Abdominal-B

DEVELOPMENTAL BIOLOGY

Embryonic

The pattern of the Abd-B regulatory (r) protein expression, as deduced by analysis of Abd-B mutants, is restricted to ps14 and 15 in all germ layers and observes a parasegmental boundary at its anterior margin of expression. In contrast, the pattern of morphogenetic (m) protein expression is unusual as its level in the ectoderm increases from ps10 to ps13 in parasegmental steps. Its anterior margin of expression is highly dynamic shifting anteriorly across more than 3 parasegments during midembryonic development. Evidently, the control mechanisms of m and r protein expression are considerably different. M protein expression and regulation varies to some extent in individual germ layers (De Lorenzi, 1990a).

To gain further insights into homeotic gene action during CNS development, the role of the homeotic genes was characterized in embryonic brain development of Drosophila. Neuroanatomical techniques were used to map the entire anteroposterior order of homeotic gene expression in the Drosophila CNS. This order is virtually identical in the CNS of Drosophila and mammals. All five genes of the Antennapedia Complex are expressed in specific domains of the developing brain. The labial gene has the smallest spatial expression domain; it is only expressed in the posterior part of the tritocerebral anlage. This contrasts with previous reports that lab is expressed throughout the tritocerebral (intercalary) neuromere. The proboscipedia gene has the largest anteroposterior extent of expression, however, in contrast to other homeotic genes, pb is only found in small segmentally repeated groups of 15-20 cells per neuromere. These groups of pb-expressing cells range from the posterior deutocerebrum toward the end of the VNC. Since pb-expressing cells are found anterior to the lab-expressing cells in the brain, this is an exception to the spatial colinearity rule. (Spatial colinearity is conserved in the epidermis, where pb expression is posterior to lab expression). The Deformed gene is expressed in the mandibular neuromere and the anterior half of the maxillary neuromere and the Sex combs reduced gene is expressed in the posterior half of the maxillary neuromere and the anterior half of the labial neuromere. The Antennapedia gene is expressed in a broad domain from the posterior half of the labial neuromere toward the end of the VNC. The three genes of the Bithorax Complex are expressed in the VNC. Ultrabithorax gene expression extends in a broad domain from the posterior half of the T2 neuromere to the anterior half of the A7 neuromere, with highest expression levels in the posterior T3/anterior A1 neuromeres. The abdominal-A gene is expressed from the posterior half of the A1 neuromere to the posterior half of the A7 neuromere. For the above mentioned genes, the anterior border of CNS expression remains stable from stage 11/12 until the end of embryogenesis. In contrast, the anterior border of CNS expression for the Abdominal-B gene shifts at stage 14. Before this stage Abd-B expression extends from the posterior half of neuromere A7 to the end of the VNC; afterwards, it extends from the posterior half of neuromere A5 to the end of the VNC with the most intense expression localized to the terminal neuromeres. With the exception of the Dfd gene, the anterior limit of homeotic gene expression in the CNS is always parasegmental (Hirth, 1998).

Each of the somatic cell types of the gonad arises from mesodermal cells that constitute the embryonic gonad. Using markers for the precursors of the somatic cells of the gonad, five discrete steps have been identified in gonadal development:

1. First, somatic gonadal precursor cells are specified within the mesoderm in parasegments 10 through 12.
2. After pole cells traverse and exit the midgut they recognize and associate primarily with specific mesodermal cells laterally positioned in the mesoderm of parasegments 11 and 12. These are the migratory gonadal precursors that delaminate from the mesodermal cell sheet.
3. In a third step, gonadal precursors and pole cells migrate anteriorly, where they contact cells in parasegment 10.
4. Next, gonadal precursors and pole cells arrest migration at parasegment 10.
5. Finally, the mesodermal cells partially ensheath the arriving cells, and the cluster coalesces into the gonad.

The functions of the homeotic genes abdominal A and Abdominal B are both required for the development of gonadal precursors. Each plays a distinct role. abd A activity alone specifies anterior gonadal precursor fates, whereas abd A and Abd B act together to specify a posterior subpopulation of gonadal precursors. Once specified, gonadal precursors born within posterior parasegments move to the site of gonad formation. The proper regional identities, as established by homeotic gene function, are required for the arrest of migration at the correct position. abd A is required in a population of cells within parasegments 10 and 11 that partially ensheath the coalescing gonad. Mutations in iab-4, a distal enhancer element, abolish expression of abd A within these cells, blocking the coalescence of the gonad (Boyle, 1995).

The embryonic dorsal vessel in Drosophila possesses anteroposterior polarity and is subdivided into two chamber-like portions, the aorta in the anterior and the heart in the posterior. The heart portion features a wider bore as compared with the aorta and develops inflow valves (ostia) that allow the pumping of hemolymph from posterior toward the anterior. Homeotic selector genes provide positional information that determines the anteroposterior subdivision of the dorsal vessel. Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) are expressed in distinct domains along the anteroposterior axis within the dorsal vessel, and, in particular, the domain of abd-A expression in cardioblasts and pericardial cells coincides with the heart portion. Evidence is provided that loss of abd-A function causes a transformation of the heart into aorta, whereas ectopic expression of abd-A in more anterior cardioblasts causes the aorta to assume heart-like features. These observations suggest that the spatially restricted expression and activity of abd-A determine heart identities in cells of the posterior portion of the dorsal vessel. Abd-B, which at earlier stages is expressed posteriorly to the cardiogenic mesoderm, represses cardiogenesis. In light of the developmental and morphological similarities between the Drosophila dorsal vessel and the primitive heart tube in early vertebrate embryos, these data suggest that Hox genes may also provide important anteroposterior cues during chamber specification in the developing vertebrate heart (Lo, 2002).

