Abdominal-B
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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