Interactive Fly, Drosophila

DEVELOPMENTAL BIOLOGY

Uniform expression of abd-A under heat shock control transforms embryonic segments anterior to the abd-A domain into an abdominal segment of the A2-A6 type [Image]. Posterior abdominal segments and telson undergo little or no transformation, that is, abd-A is phenotypically suppressed posterior to its realm of expression. The comparison of wildtype embryos with embryos carrying the heat shock-abd-A construct but no abd-A endogenous transcript indicates that some elements of the pattern-like shape of denticle belts or ventral pits depend on the amount of ABD-A protein. (Sanchez-Herrero, 1994).

It is not yet known exactly how homeotic genes transform the fate of a complete organ. Two classical homeotic transformations are transformation of antenna to leg caused by expression of Antennapedia in antenna, and transformation of wings into halteres by expression of Ultrabithorax in the second thoracic appendage. Recently it has been shown that ectopic expression of Ultrabithorax, abdominal-A and Abdominal-B cause similar transformations in some of the fruitfly appendages: antennal tissue into leg tissue and wing tissue into haltere tissue. abd-A can thus replace Ubx in haltere development. Thus the homeotic requirement to form appendages is, in some cases, non-specific (Casares, 1996).

Segment specificity of neuroblast NB1-1 is determined in the neuroectoderm at the early gastrula stage (stage 7). The activity of the homeotic genes Ubx or abd-A is required for the expression of the abdominal variant of the lineage (Prokop, 1994).

Segment-specific differences are evident in the number of neuroblasts (NBs) that persist beyond the end of embryogenesis and proliferate during larval stages. At stage 17 of embryogenesis, all NBs have stopped dividing but can still be monitored by NB-specific expression of grainyhead. Analyses of Grh expression pattern in the CNSs of wild type embryos and of mutant embryos where cell death is suppressed, strongly suggest that a number of NBs normally die towards the end of embryogenesis. The degree of cell death shows segment-specific differences: many more NBs die in the central abdomen than in the thorax and anterior abdomen. As a consequence, when NBs resume proliferation as postembryonic NBs in the larval stages, 47 NBs are detected in each thoracic segment; about 12 are detected in the two anterior abdominal neuromeres, but only six in central abdominal segments. Furthermore, postembryonic NBs in the thorax and anterior abdomen produce hundreds of daughter cells each, whereas those in abdominal neuromeres 3-A7 give rise to only five to 15 cells. In summary there are three major factors regulating the segment-specific proliferation of NBs: (1) the period and frequency of embryonic NB proliferation; (2) the number of NBs eliminated at the end of embryogenesis, and (3) the frequency and period of postembryonic proliferation (Prokop, 1998 and references).

The number and pattern of neuroblasts that initially segregate from the neuroectoderm in the early Drosophila embryo are identical in thoracic and abdominal segments. However, during late embryogenesis, differences in the numbers of NBs and in the extent of neuroblast proliferation arise between these regions. The homeotic genes Ultrabithorax and abdominal-A regulate these late differences. Abdominal NBs in Ubx and abd-A mutants continue replicating DNA, and consequently the number of NBs in these mutants resembles that of thoracic neuroblasts. In embryos lacking the Antp gene, DNA synthesis in ventrolateral/lateral NBs is normal, however, additional cells are detected in ventral positions resembling the ventral patterns of the subesophageal ganglion. Therefore abd-A function is needed to repress DNA replication in some lateral NBs of abdominal neuromeres, and Antp function is required to repress DNA replication in ventral NBs of the thorax. Misexpression of either Ubx or abd-A in thoracic neuroblasts, after segregation, is sufficient to induce abdominal behaviour in lateral neurons and subesophageal characteristics in ventral neurons. The ventral pattern appears to be due to the ability of Ubx to repress Antp expression, since the pattern of ventral neurons resembles the phenotype found in Antp mutant embryos. In wild type embryos, Abdominal-A and Ultrabithorax proteins are only detected in early neuroblasts. In stage 15 embryos no cells are found which co-express Ubx and Grh. This suggests that neither Abd-A nor Ubx are present in the NBs shortly before segment-specific differences in the numbers of cells and Grh patterns occur. Asense is expressed in NBs shortly after their segregation from the neuroectoderm and so can be used as an early marker for NBs. Ubx is detected in many NBs at stages 8-12 although there is wide variation between levels of Ubx present in different NBs and a subset of NBs contain no detectable Ubx. Similarly, Abd-A is present in many NBs at early stages. Thus both Ubx and Abd-A are present in embryonic NBs, but their expression fades before segment-specific differences become detectable (Prokop, 1998).

Transplantation experiments reveal that segment-specific behaviour is determined even prior to neuroblast segregation, that is, prior to expression of Ubx or Abd-A. When cells are heterotopically transplanted from thoracic to abdominal sites of the early gastrula neuroectoderm, 67% give rise to a large nest of postembryonic cells with postembryonic NB (pNB), consistent with the characteristics of thoracic NBs. Conversely, when cells are transplanted from abdominal to thoracic sites, all clones fail to express thoracic features and contain only embryonic cells. It is concluded that segment-specific differences in neuroblast behaviour seem to be determined in the early embryo, mediated through the expression of homeotic genes in early neuroblasts, and executed in later programs controlling neuroblast numbers and proliferation. Two models are presented for the action of the homeotic genes. They could act as transcriptional repressors that initiate a repressed state for their target genes, which can be maintained after the proteins have disappeared, or alternatively, they may activate target genes that have the capacity for autoregulation, so that the targets maintain their own expression in the absence of homeotic proteins (Prokop, 1998).

Loss of Zn finger homeodomain 1 activity disrupts the development of two distinct mesodermal populations: the caudal visceral mesoderm (along which germ cells migrate) and the gonadal mesoderm (the final destination of the germ cells). The caudal visceral mesoderm facilitates the migration of germ cells from the endoderm to the mesoderm. Zfh-1 is also expressed in the gonadal mesoderm throughout the development of this tissue. Ectopic expression of Zfh-1 is sufficient to induce additional gonadal mesodermal cells and to alter the temporal course of gene expression within these cells. Germ cell migration was also analyzed in brachyenteron mutant embryos. Like zfh-1, byn is required for the migration of the caudal visceral mesoderm, but unlike zfh-1, it is not required for gonadal mesoderm development. Since byn and zfh-1 both disrupt caudal visceral mesoderm migration and show similar defects in germ cell migration, it is proposed that in wild-type embryos, the caudal visceral mesoderm facilitates the transition of many germ cells from the endoderm to the lateral mesoderm. abdominal-A is also required for gonadal mesoderm specification. Zfh-1 expression was analyzed in abdA mutants. Zfh-1 is expressed normally in mesodermal clusters at stage 10, however, its levels are not enhanced in PS10-12 during stage ll. The loss of high Zfh-1 expression correlates with the failure of SGP specification in abdA mutants. Although abdA is required for SGP specification, the initial stages of germ cell migration are unaffected in abdA mutant embryos (Broihier, 1998).

In many animal groups, an interaction between germ and somatic lines is required for germ-line development. In Drosophila, the germ-line precursors (pole cells), which form at the posterior tip of the embryo migrate toward the mesodermal layer where they adhere to the dorsolateral mesoderm, which ensheaths the pole cells to form the embryonic gonads. These mesodermal cells may control the expression of genes that function in the development of germ cells from pole cells. However, such downstream genes have not been isolated. In this study, a novel transcript, indora(idr), is identified that is expressed only in pole cells within the gonads. The nucleotide sequence of the 1.5 kb cDNA predicts a protein of 131 amino acids. The amino acid sequence shows no significant homology to any known proteins. The putative Idr protein is highly basic (calculated isoelectric pH is 10.1). During normal development, the expression of idr transcripts become discernible in pole cells at the embryonic stage 14, when pole cells are incorporated into the gonads. Expression persisted in pole cells until the completion of embryonic development. idr expression is undetectable in the adult germ line. However, the possibility that a trace amount of IDR mRNAs is expressed in somatic cells as well as in the germ line throughout most of the life cycle cannot be excluded, because Northern blot analysis reveals that idr transcripts are detectable from late embryogenesis to adulthood (Mukai, 1998).