During early cardiogenesis at embryonic stages 10-11, peak levels of Abd-B are observed in parasegments (PS) 13 and 14, which express the m and r proteins of Abd-B, respectively. These two parasegments abut the region of PS 2-12 from which heart progenitors arise. Indeed, double stainings of stage 11 embryos for Abd-B (combined m+r) and Evenskipped (Eve), an early marker for pericardial cell and dorsal muscle progenitors, confirm that the previously known absence of mesodermal Eve cells in PS 13 coincides with the domain of peak expression of Abd-B in both ectoderm and mesoderm. This observed gap in Eve expression is compatible with the possibility that the Abd-B m variant is able to suppress the formation of Eve pericardial and somatic muscle cells in PS 13, whereas Abd-B r is not active in suppressing eve cells (with unknown fates) in PS 14. In agreement with this notion, Abd-B mutant embryos generate an additional cluster of mesodermal eve cells in PS 13. This observation suggests that Abd-B normally represses cardiogenesis, including the formation of pericardial cells, as well as the formation of somatic muscle #1, which is also derived from Eve-positive progenitor cells, in PS 13. This interpretation is further supported by the presence of supernumerary cardioblasts in the heart portion of the dorsal vessel of late stage embryos, as shown by anti-Mef2 stainings. In Abd-B mutant embryos, there are about 116 cardioblast nuclei as compared with the normal number of 104 in the wildtype. Although the heart does not appear significantly elongated in the mutant embryos, it is frequently much wider and extra cardioblasts are arranged in irregular clusters or double-rows within its posterior portion. Similar increases in the number of cardioblasts and pericardial cell within the heart portions were seen in anti-Tin stainings of late stage Abd-B mutant embryos. In addition to the observed increase in the number of heart cells, the somatic muscles in abdominal segment 8 (A8) in Abd-B mutant embryos show an increase in the number of nuclei and a Mef2 pattern that is more similar to the pattern normally found in A7. Together with the Eve expression data at earlier stages and in agreement with the known muscle pattern, this observation indicates that Abd-B functions also to suppress the formation of the majority of dorsal body wall muscles in A8, including the Eve-expressing muscle #1 (Lo, 2002).

The results of ectopic expression experiments with Abd-B are fully consistent with these proposed functions of Abd-B in early heart and somatic muscle development. Specifically, ectopic expression of Abd-B (m) that is driven by the twist promoter in the entire mesoderm completely suppresses the formation of cardioblast cells, as determined by anti-Mef2 staining. In addition, the number of Mef2-stained somatic muscle nuclei is reduced and more comparable to the number of somatic muscle nuclei normally found in A8. It appears therefore that Abd-B expression in the early mesoderm of those segments where it is not normally expressed is sufficient to suppress the development of the dorsal vessel as well as the formation of many somatic muscles (Lo, 2002).

Hox-controlled reorganisation of intrasegmental patterning cues underlies Drosophila posterior spiracle organogenesis

Hox proteins provide axial positional information and control segment morphology in development and evolution. Yet how they specify morphological traits that confer segment identity and how axial positional information interferes with intrasegmental patterning cues during organogenesis remains poorly understood. This study investigates the control of Drosophila posterior spiracle morphogenesis, a segment-specific structure that forms under Abdominal-B (AbdB) Hox control in the eighth abdominal segment (A8). The Hedgehog (Hh), Wingless (Wg) and Epidermal growth factor receptor (Egfr) pathways provide specific inputs for posterior spiracle morphogenesis and act in a genetic network made of multiple and rapidly evolving Hox/signalling interplays. A major function of AbdB during posterior spiracle organogenesis is to reset A8 intrasegmental patterning cues, first by reshaping wg and rhomboid expression patterns, then by reallocating the Hh signal and later by initiating de novo expression of the posterior compartment gene engrailed in anterior compartment cells. These changes in expression patterns confer axial specificity to otherwise reiteratively used segmental patterning cues, linking intrasegmental polarity and acquisition of segment identity (Merabet, 2005).

In the dorsal ectoderm of stage 10 embryos, hh and wg follow the same striped expression patterns in A8 as in other abdominal segments. rho expression, which marks cells secreting an active form of the Egf ligand, occurs in all primordia of tracheal pits, in A8 as in more anterior segments (Merabet, 2005).

Specification of posterior spiracle primordia occurs at early stage 11. The primordia can then be recognised by Cut expression in spiracular chamber cells and by Sal, the homogenous expression of which in A8 becomes restricted dorsally to stigmatophore cells (forming the external structure of the posterior spiracle) that form a crescent surrounding Cut-positive cells. From mid-stage 11, wg and rho adopt in the dorsal ectoderm expression patterns specific to A8, with wg transcribed in two cells only and rho in a second cell cluster, dorsal and posterior to the tracheal placode. To localise wg- and rho-expressing cells with regard to stigmatophore and spiracular chamber cells, co-labelling experiments for wg or rho transcripts and for Cut or Sal proteins were performed: the two wg cells lie between Cut- and Sal-positive cells; the second cell cluster expressing rho in A8 also expresses Cut but not Sal. This cluster is likely to produce the Egf ligand required for posterior spiracle development, since mutations that alleviate rho expression in the tracheal placodes do not abolish spiracles formation. At mid-stage 11, the hh pattern in A8, along a stripe lying posterior and adjacent to the spiracular chamber and overlapping stigmatophore presumptive cells, resembles expression in other abdominal segments. Analyses at later stages indicate that the relationships between posterior spiracle cells and hh, wg and rho patterns are maintained (Merabet, 2005).

Null mutations of wg, hh or Egfr result in the absence of posterior spiracles. The strong cuticular defects observed raise the possibility that the phenotypes result indirectly from early loss of segment polarity. Removing the Wg, Hh or Egfr signals from 5-8 hours of development using thermosensitive alleles causes strong segment polarity defects but allows filzkörpers, stigmatophores or even complete posterior spiracles to form. Thus, spiracular chamber and stigmatophore can develop in embryos that have pronounced segment polarity defects (Merabet, 2005).

