactors, and the Bab proteins to these key differentiation genes that encoded pigmentation enzymes (Camino, 2015).

The patterns of regulatory activity for the orthologous tan and yellow sequences support some additional inferences about the early events in this dimorphic trait's origin. While the t_MSE abdominal activity was strikingly lower in D. pseudoobscura and D. willistoni, the D. pseudoobscura yellow body element was active (albeit with expanded activity). These outcomes support at least two evolutionary scenarios. One scenario is a sequence of events where the origination of the t_MSE and y_BE in the lineage of D. pseudoobscura was followed by a secondary loss of the t_MSE. This scenario is supported by a previous observation of dimorphic Bab expression in the D. pseudoobscura abdomen, backing the notion that this species' broad pattern of monomorphic abdominal pigmentation evolved from a dimorphic ancestral state. For the other scenario, the body element-like regulatory activity of D. pseudoobscura could be due to this CRE's origin preceding that of the t_MSE. Distinguishing between these two scenarios will require a more rigorous comparison of the pigmentation phenotypes and networks within the melanogaster and obscura species groups. The outcomes would provide a more nuanced understanding of the early evolutionary history for the derived sexually dimorphic pigmentation network (Camino, 2015).

Tergite pigmentation evolution in the Sophophora subgenus has been relatively well-studied, and the accumulated results frame an extended perspective of trait evolution within a common network. Trans-evolution at the bric-à-brac (bab) locus has been found to be a major driver for the diversification of female tergite pigmentation. This study, in addition to previous studies, indicates that trans-evolution at as of yet unidentified loci may have played prominent roles in the diversification of male-limited tergite pigmentation. Regarding the repeated losses in male pigmentation, the current results are consistent with a scenario where both trans- and cis-evolution occurred, though the targets of cis-evolution have alternated between tan and yellow. While cis-evolution has been identified for a case of monomorphic gain (ebony) in tergite pigmentation, and for a case of monomorphic loss (ebony and tan), the full wealth of case studies portend to a more prominent role for evolutionary changes in the trans-regulatory tier of the pigmentation gene network. However, it is important to note that many of these case studies only assessed the activities of transgenes in D. melanogaster. While similarities in CRE activity might be indicative that expression divergence occurred through trans-evolution, it does not rule out the possibility that cis-changes occurred at other regions in the pigmentation enzyme gene loci, or that expression divergence results from combined cis- and trans-changes. In the future, it will be important to validate or reject the prominent role for trans-regulatory evolution by the reciprocal tests of CREs in species with the contrasting patterns of pigmentation. Two studies where CREs were tested in species with contrasting pigmentation phenotypes, showed that trans-regulatory evolution was a major driver for diversification of fruit fly wing spot patterns by modifying Distalless and wingless expression (Arnoult, 2013; Werner, 2010). Thus it appears the notion of a “conserved trans-landscape” requires more scrutiny (Camino, 2015).

In this study, and elsewhere, experiments indicate that pigmentation losses are associated with and perhaps result from both changes in the trans-regulatory tier and in the cis-regulatory regions of the yellow and tan genes. Interestingly, some instances of trans-regulatory modifications that cause loss of gene expression appear to leave perfectly good CREs intact. The current data provides a second instance in which loss of expression occurred without the loss of the encoded CRE. The yBE was found to be conserved in D. santomea, which diverged from D. yakuba ~400,000 years ago. The activity for this CRE has also remained for D. ananassae since its divergence from a pigmented ancestor. In contrast, D. kikkawai has lost pigmentation while still expressing tan in the abdomen through a perfectly active t_MSE. These results suggest that these CREs were maintained within the population for long periods of time, perhaps indicating additional functions that promote the preservation of these CREs' ancestral potential. Furthermore, the observed heterogeneity of changes in cis and trans to yellow and tan were at first surprising. However, study of the binding site architecture at the yBE and t_MSE provided key clues as to why their evolution may often be uncoupled (Camino, 2015).

The coordinated expression of genes is a ubiquitous theme in developmental biology. Gene expression is finely regulated during development through the activities of CREs that are individually encoded as evolved combinations of transcription factor binding sites (regulatory logic). A compelling question is whether such synchronized expression results from the independent evolution of CREs with similar logics. This question was previously pursued for CREs of regulatory genes coordinately expressed in the developing fruit fly neurogenic ectoderm. In this case, the coordinately activated CREs are encoded by a common regulatory logic, or a so called 'cis-regulatory module equivalence class'. However, the neurogenic ectoderm CREs are deeply conserved, and arose in the distant past (over 230 million years ago) (Camino, 2015).

The recently evolved male-specific expression patterns for tan and yellow present a case in which the evolutionary formation of coordinated regulation can be observed over shorter time-scales. Though both the t_MSE and yBE0.6 drive reporter expression in the dorsal A5 and A6 segment epidermis of males during late pupal development, this study found their regulatory logic to be surprisingly dissimilar. Whereas the yBE0.6 is directly activated by Abd-B, the results indicate that the t_MSE is indirectly activated by Abd-B and Abd-A, and is directly repressed in more anterior body segments by Abd-A and seemingly Hth. Thus, this study provides an example that illustrates how coordinated expression evolved through the evolution of very different binding site architectures and logic (Camino, 2015).

The disparity of regulatory logic governing the yBE0.6 and t_MSE sheds light on the evolutionary tendencies of gene regulatory networks. The incipient stages of the dimorphic pigmentation network's origin involved the derivation of CREs that generate similar patterns through distinct combinations of binding sites. This evolutionary history establishes a 'branched' network in which several of the possible trans-regulatory alterations are incapable of generating coordinated shifts in the expression patterns for co-expressed genes. Hence, an emerging theme from the work in this system is that the differences in regulatory logic of yBE and t_MSE may necessitate changes in one CRE or the other, but is unable to be altered through a common trans regulator that influences both CRE's patterning. Future studies are needed to substantiate the occurrence and identity of the trans changes altering this network's structure. As other recently derived morphological traits are resolved to the level of binding sites within their networks, it will be instructive to see whether similar branched networks and paths of cis and trans evolution permeate their origin and diversification. The net results may reveal general principles of gene regulatory network evolution (Camino, 2015).

Abdominal-B: Biological Overview | Evolutionary Homologs | Promoter Structure | Transcriptional Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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

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