Reduction of idr transcripts by an antisense idr expression causes the failure of pole cells to produce functional germ cells in females. Furthermore, idr expression depends on the presence of the dorsolateral mesoderm, but it does not necessarily require its specification as the gonadal mesoderm. In order to determine the source of the mesodermal cue, idr expression was analyzed in the absence of the mesodermal cells that make up the gonads. The origin and development of the somatic components of the gonads are described. The somatic gonad precursors (SGPs) are specified from the dorsolateral mesoderm within PS 10-12 at stage 11. In tin;zfh-1 double-mutants, no dorsolateral mesoderm is formed, which results in loss of SGPs. In these embryos, pole cells pass through the midgut epithelium, but subsequently they are dispersed around the midgut. idr expression is drastically reduced in tin;zfh-1 double-mutants. This result shows the requirement of the dorsolateral mesoderm for idr expression in pole cells. It was next asked whether the specification of the dorsolateral mesoderm as SGPs is needed to induce idr expression in pole cells. To examine this, abd-A and iab-4 mutations were used. abd-A function is required in the mesodermal cells for the specification of SGPs. In abd-A mutant embryos, pole cells pass through the midgut wall and are normally associated with the dorsolateral mesoderm. However, they do not coalesce with the pole cells to form the gonads due to their failure to be specified as SGPs. Consequently, pole cells are released from the mesoderm and scattered throughout the embryo. In these embryos, the dispersed pole cells express idr during stages 14-16. Furthermore, a regulatory mutation in the abd-A locus, iab-4, also has no deleterious effect on idr expression. Thus, the specification of the dorsolateral mesoderm as SGPs is dispensable for idr expression. These findings suggest that the induction of idr in pole cells by the mesodermal cells is required for germ-line development (Mukai, 1998).

The Drosophila dorsal vessel is a linear organ that pumps blood through the body. Blood enters the dorsal vessel in a posterior chamber termed the heart, and is pumped in an anterior direction through a region of the dorsal vessel termed the aorta. The dorsal vessel spans segments thoracic 2 (T2) to abdominal 8 (A8). From T2 to A5 the tube is narrow and is termed the aorta, whereas the posterior portion has a larger bore and is termed the heart. Additionally the heart is perforated by three pairs of valve-like ostia, which serve as inflow tracts for hemolymph. Although the genes that specify dorsal vessel cell fate are well understood, there is still much to be learned concerning how cell fate in this linear tube is determined in an anteroposterior manner, either in Drosophila or in any other animal. The formation of a morphologically and molecularly distinct heart depends crucially upon the homeotic segmentation gene abdominal-A (abd-A). abd-A expression in the dorsal vessel is detected only in the heart, and overexpression of abd-A induces heart fate in the aorta in a cell-autonomous manner. Mutation of abd-A results in a loss of heart-specific markers. abd-A and seven-up co-expression in cardial cells defines the location of ostia, or inflow tracts. Other genes of the Bithorax Complex do not appear to participate in heart specification, although high level expression of Ultrabithorax is capable of inducing a partial heart fate in the aorta. These findings demonstrate a specific involvement for Hox genes in patterning the muscular circulatory system, and suggest a mechanism of broad relevance for animal heart patterning (Lovato, 2002).

Markers that label the dorsal vessel can be used to illuminate the morphological differences between the heart and aorta. These markers include: (1) MEF2, which is detectable in all muscle cell nuclei; (2) the basic helix-loop-helix factor Hand, which is expressed in cardial cells and some pericardial cells, and (3) muscle myosin heavy chain (MHC), which accumulates in all muscle cells. Heart cells have a larger volume compared with aorta cells, and the lumen in the heart is larger than in the aorta. However, no markers exist that distinguish at the molecular level between the heart and aorta (Lovato, 2002).

To identify genes expressed in subpopulations of the dorsal vessel, the expression of several muscle structural gene isoforms was analyzed by in situ hybridization. A novel member of the troponin-C superfamily termed Tina-1 (for Troponin C-akin-1 -- formerly CG2803) was identified, whose expression in the dorsal vessel was detected at high levels only in the heart. Tina-1 is also expressed in a subset of other cells, including the hindgut visceral mesoderm. The identification of Tina-1 as a heart-specific marker in the dorsal vessel permitted changes in heart versus aorta fate to be followed at both the morphological and molecular levels (Lovato, 2002).

All three BX-C gene products are detected in the dorsal vessel, albeit in strikingly different patterns. Ubx is detected at low levels in dorsal vessel cells from A2 to the posterior tip of the heart, although expression is slightly lower in A5 to A8. By contrast, AbdA protein is detected in cardial and pericardial cells in the heart region from A5 to A8, and closer examination has indicated that abd-A expression corresponds exactly to the cells forming the heart. AbdB was detected in the dorsal vessel in the most posterior four nuclei in A8, in which AbdA accumulation is reduced. Therefore AbdA seems most likely to play a role in heart cell specification, and was chosen for further study. The expression of other BX-C genes in the dorsal vessel suggests that further structural and functional diversity also exists in this organ. Together, abd-A and Tina-1 represent the first two genes known whose expression patterns differentiate between the heart and the aorta cells of the dorsal vessel (Lovato, 2002).

To determine if abd-A functions as a selector gene in the Drosophila dorsal vessel to distinguish between heart and aorta cells the GAL4-UAS system was used to express abd-A ectopically in different germ layers, and the formation of the dorsal vessel was monitored by studying expression of Mef2, Hand, Mhc and the heart-specific marker Tina-1. Expression of abd-A in the mesoderm alone using either the 24B-gal4 driver or a twist-gal4 driver results in a strong a transformation into heart cell fate for all dorsal vessel cells. There was a greater distance between MEF2-postive cells in the dorsal vessel, suggesting a large lumen running the length of the dorsal vessel. In addition, visualizing Hand expression and MHC accumulation indicates that most of the dorsal vessel cells assumed a larger volume characteristic of cells of the heart. Most striking was the appearance of Tina-1 transcripts throughout the dorsal vessel, indicating that all the dorsal vessel cells had assumed a heart fate (Lovato, 2002).

These results strongly suggested that AbdA plays an important instructive role in the dorsal vessel, directing cells to take on a heart fate. To confirm this, heart formation was studied in mutants lacking abd-A function, since this would be predicted to result in a heart-to-aorta transformation in the posterior region of the dorsal vessel. Many homozygous combinations of abd-A mutants do not complete development sufficiently to answer all of these questions; however, in the absence of abd-A function, phenotypes were seen consistent with a loss of heart cell identity. Despite the lack of dorsal closure, Hand expression persists in the presumptive dorsal vessel cells; however, no size dimorphism was seen in these cells as was observed upon ectodermal expression of abd-A (in which the mutant individuals also failed to complete dorsal closure. Furthermore, there was no enrichment of MHC in the posterior group of dorsal vessel cells. Tina-1 expression in the dorsal vessel was undetectable in the absence of abd-A function. Taken together, the gain- and loss-of function experiments described here identify the homeotic selector gene abd-A as specifying heart cell identity in the Drosophila dorsal vessel (Lovato, 2002).

A unique characteristic of the Drosophila heart is the presence of inflow tracts, termed 'ostia'. There are three pairs of ostia located at the segmental boundaries of A5/A6, A6/A7 and A7/A8, and each ostium is visible in larvae as a broadening of the width of the heart, at the peak of which are small openings. No ostia form in the aorta during embryonic or larval development. The ostia, which form at each segmental boundary, develop from two pairs of cells expressing the orphan nuclear receptor gene sevenup (svp). The remaining four pairs of cardial cells in each segment express the homeobox-containing gene tinman (tin) and form the heart wall (Lovato, 2002).

Close examination of MHC-stained wild-type hearts from embryos indicated that the wall of the heart curved sharply outwards close to the segment borders, whereas no such broadening occurred in the aorta. At these locations, two cardial cells are morphologically distinct in that they have oval-shaped nuclei, rather than the round nuclei of the remaining cells. Given the locations of these morphologically distinct cells close to the segmental boundary and the similarity of this structure to the organization of the larval heart, it was reasoned that the sharp curves in the outer heart wall corresponded to the locations of the ostia. In support of this, indentations were occasionally seen at the tip of these cell pairs, suggesting that the heart wall is perforated at these locations. To confirm that these cells are ostia, wild-type embryos were double-stained with an antibody to Tin (to identify the heart wall cell nuclei) and with an antibody to muscle MHC (to visualize the shape of the heart). The sharp curves in the heart wall corresponded to the locations of ostia, since ostia are formed by the non-Tin expressing population of cardial cells. In the aorta of wild-type embryos, svp-expressing cells are still detected; however, the wall of the aorta is uniform (Lovato, 2002).