It was next asked whether defects in primordia specification could account for posterior spiracle loss, and Cut and Sal expression was examined in the dorsal A8 ectoderm of hh, wg and Egfr mutant embryos. Expression of Cut and Sal is initiated at stage 11 in all of these mutants, although the somewhat disorganised patterns, especially from late stage 11, may reveal roles for these genes in signalling in sizing or shaping the posterior spiracle primordia. Alternatively, these defects may result from altered morphology of mutant embryos. In any case, the induction of the early markers Sal and Cut in A8 dorsal ectoderm of mutant embryos indicates that posterior spiracle primordia specification does occur in the absence of signalling by Wg, Hh or Egfr. Transcription of ems, another AbdB target that is activated slightly later than Cut, although not affected in hh mutants, is lost in wg or Egfr mutants. Thus, proper regulation of AbdB downstream targets activated following primordia specification appears dependent on signalling activities (Merabet, 2005).

The role was examined of Wg, Hh and Egfr signalling pathways in posterior spiracle organogenesis (i.e., after the specification of presumptive territories). Co-labelling experiments performed on embryos expressing GFP driven by ems-Gal4 or by sal-Gal4 indicate that whereas Cut and Sal are already expressed at early stage 11, GFP is detected from late stage 11 only. These two drivers, which promote expression approximately 1 hour after primordia specification, were used to express DN molecules for each pathway, counteracting Wg (DN-TCF), Egfr (DN-Egfr) or Hh [DN-Cubitus interuptus (Ci)] signalling from that time on. Blocking either pathway in spiracular chamber cells does not perturb stigmatophore morphogenesis, but specifically leads to the loss of differentiated filzkörpers. Conversely, blockade in stigmatophore cells provokes in each case its flattening, while differentiated filzkörpers do form (Merabet, 2005).

To ask how signalling inhibition interferes with the genetic modules initiated downstream of AbdB, expression of Sal and Cut was examined from stages 11 to 13. No major defects are seen until late stage 12. Strong deviation from the wild-type patterns is, however, observed slightly later, from stage 13 onwards: Sal expression in basal cells of the stigmatophore is lost and Cut expression remains in only a few scattered cells. The 2-hour delay seen between the onset of DN molecules expression and the detection of Sal and Cut could reflect the time required for shutting down the pathways. Alternatively, Sal and Cut expression may not require signalling activities before stage 13. To discriminate between these possibilities, an earlier expression of the DN molecules was forced, using the 69B-Gal4, known to promote protein accumulation by the onset of stage 11 (i.e., slightly before posterior spiracle primordia specification). Strong defects in Sal and Cut expression were again seen only in stage 13 embryos, supporting the notion that signalling activities are dispensable before the end of stage 12, but are required from stage 13 onwards to maintain Sal in basal stigmatophore cells and Cut in the spiracle chamber (Merabet, 2005).

A8-specific modulation of rho and wg patterns at mid-stage 11 suggests a regulation by AbdB. In AbdB mutants, rho expression in the spiracle-specific cell cluster is lost, and wg transcription does not evolve towards an A8-specific pattern. In embryos expressing AbdB ubiquitously, ectopic posterior spiracle formation in the trunk can be identified as ectopic sites of Cut accumulation. In such embryos, rho and wg are induced in trunk segments following patterns that resemble their expression in A8: rho in a cluster that overlaps the Cut domain, and wg in few cells abutting ectopic Cut-positive cells. These transcriptional responses to loss and gain of function of AbdB indicate that the Hox protein controls the A8-specific expression patterns of wg and rho. The lines gene (lin), which is known to be required for Cut and Sal activation by AbdB, also controls wg and rho patterns respecification (Merabet, 2005).

In contrast to wg and rho, hh does not adopt an A8-specific expression pattern at mid-stage 11. At that stage, hh expression pattern is not affected upon AbdB mutation. The hh stripe in A8 lies posterior and adjacent to spiracular chamber cells and overlaps stigmatophore cells, suggesting that Hh signalling may participate in the regulation of rho and wg transcription by AbdB. In support of this, it was found that the AbdB-dependent aspects of rho and wg transcription patterns are missing in hh mutant embryos. Thus, inputs from both Hh and AbdB are required to remodel Wg and Egfr signalling in A8 (Merabet, 2005).

The dependence of wg and rho A8 expression patterns on Hh, and the loss of ems expression in wg and rho but not in hh mutants, suggest that transcription of ems requires Wg and Egfr signalling prior to wg and rho pattern respecification by AbdB and Hh. To explore this point further, the time course of ems, wg and rho expression was comparatively analyzed. Embryos bearing an ems-lacZ construct stained for ß-Gal and for wg or rho transcripts show that ems expression precedes wg pattern respecification, and occurs at the same time as rho acquires an A8-specific pattern. Importantly, A8-specific rho clusters were never observed before the onset of ems expression. Thus, ems transcription starts before wg and at the same time as rho pattern respecification, supporting that signalling by Wg and Egfr is required prior to mid-stage 11. These observations also indicate that respecification of the wg pattern occurs slightly later than that of rho, which could not been concluded from changes in embryo morphology (Merabet, 2005).

To determine whether signalling by Wg and Egfr from local sources is important for posterior spiracle organogenesis, the production of Wg and Spi^S (the mature form of Spi) ligands was forced from domains broader than normal in A8 dorsal ectoderm. This was performed after posterior spiracle specification, using the ems-Gal4 and sal-Gal4 drivers. Ectopic signalling results in abnormally shaped posterior spiracles: stigmatophores are reduced in size and filzkörpers do not elongate properly. Ectopic signalling from all presumptive stigmatophore cells results in stronger defects than those produced when ectopic signals emanate from all spiracular chamber cells. This can be correlated to the fact that sal-Gal4 drives expression in a pattern that more strongly diverges from the wild-type situation than ems-Gal4 does. Thus, restricted delivery of Wg and Spi^S signals is required for accurate posterior spiracle organogenesis (Merabet, 2005).

It was next asked whether, downstream of Hh, the Wg and Egfr pathways provide separate inputs for posterior spiracle organogenesis. Two sets of experiments were conducted and it was found that: (1) in embryos respectively mutant for Egfr or wg, wg and rho acquire A8-specific patterns; (2) epistasis experiments performed by forcing in spiracular or stigmatophores cells the activity of one pathway while inhibiting the other indicate that loss of one pathway could not be rescued by the other. Thus, Egfr and Wg pathways do not act as hierarchically organised modules, but provide independent inputs for posterior spiracle organogenesis (Merabet, 2005).