Since ectopic mesodermal expression of abd-A results in ectopic heart formation, these ectopic heart structures were studied for the presence of cells forming ostia. In many cases, sharp curves in the wall of the heart tube in locations more anterior to those found in wild type indicated the presence of ectopic ostia, formed by cells more elongated than their neighbors. Furthermore, by staining these embryos with anti-Tin and anti-MHC, as was done for wild type, these elongated cells were found to precisely correspond to those expressing svp. Although it is difficult to visualize the openings of the ostia, the most likely conclusion from these observations is that ectopic ostia are formed in the presence of ectopic Abd-A. Furthermore, these ostia are positioned appropriately within the segment, only at the coincidence of abd-A expression and svp expression (Lovato, 2002).

To quantify more precisely the alteration in Svp cell morphology upon the induction of ectopic heart structures, the size of each svp-expressing cell was determined by measuring the distance from the luminal surface of the Svp cells to the outer wall of the dorsal vessel. In wild-type embryos there are seven segmentally repeating groups of Svp cardial cells in the dorsal vessel, four cells in each group. To distinguish between groups located at unique positions along the AP axis, the groups are referred to as S1 to S7, from anterior to posterior in the embryo. Thus, the Svp cells of clusters S1 to S4 do not form ostia in wild type, whereas S5 to S7 form the ostia of the heart (Lovato, 2002).

In control embryos, clusters S1 to S4 contained cells measuring approximately 5 µm, whereas the Svp cells of the heart were significantly larger (7-8 µm). Upon overexpression of abd-A in the mesoderm there was a large increase in the sizes of cells in groups S1 to S4, many of which were indistinguishable from those in the wild-type heart. These results clearly show the effects of abd-A expression upon aorta cell fate, transforming Svp cells of the aorta into ostia (Lovato, 2002).

Does the mechanism of AP heart patterning uncovered in Drosophila apply to higher animals? The vertebrate heart initially forms as a linear tube in much the same manner as the Drosophila heart, and numerous genes are known to be expressed in unique domains along the AP axis in the developing vertebrate heart. However, there is much to learn concerning the factors that determine this AP pattern. Treatment of chick and zebrafish embryos with retinoic acid results in a loss of anterior heart structures and a broadening of the domain forming more posterior structures, suggesting that retinoic acid can influence the AP patterning of the heart. Retinoic acid also directly activates a number of Hox genes in the trunk of the embryo. Taking these findings together, it is tempting to speculate that Hox segmentation genes in vertebrates also function to control cell identity in the heart. In support of this are recent demonstrations of Hox gene expression in the developing heart and the finding that treatment of cardiogenic explants with retinoic acid can alter the expression of Hox genes (Lovato, 2002).

The results of this study also indicate that two distinct patterns of gene expression converge to control the differentiation of the Drosophila dorsal vessel. Superimposed upon the expression of abd-A in the heart segments, is the pattern of tin-expressing versus svp-expressing cells observed in cardial cells in every segment. Formation of the ostia in the heart occurs only at the intersection of abd-A expression and svp expression, and ectopic ostia form in the presence of ectopic AbdA, but only in svp-expressing populations of cells (Lovato, 2002).

Whether svp function is required for ostium formation in Drosophila remains to be determined. A vertebrate homolog of the Svp protein is chick ovalbumin upstream promoter transcription factor II (COUP-TF II), which in mice is expressed in and is required for the formation of the atria and sinus venosus. The atria and sinus venosus carry out functions in the mouse analogous to the ostia in Drosophila, acting as the inflow tracts for blood to enter the heart. It will be interesting to determine whether the homologous expression patterns of svp and COUP-TF II reflect a homologous function in development (Lovato, 2002).

It is interesting that Ubx and Abd-B are also expressed in unique cells in the dorsal vessel. Although loss-of-function experiments have not demonstrated a role for Ubx in the formation of the heart, it is still possible that Ubx plays a role in the specification of more anterior structures in the dorsal vessel. There are a number of cardial cells in an anterior location that do not express Ubx, suggesting an as yet undetermined function for Ubx in the dorsal vessel. Along these lines, it is interesting to note that the domain of Ubx expression in the aorta roughly corresponds to the region of the dorsal vessel remodeled during pupal development to form the adult heart. Furthermore, Ubx is required to repress lymph gland fate in the pericardial cells adjacent to the dorsal vessel, suggesting a broad requirement for members of the BX-C in patterning the dorsal vessel and its associated cells (Lovato, 2002).

Genetic analysis shows that Engrailed has both negative and positive targets. Negative regulation is expected from a factor that has a well-defined repressor domain but activation is harder to comprehend. VP16En, a form of En that has its repressor domain replaced by the activation domain of VP16, has been used to show that En activates targets using two parallel routes, by repressing a repressor and by being a bona fide activator. The intermediate repressor activity has been identified as being encoded by sloppy paired 1 and 2 and bona fide activation is dramatically enhanced by Wingless signaling. Thus, En is a bifunctional transcription factor and the recruitment of additional cofactors presumably specifies which function prevails on an individual promoter. Extradenticle (Exd) is a cofactor thought to be required for activation by Hox proteins. However, in thoracic segments, Exd is required for repression (as well as activation) by En. This is consistent with in vitro results showing that Exd is involved in recognition of positive and negative targets. Moreover, genetic evidence is provided that, in abdominal segments, Ubx and Abd-A, two homeotic proteins not previously thought to participate in the segmentation cascade, are also involved in the repression of target genes by En. It is suggested that, like Exd, Ubx and Abd-A could help En recognize target genes or activate the expression of factors that do so (Alexandre, 2003).

The most unexpected aspect of these results is that, in abdominal segments, the Hox proteins Ubx and Abd-A are involved in repression by En. In formal genetic assays, Ubx and Abd-A can substitute for Exd in helping En act on negative targets. In the absence of Ubx, Abd-A and Exd, En can no longer repress target genes. By contrast, two other Hox proteins (Antp and Abd-B) appear not to be involved in En function. Antp does not help En repress targets in vivo even though its homeodomain differs from that of Abd-A at only five positions. Likewise, Abd-B, a more distantly related Hox protein, is also unlikely to participate in En function. It is concluded that the role of Ubx and Abd-A in repression by En is specific (Alexandre, 2003).

How could ectopic Ubx or Abd-A allow En to repress targets in the absence of Exd? It could be that this is mediated by wholesale transformation of segmental identity [although such transformation would have to be exd/hth-independent. Alternatively, Ubx and Abd-A could have a more immediate involvement in En function. One can envisage that they could regulate an as yet unidentified corepressor of En (although such regulation would not require Exd). Alternatively, and more speculatively, Ubx and Abd-A could serve as cofactors themselves in regions of the embryo where Exd levels are low. Again, molecular analysis of negative targets will be needed to discriminate these possibilities (Alexandre, 2003).

Homeotic genes have not been previously implicated in En function despite many years of genetic analysis of the Bithorax complex. It is suggested that the role of Ubx and Abd-A in En function has been overlooked previously because, in the absence of these two genes, Exd is upregulated in the presumptive abdomen and thus takes over as a repression cofactor. However, the present results establish that homeotic genes do participate in the segmentation cascade and link two regulatory networks previously thought to be independent (Alexandre, 2003).

Pleiotropic functions of a conserved insect-specific Hox peptide motif

The proteins that regulate developmental processes in animals have generally been well conserved during evolution. A few cases are known where protein activities have functionally evolved. These rare examples raise the issue of how highly conserved regulatory proteins with many roles evolve new functions while maintaining old functions. This was investigated by analyzing the function of the 'QA' peptide motif of the Hox protein Ultrabithorax (Ubx), a motif that has been conserved throughout insect evolution since its establishment early in the lineage. The QA motif was precisely deleted at the endogenous locus via allelic replacement in Drosophila melanogaster. Although the QA motif was originally characterized as involved in the repression of limb formation, it was found to be highly pleiotropic. Curiously, deleting the QA motif had strong effects in some tissues while barely affecting others, suggesting that QA function is preferentially required for a subset of Ubx target genes. QA deletion homozygotes had a normal complement of limbs, but, at reduced doses of Ubx and the abdominal-A (abd-A) Hox gene, ectopic limb primordia and adult abdominal limbs formed when the QA motif was absent. These results show that redundancy and the additive contributions of activity-regulating peptide motifs play important roles in moderating the phenotypic consequences of Hox protein evolution, and that pleiotropic peptide motifs that contribute quantitatively to several functions are subject to intense purifying selection (Hittinger, 2005).