The expression of the posterior compartment selector gene engrailed (en) until stage 12 follows a striped pattern identical in all trunk segments. Later on, En adopts a pattern that is specific to A8: it is no longer detected in the ventral part of the segment; dorsally, the En stripe has turned to a circle of cells that surround the future posterior spiracle opening and express the stigmatophore marker Sal. The transition from a striped to a circular pattern depends on AbdB. This transition could result either from a migration of en posterior cells towards the anterior, or from transcriptional initiation in cells that were not expressing en before stage 12, and that can therefore be defined as anterior compartment cells (Merabet, 2005).

To distinguish between the two possibilities, en-Gal4/UAS-lacZ embryos were simultaneously stained with anti ß-Gal and anti-En antibodies. If circle formation results from cell migration, one would expect ß-Gal and En to be simultaneously detected in all cells of the circle since the two proteins are already co-expressed in the posterior compartment stripe earlier on. Conversely, if the circle results from de novo expression, one would expect anterior cells in the circle to express En before ß-Gal, since ß-Gal production requires two rounds of transcription/translation compared with one for En. It was found that cells from the anterior part of the circle express En but not ß-Gal in stage 13 embryos, which demonstrates that de novo expression of En occurs in anterior compartment cells. Further supporting En expression in anterior compartment cells, it was found that precursors of anterior spiracle hairs that do not express En at stage 12 do so at stage 13. Engrailed function in A8 is essential for posterior spiracle development, since stigmatophores do not form in en mutants, and are restored if En is provided in stigmatophore cells (Merabet, 2005).

It was also found that although identical in all abdominal segments at stage 11, hh transcription adopts an A8-specific pattern from stage 12 onwards: transcripts are then localised only at the anterior border of the En stripe. This expression of hh is lost in AbdB mutants and still occurs in en mutant. The uncoupling of hh transcription from En activity in the dorsal A8 ectoderm correlates with the distinct phenotypes seen for en mutants, which do differentiate filzkörper like structures, and for hh mutants, which do not (Merabet, 2005).

Data in this paper allow the distinguishing of four phases in functional interactions between AbdB and signalling by Wg, Hh and Egfr during posterior spiracle formation. The first phase corresponds to the specification of presumptive territories of the organ. The signalling activities are not involved in this AbdB-dependent process, since they are not required for the induction of the earliest markers of spiracular chamber and stigmatophore cells, Cut and Sal, in the dorsal ectoderm of A8. The second phase, which immediately follows primordia specification, concerns the regulation of AbdB target genes activated slightly later. Inputs from the Hox protein and the Wg and Egfr pathways are then simultaneously needed, as seen for transcriptional initiation of the ems downstream target. This function of Wg and Egfr signalling precedes and does not require the reallocation of signalling sources in A8-specific patterns; impairing A8-specific expression of wg and rho by loss of hh signalling does not affect ems expression. Within the third phase, AbdB and Hh activities converge to reset wg and rho expression patterns. The three phases take place in a narrow time window, less than 1 hour during stage 11, and could only be distinguished by studying the functional requirements of Wg, Hh and Egfr for transcriptional regulation in the posterior spiracle (Merabet, 2005).

The fourth phase is referred to as an organogenetic phase. Data obtained using DN variants to inhibit the pathways in cells already committed to stigmatophore or filzkörper fates, indicate that Wg, Egfr and Hh pathways are required for organ formation after specification and early patterning of the primordia. Their roles are then to maintain the AbdB downstream targets' expression in posterior spiracle cells as development proceeds, as shown for Cut and Sal at stage 13 (Merabet, 2005).

A salient feature of AbdB function during posterior spiracle development is to relocate Wg and Egfr signalling sources in the dorsal ectoderm at mid-stage 11. wg and rho then adopt expression patterns that differ from expressions in other abdominal segments, conferring axial properties unique to A8 to otherwise segmentally reiterated patterning cues. Resetting Wg and Egfr signalling sources into restricted territories is of functional importance for organogenesis, as revealed by the morphological defects that result from the delivery of Wg or Spi^S signals in all spiracular chamber or stigmatophore cells after the specification phase. During stage 12, AbdB also relocates the Hh signalling source by inducing En-independent expression of hh in the dorsal ectoderm. Thus, later than Wg and Egfr signalling, the Hh signal also acquires properties unique to A8. In generating this pattern, AbdB plays a fundamental role in uncoupling hh transcription from En activity, providing a context that prevents anterior compartment En-positive cells to turn on hh transcription, and that allows hh expression in the absence of En in other cells. Slightly later, at stage 13, AbdB modifies the expression of the posterior selector gene en, initiating de novo transcription in anterior compartment cells. In these cells, En fulfils different regulatory functions than in posterior cells, as discussed above for hh regulation. Changes in En expression and function can be interpreted as a requisite to loosen AP polarity in A8 and gain circular coordinates required for stigmatophore formation (Merabet, 2005).

Postmitotic specification of Drosophila insulinergic neurons from pioneer neurons

Insulin and related peptides play important and conserved functions in growth and metabolism. Although Drosophila has proved useful for the genetic analysis of insulin functions, little is known about the transcription factors and cell lineages involved in insulin production. Within the embryonic central nervous system, the MP2 neuroblast divides once to generate a dMP2 neuron that initially functions as a pioneer, guiding the axons of other later-born embryonic neurons. Later during development, dMP2 neurons in anterior segments undergo apoptosis but their posterior counterparts persist. Surviving posterior dMP2 neurons no longer function in axonal scaffolding but differentiate into neuroendocrine cells that express insulin-like peptide 7 (Ilp7) and innervate the hindgut. The find that the postmitotic transition from pioneer to insulin-producing neuron is a multistep process requiring retrograde bone morphogenetic protein (BMP) signalling and four transcription factors: Abdominal-B, Hb9, Forkhead, and Dimmed. These five inputs contribute in a partially overlapping manner to combinatorial codes for dMP2 apoptosis, survival, and insulinergic differentiation. Ectopic reconstitution of this code is sufficient to activate Ilp7 expression in other postmitotic neurons. These studies reveal striking similarities between the transcription factors regulating insulin expression in insect neurons and mammalian pancreatic beta-cells (Miguel-Aliaga, 2008).