The genetic deletion of the QA motif of Ubx produced a surprisingly subtle but highly pleiotropic homozygous phenotype. The QA motif is partially redundant with Abd-A in A1 for limb repression, is one of several motifs within Ubx that quantitatively affect Ubx activity, and that reducing Ubx or Abd-A levels uncovers a requirement for the QA motif in limb repression. The QA motif is preferentially required for a subset of Ubx-regulated developmental processes, a characteristic that is termed here differential pleiotropy. The conservation of the QA motif throughout the insect lineage suggests some of its many functions are crucial for the proper patterning and fitness of insects. These findings offer a conceptual framework for understanding how pleiotropy, redundancy and selection interact to guide the evolution of selector proteins and the morphology they govern (Hittinger, 2005).

The QA motif is not strictly necessary for limb repression in A1 at any stage of development because of the additive roles played by other peptide motifs in Ubx and because it is partially redundant with the Hox protein Abd-A. Extensive limb derepression was oberved in A1 in embryos and adults when both the QA motif was absent and when the Ubx and abd-A doses were reduced but not when either was manipulated singly. The partial redundancy of the Ubx and Abd-A in limb repression is mechanistically explained by their direct repression of the Dll limb primordia enhancer through the same binding site. The absence of ectopic limb primordia or limbs on the more posterior abdominal segments of Ubx^Delta^QA/Ubx^– abd-A^– flies suggests that the higher level and broader expression of Abd-A are sufficient to repress limb formation in more posterior segments (A2-A7) (Hittinger, 2005).

Temporal and spatial expression of homeotic genes is important for segment-specific neuroblast 6-4 lineage formation in Drosophila

Different proliferation of neuroblast 6-4 (NB6-4) in the thorax and abdomen produces segmental specific expression pattern of several neuroblast marker genes. NB6-4 is divided to form four medial-most cell body glia (MM-CBG) per segment in thorax and two MM-CBG per segment in abdomen. Since homeotic genes determine the identities of embryonic segments along the A/P axis, whether temporal and specific expression of homeotic genes affects MM-CBG patterns in thorax and abdomen was ivestigated. A Ubx loss-of-function mutation was found to hardly affect MM-CBG formation, whereas abd-A and Abd-B caused the transformation of abdominal MM-CBG to their thoracic counterparts. In contrast, gain-of-function mutants of Ubx, abd-A and Abd-B genes reduced the number of thoracic MM-CBG, indicating that thoracic MM-CBG resembled abdominal MM-CBG. However, mutations in Polycomb group (PcG) genes, which are negative transregulators of homeotic genes, did not cause the thoracic to abdominal MM-CBG pattern transformation although the number of MM-CBG in a few per-cent of embryos were partially reduced or abnormally patterned. These results indicate that temporal and spatial expression of the homeotic genes is important to determine segmental-specificity of NB6-4 daughter cells along the anterior-posterior (A/P) axis (Kang, 2006).

In the Drosophila embryonic central nervous system (CNS), about 30 glia are produced in a stereotyped pattern in each hemisegment, and certain of these glia are arranged in different patterns between segments along the A/P axis. Thus, it is important to understand how the regional specificity of certain glia is determined and maintained during nervous system development. repo is essentially required for the differentiation and maintenance of glia. Moreover, some of these repo expressing cells, MM-CBG, show different patterns along the A/P axis. In the present study, MM-CBG pattern abnormalities were examined in BX-C and its negative transregulator, PcG mutant embryos (Kang, 2006).

The data showed that Ubx loss-of-function mutation did not cause the homeotic transformation of the abdominal MM-CBG pattern to the thoracic one. However, a loss-of-function mutation in the abd-A gene caused the transformation of abdominal MM-CBG into a thoracic pattern. Abd-B mutant embryos also showed transformation of MM-CBG in its functional domain. These results indicate that unlike Ubx, abd-A and Abd-B genes are involved in the segment-specific MM-CBG pattern formation. The role of BX-C on MM-CBG formation was confirmed using gain-of-function BX-C mutation. Ectopic expression of BX-C with sca-GAL4/UAS system caused thoracic MM-CBG to follow the abdominal pattern of MM-CBG. Unlike the result shown in Ubx loss-of-function mutant embryos, four thoracic MM-CBG were frequently reduced to two or three MM-CBG in Ubx gain-of-function mutant embryos, suggesting that Ubx might be involved in MM-CBG pattern formation. The Abd-A and Abd-B proteins driven by sca-GAL4 driver changed the thoracic MM-CBG pattern to the abdominal one. It was suggested that Abd-A and Abd-B proteins repress the proliferation of MM-CBG through inhibition of CycE in the abdomen, which makes two MMCBG per abdominal segment and four MM-CBG per thoracic segment (Kang, 2006).

PcG mutation causes the ectopic expressions of abd-A and Abd-B genes in the anterior of their functional domains. It is presumed that the ectopic thoracic expressions of abd-A and Abd-B genes would transform thoracic MM-CBG to an abdominal one as shown in the gain-of-function BX-C mutation, because the thoracic pattern of the epidermis and central nervous system are transformed to the abdominal segments in these two mutants. However, PcG mutant embryos showed little evidence of an abnormal MM-CBG pattern in the thorax because most PcG mutant embryos showed wild type thoracic MM-CBG pattern. This was confirmed using a gcm enhancer trap line. A gmc driven reporter was expressed only in the MM-CBG of the abd-A domain. Although Pc zygotic, esc and pho maternal effect mutations caused the ectopic expressions of abd-A and Abd-B in the CNS from head to tail, the anterior boundary of gcm-lacZ expression did not move to more anterior segments. In addition, thorax-specific eg expression pattern was unchanged in PcG mutant embryos (Kang, 2006).

These observations indicate that temporal and spatial homeotic gene expression is important in MM-CBG pattern formation. The homeotic gene products driven by sca- GAL4 driver are present in the neuroectoderm from embryonic stage 8, which clearly changes the thoracic MM-CBG pattern. However, derepressed BX-C gene products caused by PcG mutations do not affect MM-CBG pattern. Ubx, abd-A and Abd-B genes begin to be weakly misexpressed from stage 11 and shows strong ectopic expression at stage 13 in Pc and esc mutant embryos. In wild type embryos MM-CBG appears to proliferate once between stage 11 and 12, and become four cells per segment in the thorax, while there is no cell division of MM-CBG in the abdomen because Abd- A and Abd-B proteins repress CycE expression. So PcG mutants seems to cause the ectopic expression of the BX-C genes after MMCBG are already determined to be prolifered in the thorax. Early segment-specific commitment of NB6-4 progeny cells also supports this conclusion. When BX-C genes are overexpressed from stage 10 using eg-GAL4, thoracic MM-CBG pattern was not changed. Taken together, temporal and spatial expression of the homeotic genes is important to determine segmental-specificity of MM-CBG along the anterior-posterior (A/P) axis (Kang, 2006).

Requirement of Abdominal-A and Abdominal-B in the developing genitalia of Drosophila breaks the posterior downregulation rule

The genitalia of Drosophila derive from the genital disc and require the activity of the Abdominal-B (Abd-B) Hox gene. This gene encodes two different proteins, Abd-B M and Abd-B R. The embryonic genital disc, like the larval genital disc, is formed by cells from the eighth (A8), ninth (A9) and tenth (A10) abdominal segments, which most likely express the Abd-B M, Abd-B R and Caudal products, respectively. Abd-B m is needed for the development of A8 derivatives such as the external and internal female genitalia, the latter also requiring abdominal-A (abd-A), whereas Abd-B r shapes male genitalia (A9 in males). Although Abd-B r represses Abd-B m in the embryo, in at least part of the male A9 such regulation does not occur. In the male A9, some Abd-B m^–r^– or Abd-B r^– clones activate Distal-less and transform part of the genitalia into leg or antenna. In the female A8, many Abd-B m^–r^– mutant clones produce similar effects, and also downregulate or eliminate abdominal-A expression. By contrast, although Abd-B m is the main or only Abd-B transcript present in the female A8, Abd-B m^– clones induced in this primordium do not alter Distal-less or abd-A expression, and transform the A8 segment into the A4. The relationship between Abd-B and abd-A in the female genital disc is opposite that of the embryonic epidermis, and contravenes the rule that posteriorly expressed Hox genes downregulate more anterior ones (Foronda, 2006).