The observed death of some Drosophila pioneer neurons has been used to argue that their function is transient, but persistence in other cases suggested that, either they continue to play an axonal-scaffolding role, or that they adopt some other identity. The current findings resolve this long-standing issue by clearly demonstrating that, for dMP2 neurons, the axonal scaffolding function is only transient. After this role is no longer required, surviving dMP2 neurons become insulinergic and innervate the hindgut. The other known innervation of the Drosophila gut occurs much more anteriorly, in the foregut and anterior midgut, from neuronal cell bodies located in the peripheral ganglia of the stomatogastric nervous system. Unlike dMP2 neurons, however, the individual identities of the stomatogastric neurons and their cell lineages remain to be clearly defined. Thus, dMP2 neurons may provide a simple and well-characterised system for studies of the guidance cues involved in enteric innervation. Future studies, however, will be needed to determine the functions of Ilp7 in dMP2 neurons. It will be important to distinguish if this posterior neural source of insulin acts humorally to promote growth, like the more anterior brain mNSCs, or if it has more local effects in abdominal tissues. In this regard, the presence of Ilp7-expressing neurites in close proximity to the Ilp2-producing mNSCs is intriguing (Miguel-Aliaga, 2008).

The transition from pioneer to neuroendocrine neuron is not unique to dMP2 neurons, as Drosophila MP1 pioneer neurons also become neuropeptidergic at larval stages (Wheeler, 2006). In the grasshopper, segment-specific survival of pioneer neurons has also been reported, raising the possibility that they too may become neuroendocrine. Studies in other species, including vertebrates, will be needed to reveal the extent to which the linkage between pioneer and neuroendocrine functions is conserved. Identifying pioneer neurons with an 'ancestral' neuroendocrine identity in other phyla would lend further support to the proposal that pioneer neurons are highly conserved in evolution (Miguel-Aliaga, 2008).

Apoptosis of postmitotic neurons is a widespread feature of normal VNC development, but few developmental regulators of core pro-apoptotic genes such as grim, hid, and rpr have been identified. This study uncovers roles for Fkh and Hb9. Hb9, at least, appears linked to cell death in neurons other than dMP2: in Df(3L)H99 mutant embryos, where apoptosis is blocked, ectopic Hb9-positive RP motor neurons are observed in segments A7-A8. Hb9 is an important regulator of motor neuron identity in both Drosophila and vertebrates. Finding of a pro-apoptotic function for Hb9 in Drosophila, together with the neurotrophic requirement for motor neuron survival in vertebrates, raises the possibility that the same genetic programs specifying the identities of motor neurons also sensitize them for postmitotic editing via apoptosis (Miguel-Aliaga, 2008).

Fkh function in CNS development has not been characterized. Fkh is expressed in segmentally repeated clusters of midline neurons, including dMP2, vMP2, MP1 neurons, and the VUM interneurons. Within the MP2 lineage, Fkh is first expressed in the MP2 neuroblast at stage 9-10 and continues to be expressed in both the dMP2 and vMP2 daughters throughout embryonic and larval stages. In fkh mutants, 95% of anterior dMP2 neurons fail to undergo apoptosis, and 95.3% of posterior dMP2 neurons (and 100% of ectopic anterior counterparts) fail to express Ilp7. Both of these dramatic phenotypes could be rescued to near wild-type levels by reintroducing Fkh under odd-GAL4 regulation, indicating a cell-autonomous requirement for promoting dMP2 apoptosis and Ilp7 expression (Miguel-Aliaga, 2008).

Hb9 and Fkh expression in many neurons that do not die suggests a combinatorial mechanism for the control of developmental apoptosis. One possibility is that several transcription factors function in combination to activate the core pro-apoptotic genes. Given the proposed role for Foxa proteins in chromatin accessibility, Fkh expression in dMP2 neurons may render the promoters of core pro-apoptotic genes responsive to activation by Hb9. An alternative but not mutually exclusive mechanism involves individual transcription factors activating different pro-apoptotic genes such that a combination of these would then be required to trigger neuronal death. For example, Hb9 could be required for rpr/skl but not grim expression. Some support for this idea comes from the observation that loss of hb9 activity blocks rpr/skl-mediated death of dMP2 neurons but not the largely grim-dependent apoptosis of anterior MP1 neurons (Miguel-Aliaga, 2008).

An important conclusion from this study is that the combinatorial transcription factor code controlling apoptosis partially overlaps with that regulating insulinergic identity. Thus, Fkh and Hb9 are both essential components of the codes for anterior apoptosis and also Ilp7 expression, illustrating that these transcription factors play surprising dual roles as pro-apoptotic and pro-differentiation factors within the same neuronal subtype. Importantly, the results also show that the segment-specific Hox protein Abd-B acts as a postmitotic switch, converting the pro-apoptotic Fkh⁺ Hb9⁺ code into an insulinergic Fkh⁺ Hb9⁺ Abd-B⁺ code (Miguel-Aliaga, 2008).

Three Ilp7 regulators (Hb9, Abd-B, and Fkh) are expressed at least 12 h before Ilp7 is first activated: from the time when the MP2 neuroblast exits the cell cycle. In the case of Hb9, it was not possible to uncouple two temporally separable functions. Early postmitotic expression of Hb9 is important for its death-activating function, whereas later expression suffices for activating Ilp7. Similarly, the Hox protein Abd-B generates a segment-specific neuropeptide pattern via postmitotic regulation of posterior dMP2 survival and also Ilp7 activation. As vertebrate neuropeptides are also expressed in restricted neuronal populations within specific rostrocaudal domains, they may be similarly regulated by Hox survival/neuroendocrine inputs. In the case of Fkh, it is required for many different aspects of the progression from the early to the late postmitotic dMP2 fate. Fkh expression is restricted to VNC midline neurons and its vertebrate orthologue Foxa2 functions in the differentiation of the floor plate and ventral dopaminergic and serotonergic neurons (Ferri, 2007; Jacob, 2007; Norton, 2005). Thus, in both the Drosophila midline and its vertebrate counterpart, the floor plate, Fkh proteins play a conserved role in the differentiation of ventral neuronal subtypes (Miguel-Aliaga, 2008).