Abd-B is a complex gene: the use of four different promoters and the existence of specific exons give rise to several transcripts that encode two different proteins. The A (m) transcript encodes the Abd-B M (or Abd-B I) protein, and the B, C (r) and gamma RNAs encode the Abd-B R (or Abd-B II) protein. The Abd-B M protein has 221 amino acids more than the Abd-B R product does in its N-terminal domain but both proteins share a common C-terminal region, which includes the homeodomain. In the embryonic epidermis, the Abd-B M transcript and protein are expressed in parasegments (PS) 10-13 (A5-A8 segments), whereas the Abd-B R transcript and protein are present in PS14-PS15 (A9-A10) initially, and in PS14 (A9) at late stages. The gamma RNA is transcribed in just a few cells of PS14 or PS15 (Foronda, 2006 and references therein).

The role of Abd-B M and Abd-B R products in genital development remains unclear. Abd-B m mutations transform the A5-A8 segments into the A4 segment, both in males and females; the female genitalia are lost whereas male genitalia remain intact. Significantly, the transformations obtained in either Abd-B m or Abd-B r mutants clearly differ from those observed when all Abd-B functions are eliminated: in some of the clones mutant for Abd-B (m and r), part of the male or female genitalia are transformed into leg or antenna. Therefore, the precise role of abd-A, Abd-B m and Abd-B r in genitalia development is not well defined (Foronda, 2006).

This study has analyzed homeotic expression and requirement in terminalia development. It is proposed that in the embryonic genital disc, as in the larval discs, Abd-B m, Abd-B r and cad are expressed in the A8, A9 and A10, respectively. It is also reported that abd-A, Abd-B m and Abd-B r are needed for development of the internal female genitalia, Abd-B m for the development of female external genitalia and Abd-B r for the development of male genitalia. Strikingly, abd-A and Abd-B bear unexpected relationships in mature genital discs. In the A8 of the female genital disc, Abd-B M maintains abd-A expression. In Abd-B m mutant clones, however, another Abd-B protein maintains abd-A expression but does not prevent abd-A function, since these clones transform the A8 segment into the A4. In the male A9, Abd-B r function does not repress the Abd-B m transcript, at least in part of the primordium, and some Abd-B r mutant clones transform male genitalia into leg or antenna. These relationships between Hox genes are different from those reported in the embryonic epidermis and contravene the rule that posteriorly expressed Hox genes repress those expressed more anteriorly (Foronda, 2006).

In the third instar genital disc of Drosophila, Abd-B is expressed in the A8 and A9 segments, and cad in the A10. To study whether these expression domains are established early in development, Abd-B and cad transcription were examined in the embryonic genital disc. This disc is identified by the expression of genes like snail, escargot or headcase (hdc), and the hdc-lacZ B5 line, which reproduces the pattern of hdc RNA expression, was selected to mark the genital disc. At about stage 15, hdc is expressed in three clusters of cells, two anterior ones placed bilaterally, and a third one located in a more posterior and central position. The three clusters fuse later in development to form the genital disc. At stage 15, six to seven cells were counted at each of the two anterior groups, and two to three cells in the posterior one, making up a total of 14-17 cells. Double staining with anti-Abd-B and anti-ß-galactosidase antibodies (in hdc-lacZ embryos), or with GFP and anti-ß-galactosidase antibody (in cad-Gal4/UAS-GFP; hdc-lacZ/+ embryos), shows that Abd-B is expressed in the two anterior clusters and cad in the posterior one (Foronda, 2006).

To ascertain whether the two Abd-B products (Abd-B M and Abd-B R) are present in the genital disc primordium, the expression driven by an Abd-B m-Gal4 line was compared with the signal detected with an antibody that recognizes both Abd-B M and Abd-B R proteins. In UAS-myc-EGFP^F/+; Abd-B-Gal4^LDN/hdc-lacZ embryos, a GFP signal was seen in about two cells located laterally in each of the two anterior clusters; these cells most likely express Abd-B m, and, therefore, are also labelled with the anti-Abd-B antibody. There are also 8-10 Abd-B-expressing cells not labelled with GFP, and these, probably, correspond to those expressing the Abd-B R protein. Taken together, these results suggest that the embryonic genital primordium includes three groups of cells that probably express Abd-B m, Abd-B r and cad, respectively (Foronda, 2006).

Study of mutant phenotypes reveals that as in the embryonic cuticle, abd-A and Abd-B m are needed in the A8 whereas Abd-B r is required in the A9. The relationship between these homeotic products in the mature genital discs, however, clearly differs from what is observed in the embryonic epidermis. The embryonic genital disc has three distinct cell populations at stages 15/16: some anterior-lateral cells transcribe Abd-B m, anterior-central and middle cells express Abd-B r and posterior cells transcribe cad, although the expression of these products may overlap. Because the genital disc is formed by the fusion of cells coming from the A8, A9 and A10 segments, and by analogy to the expression of these genes in the mature genital discs, it is concluded that Abd-B m, Abd-B r and cad are probably expressed in the A8, A9 and A10 segments, respectively, of the embryonic genital disc (Foronda, 2006). Abd-B is not only expressed, but also required in the embryonic genital primordium. In the absence of Abd-B m, the number of hdc-expressing cells in the disc is reduced, most likely because these cells adopt now a more anterior fate, as occurs in the cuticle. When Abd-B r is absent, the genital primordium lacks some cells and is disorganized, and when both Abd-B products are absent, the primordium is reduced to a few, dispersed cells, some of which express Dll ectopically, suggesting a transformation into a leg primordium (Foronda, 2006).

The A8, A9 and A10 primordia of the mature genital discs bear anterior and posterior compartments, with expression of en and wg in each of these three primordia. Curiously, although three primordia in the embryonic disc can be defined, based on the expression of Abd-B m, Abd-B r and cad, neither en nor wg is expressed in the three separate domains at this stage. This may suggest, as was also recently proposed, that new bands of en and wg expression may be formed later in development, in precise concordance with the three primordia defined by the Abd-B m, Abd-B r and cad genes. It is noted that late en expression is also characteristic of the antennal primordium of the eye-antennal disc (Foronda, 2006).

abd-A is expressed in the whole internal female genitalia except for the parovaria, and this is consistent with experiments indicating that parovaria derive from the female A9 segment. abd-A has been shown to be required for gonad development, and in the abd-A^iab-3/Df mutant, combinations ovaries are also absent. However, the defects observed in the female internal genitalia are not simply due to an indirect effect of the lack of gonads, since iab-4 mutations prevent the formation of the ovaries but do not alter internal genitalia formation (Foronda, 2006).

The results indicate that Abd-B m is required for the development of female external and internal genitalia, both derived from the female A8. The internal genitalia of Abd-B-Gal4^LDN/UAS-lacZ females (driving expression only where Abd-B m levels are high) were stained with X-gal except in two structures, the oviducts and parovaria. The absence of oviduct staining in Abd-B-Gal 4^LDN/UAS-lacZ females is probably due to the particular expression driven by this reporter, and does not imply an absence of Abd-B m transcription in these organs, for two reasons: (1) Abd-B m transcripts are present in the whole A8 segment of the female genital disc, and (2) oviduct development is affected in Abd-B m mutant females. Parovaria, by contrast, are not stained in Abd-B-Gal 4^LDN/UAS-lacZ or abd-A-lacZ females, and this agrees with their A9 provenance. This is supported by the observation that in some Abd-B m mutant females parovaria are the only structures that remain in the internal female genitalia (Foronda, 2006).

Abd-B M seems to be the main or only Abd-B product present in the female A8, so it was expected that elimination in this segment of just Abd-B M or of all Abd-B proteins would give similar results. This is not so. Some Abd-B^– clones transform part of the female genitalia into leg or antenna, whereas Abd-B m mutant clones convert the eighth tergite, and probably the female genitalia, into an anterior abdominal segment. The differences between Abd-B m^– and AbdB^– clones in the A8 of the female genital disc reveal the existence of unsuspected regulatory interactions between the abd-A and Abd-B genes: whereas Abd-B m^– clones do not affect abd-A, in AbdB^– clones abd-A expression is eliminated. This is a surprising result, because it is contrary to what is observed in the embryo, where Abd-B represses abd-A (Foronda, 2006).

Abd-B m^– clones induced in the female A8 do not alter abd-A expression but do not change Abd-B expression levels either. This is observed with mutations that do not make Abd-B M protein, so the Abd-B protein detected is not the Abd-B M product. Surprisingly, although some Abd-B r expression is detected in the female A8, uniform Abd-B r expression is not seen throughout this primordium and Abd-B r transcripts seem not to be derepressed in Abd-B^M5 mutant clones. No explanation is available for this conundrum. Perhaps the probe used, although it includes sequences complementary to all of the Abd-B r cDNA sequences that have been published, does not efficiently detect all of the non-Abd-B m transcripts (Foronda, 2006).

The differences in regulatory and functional interactions among gene products in the embryo and the genital discs are not limited to those of Abd-B and abd-A that have been discussed above. Three other possibilities should be considered. (1) There may be changes in phenotypic suppression: the transformation of the eighth tergite to the fourth one in Abd-B m^– clones is due to abd-A. Because in these clones Abd-B protein is still present, this suggests that abd-A may phenotypically suppress Abd-B, differently from what is generally observed in the embryo. (2) Abd-B r represses Abd-B m in the embryo, but some Abd-B r^– clones do not activate Abd-B m in the male disc. (3) abd-A represses Dll in the embryo, but not in the female genital disc, and ectopic Dll can repress abd-A instead. abd-A does not repress Dll in the leg discs either, and this resembles Ubx function, which represses Dll only early in development. By contrast, Abd-B represses Dll in the embryo, in the larval genital disc, and in the leg disc when ectopically expressed (Foronda, 2006).

Abd-B r expression is restricted to the A9 segment in male genital discs, but shows expression in the A9 and in some cells of the A8 in female genital discs. In spite of this, Abd-B r^– clones in the external female genitalia (A8) are phenotypically wild type. In the male A9, some Abd-B r mutant clones eliminate Abd-B, activate Dll and transform part of the genitalia into distal leg or antenna. This is similar to the result obtained in some Abd-B^– clones, and it implies that Abd-B m is not derepressed in these mutant clones. However, Abd-B m is perhaps derepressed in those Abd-B r mutant clones where Abd-B signal remains (Foronda, 2006).

Although Abd-B r^– clones affect, almost exclusively, male genitalia development, Abd-B r hemizygous or trans-heterozygous flies lack genitalia and analia in both sexes. This probably reflects the absence of proper interactions between the different primordia needed for the growth of the genital disc. In Abd-B r mutant females, the internal genitalia are abnormal, and in some of these females, an absence of parovaria and the presence of three or four spermathecae is observed. This phenotype is consistent with a segment-autonomous transformation of A9 derivatives (parovaria) into A8 structures (spermathecae), similar to the embryonic cuticular transformation of A9 into A8 observed in Abd-B r mutations. A transformation of parovaria into spermathecae has been described in Polycomblike mutants, and may also indicate a transformation of A9 to A8 (Foronda, 2006).

These results illustrate that there are quite different Hox cross-regulatory interactions in the embryo and in the genital disc. The effects in the genital discs contradict the general rule that genes transcribed more posteriorly suppress or downregulate the expression of more anterior ones. This rule has, nevertheless, some exceptions in genes of the Antennapedia complex. Further, differences in Hox cross-regulation between the embryo and imaginal discs are not unprecedented: the proboscipedia (pb) Hox gene is positively regulated by Sex combs reduced in the embryo, but pb activates Sex combs reduced in the labial imaginal disc (Foronda, 2006).

It has been proposed that the primordia of female and male genitalia could be subdivided into an 'appendage-like' and a 'trunk-like' region). These two regions of the female A8 can now be defined more precisely. The 'appendage-like' region would be that expressing abd-A and low levels of Abd-B, and corresponds approximately to the presumptive internal female genitalia. This domain is roughly coincident with the region of expression of a reporter insertion in buttonhead, the gene that defines ventral appendage development, and this is also, approximately, the domain where Abd-B^– clones may activate Dll. If this subdivision is correct, the 'appendage' specification defined by buttonhead would be repressed in the wild type by Abd-B, which both limits Dll expression to a few cells of the A8 primordium and prevents Dll function. Abd-B^– clones in this region eliminate abd-A expression and promote leg or antenna development. This subdivision may also apply to the male disc, the penis apparatus presumptive region being the main 'appendage' domain. Similar to what is described in this study, the labial disc possesses a large 'appendage' region that is revealed by Dll derepression in pb mutations. This characteristic, and the changes in Hox gene cross-regulation between the embryo and the imaginal disc, are two features shared by pb/labial disc and Abd-B/genital disc (Foronda, 2006).

Bithorax complex genes control alary muscle patterning along the cardiac tube of Drosophila

Cardiac specification models are widely utilized to provide insight into the expression and function of homologous genes and structures in humans. In Drosophila, contractions of the alary muscles control hemolymph inflow and support the cardiac tube, however embryonic development of these muscles remain largely understudied. This study found that alary muscles in Drosophila embryos appear as segmental pairs, attaching dorsally at the seven-up (svp) expressing pericardial cells along the cardiac dorsal vessel, and laterally to the body wall. Normal patterning of alary muscles along the dorsal vessel was found to be a function of the Bithorax Complex genes abdominal-A (abd-A) and Ultrabithorax (Ubx) but not of the orphan nuclear receptor gene svp. Ectopic expression of either abd-A or Ubx resulted in an increase in the number of alary muscle pairs from seven to 10, and also produced a general elongation of the dorsal vessel. A single knockout of Ubx resulted in a reduced number of alary muscles. Double knockouts of both Ubx and abd-A prevented alary muscles from developing normally and from attaching to the dorsal vessel. These studies demonstrate an additional facet of muscle development that depends upon the Hox genes, and define for the first time mechanisms that impact development of this important subset of muscles (LaBeau, 2009).

In Drosophila, the seven pairs of embryonic alary muscles attach to Svp pericardial cells along the dorsal vessel as it migrates dorsally towards its final location. The alary muscles persist throughout larval development, playing what are thought to be important roles in stabilizing the location of the heart in the body cavity. In addition, modified alary muscles are also found in the adult, and there is evidence from some insects that contraction of these adult muscles is concordant with heart beating. These data suggest important functions for the alary muscles throughout the life cycle (LaBeau, 2009).

Attachment of the alary muscles to the cardiac tube occurs in the vicinity of the Svp pericardial cells. The data clearly identify processes emanating from the alary muscles towards the pericardial cells. It is reasonable to propose that the Svp pericardial cells produce or present some molecule(s) to which the alary muscles attach, although the nature of this molecule has yet to be defined. This suggestion is consistent with observations that in the developing pupa the pericardial cells and alary muscles are connected by significant amounts of connective tissue. In addition, the expression of this unknown molecule must be independent of svp function, since in svp mutants the patterning of the alary muscles appears largely normal. The cardiac tube expresses several secreted molecules which are known to function in cell attraction. However an enrichment for any of these in the Svp pericardial cells has not been reported (LaBeau, 2009).

The data also demonstrate that normal alary muscle patterning is under the direct control of the Hox genes Ubx and abd-A. This finding is consistent with previous research on the role of the Bithorax Complex in patterning of cardiac and skeletal muscle within the mesoderm, and the more general function of the Hox genes in controlling AP diversity. It is noted that the domains of Hox gene function in alary muscles bear a closer resemblance to their expression in developing skeletal muscles rather than Hox gene expression in the cardiac tube. This observation is consistent with the conclusion that the alary muscles are skeletal muscle derivatives based upon their multinucleate nature (LaBeau, 2009).

Previous research further illustrates the requirement of the Hox genes abd-A and Ubx in the normal development and patterning of Svp cardial and pericardial cells. Experiments manipulated both abd-A and Ubx in knockout as well as over-expression conditions, and the nature of the current results are in general consistent with previous findings for the cardiac Svp cells: over-expression of abd-A and Ubx produced three additional sets of Svp expressing cardial cells; and knockout conditions produced either no change in Svp cardial cell number (for abd-A mutants), loss of anterior Svp cardial cells (for Ubx mutants), or almost no sets of Svp cardial cells (for Ubx abd-A mutants) (LaBeau, 2009).

Orthologs of Drosophila Hox genes are detected in the developing human heart, as well as being widely expressed in the neighboring viscera such as the lungs, spleen, liver, pancreas and epidermis. In other vertebrates, Hox genes have been generally (although not specifically) implicated in cardiac development. A recent genome-scale study of skeletal muscle development also established an AP pattern of Hox gene expression in the developing embryonic skeletal myoblasts. Together, these studies support a general role for Hox gene patterning of muscle derivatives broadly across the Animal Kingdom (LaBeau, 2009).