The other two dMP2 regulators identified in this study, Dimm and the BMP pathway, are switched on shortly before the onset of Ilp7 expression. The timing of onset of these two broad neuroendocrine regulators is likely to specify when Ilp7 is first activated, whereas the earlier factors Fkh, Hb9, and Abd-B may contribute more specifically to insulinergic identity. Together, the genetic and expression analyses in this study demonstrate that the combinatorial code of genetic inputs required for Ilp7 expression is assembled in a step-wise manner during postmitotic maturation. Importantly, this allows a subset of the components to be shared (such as Fkh and Hb9) between sequential neuronal programmes (survival and Ilp7 expression) without losing output specificity (Miguel-Aliaga, 2008).

Two observations from this study indicate that insulinergic combinatorial codes can vary from cell-to-cell and also from one Ilp to another. (1) None of the regulators of Ilp7 in dMP2 neurons appear to regulate it in DP neurons. (2) The dMP2 insulinergic code is sufficient to trigger ectopic expression of Ilp7 but not Ilp2 or other neuropeptides such as FMRFa. These findings suggest the existence of additional, as yet unidentified, insulinergic factors in DP neurons and also in the brain mNSCs where Ilp2 is expressed. Identification of the neural progenitor for these mNSCs (Wang, 2007) should facilitate characterization of the Ilp1/Ilp2/Ilp3/Ilp5 combinatorial codes and thus clarify the extent to which different insulinergic transcriptional programmes overlap (Miguel-Aliaga, 2008).

The finding that an Ilp7-expressing neuron derives from the MP2 lineage reveals that at least some insulinergic regulators are similar in insects and mammals. Three apparent similarities may not be very insulin-specific but reflect more general processes shared by neural and endocrine programmes in many species. (1) Notch signalling singles out the MP2 neuroblast and distinguishes its two progeny neurons, while in mammals, it limits pancreatic expression of the 'proneural' gene Ngn3 to prospective endocrine cells. (2) The survival and pro-Ilp7 functions mediated by Abd-B in the dMP2 neuron could also have their postmitotic counterparts in ß-cells, either mediated by related Hox genes or via another homeobox gene, Pdx-1, following its early input into pancreatic induction. (3) Nerfin-1 is required for dMP2 pioneer function (Kuzin, 2005), while its mammalian orthologue Insm1/IA1 is important for pancreatic ß-cell specification (Miguel-Aliaga, 2008).

Several more specific regulatory similarities exist between the insulinergic differentiation factors active in postmitotic dMP2 neurons. For example, the role of fkh in dMP2 neurosecretory differentiation described in this study is similar to the functions of HNF3b/Foxa2 in islet maturation and insulin secretion (Sund, 2001). In addition, mammalian Nkx2.2 is important for pancreatic ß-cell specification and is known to activate transcription of the insulin regulator Nkx6.1: an important late event in ß-cell differentiation. Intriguingly, the Drosophila orthologue of Nkx2.2, Vnd, is required for dMP2 formation. Drosophila Nkx6.1, the orthologue of mammalian Nkx6 (FlyBase name HGTX), is expressed by postmitotic dMP2 neurons, and it will be interesting to determine whether it too functions downstream of Vnd during Ilp7 regulation. Most strikingly, mammalian equivalents of two of the insulinergic inputs identified in this study, Hb9 and BMP signalling, are also required for several aspects of late ß-cell differentiation including the expression of Nkx6.1 and insulin. Together, these insect-mammalian comparisons provide evidence that, although the cell types involved look very different, some of the genetic circuitry regulating insulin is conserved between arthropods and chordates. This suggests that the power of fly genetics can now be harnessed to identify additional mammalian regulators of neuroendocrine cell fates and insulin expression (Miguel-Aliaga, 2008).

Larval

The level of polyteny of the Drosophila salivary gland chromosomes was determined throughout the chromosome region 89E1-4, the locus of the Bithorax Complex. A zone of underreplication spans the 300 kb of DNA from the Ubx to Abd-B loci. From the centromere proximal end of the complex, a 70-kb-long gradual decrease of polytenization starts with the Ubx transcription unit and, after a floor corresponding to the abd-A locus, raises gradually back to the maximum over 70 kb in the region of the Abd-B transcription unit. The maximum relative level of underreplication is about 10-fold. The level of polyteny of chromosomes in a gland is estimated at about 1,000. Therefore, even at the lowest point of polyteny, the number of DNA duplexes assuring the continuity of the chromosomes can be estimated at 100 and certainly not limited to a unique double helix. In flies carrying the mutation Suppressor of DNA Underreplication [Su(UR)ES], the underreplication of the Bithorax Complex is fully suppressed. In the wild type, the Bithorax Complex forms a weak point featuring thinner bands separated by clefts or constrictions. In Su(UR)ES strain in contrast, the 89E1-4 band looks like a single solid band consisting of homogenous dense material. It is speculated that the wild-type Su(UR)ES protein hampers DNA replication of silenced domains and leads to their underreplication in salivary gland polytene chromosomes (Moshkin, 2001).

The expression of homeotic Bithorax Complex proteins in the fat bodies of Drosophila larvae was analyzed by staining with specific antibodies. These proteins are differentially expressed along the anteroposterior (AP) axis of the fat body, with patterns parallel to those characterized for the larval and adult epidermis. Since fat body nuclei have polytene chromosomes, it was possible to identify the BX-C locus and show that it assumes a strongly puffed conformation in cells actively expressing the genes of the BX-C. Immunostaining of these polytene chromosomes provided the resolution to cytologically map binding sites of the three proteins: Ubx, Abd-A and Abd-B. The results of this work provide a system with which to study the positioning of chromatin regulatory proteins in either a repressed and/or active BXC at the cytological level. In addition, the results of this work provide a map of homeotic target loci and thus constitute the basis for a systematic identification of genes that are direct in vivo targets of the BX-C genes (Marchetti, 2003).