What are the embryonic origins of the alary muscles? As indicated previously, this question is difficult to answer absent a specific marker for the alary muscles early in embryonic development, and it must be noted that these analyses are therefore by necessity end-point assays carried out at stage 16. Nevertheless given the syncytial nature of the alary muscles, they likely arise from specific founders cells specified at particular locations in the somatic mesoderm. Furthermore, since there is only one alary muscle which arises in each hemisegment, it can be proposed that the founder for this muscle arises from an asymmetrical cell division. Experiments were carried out to test this hypothesis using mutants for sanpodo and numb, which are genes in the asymmetric cell division pathway. While sanpodo mutants produced individuals with a partial loss of alary muscles, the numb mutants were so disrupted at the level of the whole organism that it was not possible to discern any sign of the forming alary muscles if present. This issue might be addressed in the future via analysis of alary muscle precursor formation in these mutants, once suitable markers become available. Alternatively, a strategy for following cell lineages in founder cells might prove useful (LaBeau, 2009).

Are there mammalian versions of the alary muscles? While in some cases it is difficult to assign directly homologous structures between insects and mammals, there are a number of ligaments known to stabilize the location of the heart in mammals. These include in particular the ligaments which attach the outer pericardial layer to the diaphragm and spinal column, as well as the sternopericardiac ligaments which connect the pericardium to the sternum. Since the requirement for structures to stabilize the heart within the body cavity appears to be conserved, the molecular mechanisms responsible for their development might also bear some resemblances to each other (LaBeau, 2009).

Segment-specific generation of Drosophila Capability neuropeptide neurons by multi-faceted Hox cues

In the Drosophila ventral nerve cord, the three pairs of Capability neuropeptide-expressing Va neurons are exclusively found in the second, third and fourth abdominal segments (A2-A4). To address the underlying mechanisms behind such segment-specific cell specification, the developmental specification of these neurons was followed. Va neurons are initially generated in all ventral nerve cord segments and progress along a common differentiation path. However, their terminal differentiation only manifests itself in A2-A4, due to two distinct mechanisms: segment-specific programmed cell death (PCD) in posterior segments, and differentiation to an alternative identity in segments anterior to A2. Genetic analyses reveal that the Hox homeotic genes are involved in the segment-specific appearance of Va neurons. In posterior segments, the Hox gene Abdominal-B exerts a pro-apoptotic role on Va neurons, which involves the function of several RHG genes. Strikingly, this role of Abd-B is completely opposite to its role in the segment-specific apoptosis of other classes of neuropeptide neurons, the dMP2 and MP1 neurons, where Abd-B acts in an anti-apoptotic manner. In segments A2-A4 abdominal A was found to be important for the terminal differentiation of Va cell fate. In the A1 segment, Ultrabithorax acts to specify an alternate Va neuron fate. In contrast, in thoracic segments, Antennapedia suppresses the Va cell fate. Thus, Hox genes act in a multi-faceted manner to control the segment-specific appearance of the Va neuropeptide neurons in the ventral nerve cord (Suska, 2011).

Addressed here is the segment-specific appearance of one peptidergic neuronal subtype, the Capa-expressing Va neurons. One pair of Va neurons is initially generated in each segment of the VNC. At embryonic stage 14, differentiation begins and the cells commence the expression of the transcription factors Dac and Dimm. Only after this process is initiated, at stage 16, the posteriorly expressed Hox gene Abd-B triggers PCD in segments A5 to A8. This PCD involves the RHG motif genes, and mutant analysis indicates that grim, or grim and hid play the most important roles. As development progresses, the Va neurons in abdominal segments A2-A4 are further specialized under the influence of abd-A, which results in expression of the Capa neuropeptide at stage 17. The single pair of Dimm/Dac-expressing Va neurons in the first abdominal segment is present into larval stages, but does not express Capa. These alternate Va neurons depend upon Ubx for their Dimm expression, but it is unclear if they differentiate into peptidergic neurons, and if so, which neuropeptide gene they express. In thoracic segments, Antp is involved in the down-regulation of Dac and Dimm. These studies unravel a complex interplay of Hox gene input critical for the segment-specific survival and differentiation of the Va neurons and thereby highlight the involvement of Hox genes during the process of shaping the segment-specific structures of the nervous system (Suska, 2011).

Ectopic appearance of Capa expression through ectopic expression of abd-A indicates that abd-A is an important partner in the combinatorial code of transcription factors necessary for initiating the expression of Capa. The roles of Ubx and Antp are not as straightforward to assess. Ubx showed a participation in the specification of the Va neurons in more anterior segments of the VNC, mainly the thoracic area. Ectopic Ubx expression resulted in maintained Dac/Dimm expression in thoracic Va cells into late embryonic stages (18hAEL). Its endogenous role seems to be confined to segment A1, which is characterized by co-expression of Dac/Dimm and a lack of Capa. The role this pair of neurons plays is unknown, as they are not known to express any neuropeptide. The mutant analysis indicates a possible role of Antp in the down-regulation of Dac/Dimm in thoracic Va neurons. The ectopic expression of Antp however could not override specification signals provided by the other factors (Suska, 2011).

Several studies have identified roles for Hox genes in specifying neuronal subtypes. Of particular interest for the current study are previous findings that Antp acts at a late stage to specify two other neuropeptide cells; the thoracic Nplp1 and FMRFa neurons of the Apterous (Ap) cluster. In this study, Antp first acts together with the temporal gene castor to activate expression of the collier gene, an EBF family member, thus triggering specification of a transient 'generic' Ap cluster neurons identity. Subsequently, Antp acts in a feedforward manner with collier to activate late cell fate determinants, such as dimm, and ultimately the Nplp1 and FMRFa neuropeptide genes. Currently, the neuroblast origin of the Va neurons is unclear. Double-labeling with the neuroblast row 5-6 marker GooseberryNeuro indicates that Va neurons originate from a row 5 neuroblast. As the neuroblast origin of the Va neurons is established, and this lineage mapped, it will be possible to place the generation of Va neurons within a lineage tree. This will furthermore allow identification of the temporal window that generates Va neurons (Suska, 2011).

Programmed cell death plays a critical role in the generation of segmental diversity. Studies in the Drosophila embryo have revealed that this can act both at the level of progenitor and postmitotic, even differentiated cells. In progenitors, PCD acts to remove many abdominal neuroblasts after they have completed their lineages and become quiescent. This ensures that as neuroblasts re-enter proliferative states in the larvae, the abdomen has very few quiescent neuroblasts that can enter the cell cycle. Thus, in the adult CNS, the abdomen will end up containing substantially fewer neurons and glia. In postmitotic cells, PCD acts in two apparently different ways: (1) to remove certain postmitotic cells immediately after mitosis, or (2) to remove differentiated neurons. A particularly relevant case to the studies presented is the removal of the peptidergic dMP2 and MP1 neurons. These cells are generated in all VNC segments, extend axons to pioneer critical axon tracts, and subsequently undergo PCD in all segments but the A6-A8 segments. Strikingly, here Abd-B has an anti-apoptotic and promotes peptidergic identity role, while in the Va neurons it has a pro-apoptotic role. Moreover, the cell death of both MP1 and Va neurons also depends upon the RHG genes. These results suggest that Abd-B acts in an opposing manner, pro- versus anti-apoptotic, by differentially controlling the same PCD pathway in related neurons. An attractive and simple model for this dual role of Abd-B would be that MP1 and Va neurons express different regulatory genes, which can act with Abd-B to trigger either survival or death. Further studies of PCD in the dMP2, MP1 and Va neurons may help shed light on the molecular genetic mechanisms behind these dual roles of Abd-B (Suska, 2011).

A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation

Trait development results from the collaboration of genes interconnected in hierarchical networks that control which genes are activated during the progression of development. While networks are understood to change over developmental time, the alterations that occur over evolutionary times are much less clear. A multitude of transcription factors and a far greater number of linkages between transcription factors and cis-regulatory elements (CREs) have been found to structure well-characterized networks, but the best understood networks control traits that are deeply conserved. Fruit fly abdominal pigmentation may represent an optimal setting to study network evolution, as this trait diversified over short evolutionary time spans. However, the current understanding of the underlying network includes a small set of transcription factor genes. This study greatly expands this network through an RNAi-screen of 558 transcription factors. Twenty-eight genes were identified, including previously implicated abd-A, Abd-B, bab1, bab2, dsx, exd, hth, and jing, as well as 20 novel factors with uncharacterized roles in pigmentation development. These include genes which promote pigmentation, suppress pigmentation, and some that have either male- or female-limited effects. Many of these transcription factors control the reciprocal expression of two key pigmentation enzymes, whereas a subset controls the expression of key factors in a female-specific circuit. Pupal Abd-A expression pattern was conserved between species with divergent pigmentation, indicating diversity resulted from changes to other loci. Collectively, these results reveal a greater complexity of the pigmentation network, presenting numerous opportunities to map transcription factor-CRE interactions that structure trait development and numerous candidate loci to investigate as potential targets of evolution (Rogers, 2014).

Signalling crosstalk at the leading edge controls tissue closure dynamics in the Drosophila embryo

During Dorsal closure (DC), JNK (JUN N-terminal Kinase) signalling controls leading edge (LE) differentiation generating local forces and cell shape changes essential for DC. The LE represents a key morphogenetic domain in which, in addition to JNK, a number of signalling pathways converge and interact (anterior/posterior -AP- determination; segmentation genes, such as Wingless; Decapentaplegic). To better characterize properties of the LE morphogenetic domain, this study sought new JNK target genes through a genomic approach: 25 were identified, of which eight are specifically expressed in the LE, similar to decapentaplegic or puckered. Quantitative in situ gene profiling of this new set of LE genes reveals complex patterning of the LE along the AP axis, involving a three-way interplay between the JNK pathway, segmentation and HOX genes. Patterning of the LE into discrete domains appears essential for coordination of tissue sealing dynamics. Loss of anterior or posterior HOX gene function leads to strongly delayed and asymmetric DC, due to incorrect zipping in their respective functional domains. Therefore, in addition to significantly increasing the number of JNK target genes identified so far, the results reveal that the LE is a highly heterogeneous morphogenetic organizer, sculpted through crosstalk between JNK, segmental and AP signalling. This fine-tuning regulatory mechanism is essential to coordinate morphogenesis and dynamics of tissue sealing (Rousset, 2017).

This identification of several new JNK target genes during DC and analysis of their quantitative expression patterns uncovers the complex transcriptional response taking place in the LE morphogenetic domain. Results reveal an intricate regulatory network integrating multiple signalling layers. In this process, AP positional information and JNK signalling cooperate to generate a highly patterned, yet apparently smooth and regular LE. Mutant analysis shows that LE partitioning into discrete domains is important to control the coordination, and hence the dynamics of the whole closure process (Rousset, 2017).

The LE is a major component of DC, being the site of JNK activity and actin cable assembly; it also provides an active boundary with the amnioserosa, driving epidermal spreading and seamless tissue sealing. Therefore, it is important to determine its morphogenetic and signalling features and how these are dynamically controlled. To this end, a new set of target genes was identified whose expression in the dorsal ectoderm is dependent on JNK activity during DC. Transcriptome analysis allowed identification of 1648 independent genes which are up- or down-regulated in JNK activated embryos. Filtering of this large set yielded a group of 194 genes whose expression was analysed by quantitative in situ hybridization under different genetic conditions. Transcriptional profiling unveiled 31 Drosophila JNK target genes, of which only a fraction were already known, including jra/jun, reaper, Zasp52 and scab. Amongst novel targets were also Scaf and Rab30 the roles of which during DC have previously been described. Two categories of JNK target genes were distinguished: genes that are specifically expressed in the LE and genes whose expression is more ubiquitous in the dorsal ectoderm. Genes belonging to the latter category may play a general role in the ectoderm under the control of different pathways, for example in the case of Rab30. In contrast, LE-specific genes likely play a specific role during DC, as is the case for puc, dpp and scaf. However, it is also possible that some of the new genes, despite being expressed in the embryo in a JNK-dependent manner, are not involved in DC. These target genes thus remain under the control of JNK, but are functionally ‘silent’ during DC. This behaviour is best illustrated by reaper, whose expression is JNK-dependent in the embryo, but which does not seem to have any function in the LE, acting only later during development or at the adult stage (Rousset, 2017).

Surprisingly, quantitative analysis of LE-specific gene expression profiles showed a variety of previously uncharacterized expression patterns along the LE, with two levels of regulation, AP and segmental. These observations reveal a new property of the LE which appears highly patterned along the AP axis, contrasting with the homogenous and linear structure previously envisioned. In addition, the higher order regulation that emerges from these results provides every LE cell with its own identity through the cross-talk between JNK, AP and segmental information. Such cell-level patterning through signalling crosstalk is likely essential for coordination and robustness of closure as well as segment matching. In this view, recent work showed that Wg and JNK interact at the LE to control the formation of specific mixer cells at segment boundaries (Rousset, 2017).

Previous work showed that, instead of acting independently, HOX and segmentation genes can be coupled to regulate target genes in the embryo. This study revealed an additional layer of regulation involving the 'morphogenetic' JNK signalling pathway. During DC, JNK acts as a tissue-specific switch whose activity can be regulated by HOX and segmentation pathways, providing positional information an 'onion-like' regulatory model allows for several levels of regulation/information to pile up in order to regulate individual cellular behaviours important for tissue morphogenesis. Each layer can act positively or negatively on LE target gene expression, generating a complex repertoire of regulatory pathways. Distinct categories of expression profiles were identified in this study through the analysis of individual target genes, with the likelihood of more gene-specific patterns to be discovered. For example, the same HOX gene (abd-A or Abd-B) can have activating or repressive activity according to the target gene, as is the case for the transcription factor En. Molecular functional characterization of cis-regulatory elements controlling LE gene expression will bring a more detailed view of how transcription factor complexes are formed, how specificity of DNA recognition is achieved and how activating or repressive activities are regulated to generate LE patterning (Rousset, 2017).

scaf proves to be a remarkable case among the JNK target genes, showing the different levels of regulation that can be integrated into a single promoter. Not only is it strongly expressed in the LE in a JNK-dependent manner, but it is also regulated by both the segmentation gene en and the HOX genes. In particular scaf displays a transcriptional response induced by all the trunk HOX genes tested, being positively controlled by Scr, Antp, Ubx and Abd-B and negatively by abd-A. It can therefore be considered as a general HOX target gene, i.e. regulated by most Hox paralogs, as previously defined. Another example of a general target is the Drosophila gene optix, which is activated by the head HOX genes labial and Deformed (Dfd) and inhibited by the trunk HOX genes. Nonetheless the general HOX target genes do not represent the majority. A genomic analysis in the Drosophila embryo identified more than 1500 genes regulated by at least one of the six HOX paralogs tested (Dfd, Scr, Antp, Ubx, abd-A, Abd-B). Only 1.3% of these genes are regulated by the six paralogs and 1.5% by the five paralogs that were used in this study. Interestingly more than 40% of the ~1500 HOX target genes are also present in the JNK genomic data set that was obtained. This strong overlap well reflects the fact that the LE runs along most of the body AP axis encompassing the thorax and abdomen. More importantly, it also indicates that AP patterning plays a crucial role in the regulation of DC, as shown in this study (Rousset, 2017).

Live imaging and mathematical modelling revealed asymmetries in the geometry and zipping process along the AP axis; these can be attributed to local constraints induced by head involution and apoptosis. Head involution is concomitant with DC and induces tension in the anterior part of the embryo, explaining why the DC phenotypes are almost exclusively observed in the anterior part, leading to the so-called 'anterior-open phenotype'. The exception to this rule is the experimental manipulation of the posterior zipping rate through localized laser ablation of the amnioserosa close to the canthus, which induces a strong delay of posterior closure. The results with the abd-A and Abd-B mutants show that posterior delay can also be obtained in genetically-perturbed embryos. However, while anterior zipping is slightly up-regulated when posterior zipping is laser-targeted, it was shown that the anterior speed of closure is diminished in the Abd-B embryo. Thus, compensatory mechanisms may only appear when tissue integrity is severely impaired. Apoptosis was also proposed to participate in the asymmetric properties of DC. Delamination of apoptotic cells in the anterior amnioserosa produces forces that are responsible for a higher rate of anterior zipping. However, the phenotype that was observed with the abd-A or Abd-B mutation cannot be attributed to defects in this mechanism, as the rate of apoptosis is already very low in the posterior amnioserosa. In summary, the data reveal a genetic control of zipping through precise transcriptional regulation in the LE. Overall, this work provides a framework for apprehending how the HOX selector genes and their cofactors collaborate with other signalling pathways to generate specific transcriptional responses allowing morphogenetic patterning and proper coordinated development (Rousset, 2017).

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