Ubx is intensely expressed in a contiguous region, with an anterior limit distal to, but near, the anterior crossbridge in the third thoracic segment (T3). The domain includes the gonad, and the posterior limit falls in a region corresponding approximately to segments A6/A7. The Abd-A protein is expressed anteriorly in a longitudinal line of cells in a region corresponding to the A2 segment. From that point posteriorly it is accumulated in almost all of the cells in a region that is co-extensive with abdominal segments A3-A7. Finally, the Abd-B protein is expressed to the posterior end of the fat body with an anterior limit in the middle of A4. It is interesting to note that although Ubx is detected in all the nuclei of its domain, Abd-A and Abd-B are only expressed in subsets of nuclei in their respective domains. However, in the region corresponding to segments A4-A6 all of the proteins are co-expressed in most nuclei. These observations demonstrate that the protein products of the BX-C are differentially expressed along the AP axis of the fat body in a manner reminiscent of their accumulation patterns in the epidermis. However, the similarity of expression patterns of the proteins between the two tissues is more evident at their anterior limits than in their posterior extent. Perhaps the most striking result is the overlap of the three proteins in the region around the gonads. It will be interesting to determine if this overlap of domains has some operational significance, or if it is functionally irrelevant (Marchetti, 2003).

Pupal

The cuticle of the adult abdomen of Drosophila is produced by nests of imaginal histoblasts, which proliferate and migrate during metamorphosis to replace the polyploid larval epidermal cells. In this report, a detailed description is presented of the expression of four key patterning genes, engrailed (en), hedgehog (hh), patched (ptc), and optomotor-blind (omb), in abdominal histoblasts during the first 42 h after pupariation, a period in which the adult pattern is established. In addition, the expression is described of the homeotic genes Ultrabithorax, abdominal-A, and Abdominal-B, which specify the fates of adult abdominal segments. The results indicate that abdominal segments develop in isolation from one another during early pupal stages, and that some patterning events are independent of hh, wg, and dpp signaling. Pattern and polarity in a large anterior portion of the segment are specified without input from Hh, and evidence is presented that abdominal tergites possess an underlying symmetric pattern upon which patterning by Hh is superimposed. The signals responsible for this underlying symmetry remain to be identified (Kopp, 2002).

The dorsal cuticle of a typical abdominal segment contains a stereotyped sequence of pattern elements. At the anterior edge of each segment is the acrotergite, a narrow strip of naked sclerotized cuticle (a1). The remainder of the tergite is covered by trichomes, and can be subdivided into four regions. From anterior to posterior these regions are: a lightly pigmented region with no bristles (a2 fate); a lightly pigmented region that contains two to three rows of microchaetes (a3); a darkly pigmented region with one to two rows of microchaetes (a4); and a darkly pigmented region with a single row of macrochaetes (a5). The tergite is followed by the unpigmented posterior hairy zone (PHZ), which is composed of both anterior (a6) and posterior (p3) compartment cells. All trichomes and bristles in the segment are oriented uniformly from anterior to posterior. Finally, at the posterior edge of the segment is a zone of thin, naked intersegmental membrane (ISM), which can be subdivided into anterior smooth (p2) and posterior crinkled (p1) regions (Kopp, 2002).

The adult abdominal pattern is established in the first 2 days of pupal development, concurrent with the proliferation and migration of histoblasts and the destruction of the larval epidermal cells (LECs.) The spatial and temporal evolution of en, hh, ptc, and omb expression is followed during this critical period. The cuticle of each abdominal hemisegment is formed by three major histoblast nests. The anterior dorsal nest (aDHN) is composed of anterior compartment histoblasts and produces the tergite and part of the PHZ (a1-a6), whereas the posterior dorsal nest (pDHN) is composed of posterior compartment cells and produces the intersegmental membrane and the remainder of the PHZ (p1-p3). The ventral histoblast nest, which produces the sternite and pleura, contains both anterior and posterior compartment cells. en, hh, ptc, and omb are expressed in similar patterns in dorsal and ventral histoblasts, and the description is limited to the dorsal abdomen (Kopp, 2002).

Segment identities in the abdomen are specified by the Ubx, abd-A, and Abd-B genes of the bithorax complex (BX-C). More precisely, BX-C genes control the development of parasegments (ps), which are composed of the posterior compartment of one segment and the anterior compartment of the following segment. Ubx controls the identity of ps6, which includes the anterior compartment of the first abdominal segment (A1); abd-A functions primarily in ps7-ps9 (A2-A4), although it also contributes to the identities of ps10-ps12; and Abd-B is the main determinant of the identities of ps10-ps12. In the pupal abdomen, Abd-B is expressed strongly in ps12 (A7) (in females; the last abdominal segment is rudimentary in males), weaker in ps11 (A6), and at very low levels in ps10 (A5). This pattern is consistent with the view that different levels of Abd-B expression promote distinct segment identities in the posterior abdomen. abd-A is expressed in ps7 (A2) through ps12 (A7), at levels gradually increasing from the anterior to the posterior parasegments. Ubx is expressed only in the anterior compartment of A1 (ps6) in the abdominal epidermis. Double staining for Ubx and hh-lacZ shows that the posterior boundary of Ubx expression coincides precisely with the ps6/ps7 boundary. Thus, Ubx and abd-A are expressed in adjacent nonoverlapping domains, contrasting sharply with their overlapping expression in the embryo. Ubx expression is eliminated from A1 in the abd-A gain-of-function mutant Uab⁵, suggesting that abd-A represses Ubx during the pupal stage (Kopp, 2002).

Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia

In metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmental timetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct) loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia, indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the looping morphogenesis of male terminalia have been unclear. This study shows the role of apoptosis in the organogenesis of male terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerates as development proceeded, and completes a full 360° rotation. This acceleration is impaired when the activity of caspases or JNK or PVF/PVR signaling was reduced. Acceleration was induced by two distinct subcompartments of the A8 segment that form a ring shape and surround the male genitalia: the inner ring rotates with the genitalia and the outer ring rotates later, functioning as a 'moving walkway' to accelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation with single-cell tracking showed that the timing and degree of apoptosis correlates with the movement of the outer ring, and upregulation of the apoptotic signal increases the speed of genital rotation. Therefore, apoptosis coordinates the outer ring movement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitalia within a limited developmental time frame (Kuranaga, 2011).

To visualize the genitalia rotation in living animals, the His2Av-mRFP Drosophila line was used whose nuclei are ubiquitously marked by a fluorescent protein. The genital disc is a compound disc comprised of cells from three different embryonic segments: A8 (male eighth tergite), A9 (male primordium) and A10 (anal). During metamorphosis, the genital disc is partially everted, exposing its apical surface, and adopts a circular shape. The results captured the male genitalia undergoing a 360° clockwise rotation. Inhibiting apoptosis by expressing the baculovirus pan-caspase inhibitor p35 driven by engrailed-GAL4 (en-GAL4), which is expressed in the posterior compartment of each segment, results in genital mis-orientation at the adult stage (Kuranaga, 2011).

In flies expressing nuclear fluorescent protein driven by en-GAL4, it was observed that the posterior part of the A8 segment (A8p) formed a ring of cells surrounding the A9-A10 part of the disc. First, the images were recorded at a low resolution (10× objective lens) to measure the rotation speed accurately in control and p35-expressing flies, because long-term time-lapse imaging at a high resolution can cause photodamage, and thus alter pupal development. Most of the cells in the A8p that seem to disappear actually moved out of the plane of focus. The imaging results, the rotation started around 24 hours APF (after puparium formation) and stopped about 12 hours later. To confirm whether the mis-oriented genital phenotype in the caspase-inhibited flies was caused by incomplete rotation, the rotation was observed in flies expressing p35 under the en-GAL4 driver. In the p35-expressing flies, the rotation began, but it stopped before it was complete, after about 12 hours, i.e. with the same timing as in control flies. This suggested that the reduced caspase activation in A8p prevented the genitalia from completing the rotation, resulting in mis-oriented adult genitalia (Kuranaga, 2011).

To compare complete rotation with incomplete rotation, the rotation speed was calculated by measuring the angle (theta_control and theta_p35) of the A9 genitalia every 30 minutes on time-lapse images. The normal rotation was composed of at least four steps: initiation, acceleration, deceleration and stopping. The velocity of rotation V=dtheta/dt was calculated by measuring theta as a function of time t. At first, the genitalia rotated at an average velocity (V_control) of 7.67±3.72°/hour by 1 hour after initiation, then the rotation accelerated, with V_control gradually increasing to 53.83±7.11°/hour by 7 hours after initiation. Interestingly, in the p35-expressing flies, the rotation normally started at 24 hours APF, and the average velocity (V_p35) from the initial rotation to 1 hour later was 7.45± 2.98°/hour, which was not significantly different from the normal rotation. However, the acceleration of the rotation in the p35-expressing flies was lower than normal, with V_p35 gradually increasing to 21.35±7.45°/hour at 5.5 hours after initiation. The first peak of the acceleration rate, which was defined as the initiation of rotation, was observed in the p35-expressing flies (a_p35) and was the same as in the control flies (a_control). However, the duration of the acceleration period was shorter in the p35-expressing flies. These data suggest a relationship between apoptosis and the acceleration of genitalia rotation (Kuranaga, 2011).

Next, the signaling mechanism(s) involved in the acceleration of genitalia rotation wee examined. The inhibition of JNK (c-Jun N-terminal kinase) and PVF (platelet vascular factor) signaling in male flies has been shown to result in mis-oriented adult male terminalia, and it has been hypothesized that the PVF/PVR (PVF receptor) may affect the genitalia rotation via JNK-mediated apoptosis (see Benitez, 2010). Consistent with previous reports, the acceleration of genitalia rotation was significantly impaired in flies expressing dominant-negative forms of JNK (JNK-DN) and PVR (PVR-DN). These data implied that caspase activation and JNK signaling contribute to driving the acceleration of genitalia rotation (Kuranaga, 2011).

To analyze how the genitalia accelerate their rotation, the movement of A8p was traced at the single-cell level. For this experiment, live imaging was performed at a high resolution (20× objective lens), which enabled the cells in A8p to be tracked at single-cell resolution. Cells that were neighbors of A9 rotated with A9, whereas cells located in the anterior half of A8p rotated later than A9. Based on this imaging, A8p was divided into two sheets, named A8pa (anterior of A8p) and A8pp (posterior of A8p). It was found that a part of the cells in A8p underwent apoptosis (Kuranaga, 2011).

To observe caspase activation in living animals, a FRET (fluorescence resonance energy transfer)-based indicator, SCAT3 (sensor for activated caspases based on FRET) was generated. To precisely evaluate apoptosis, a nuclear localization signal-tagged SCAT3 (nls-SCAT3; UAS-nls-ECFP-venus) was used. The nls-SCAT3 signal was clearly observed in A8p. Cells exhibiting high caspase activity were extruded into the body cavity and disappeared, consistent with their apoptotic death and engulfment by circulating hemocytes. Each cell was tracked in the A8p region during the first half of the rotation, and it was found that at least three types of cellular behavior were observed: (1) cells located in A8pp moved with A9, (2) cells underwent apoptosis and (3) cells located in A8pa rotated later (Kuranaga, 2011).

Thus, to observe the behavior of the cells in A8pa, Abdominal B (AbdB) was used as an A8 marker. AbdB is a homeotic gene that is required for the correct development of the genital disc, and AbdB-GAL4^LDN is expressed in the segment A8 (in A8a and A8p) of the genital disc during the 3rd instar larval stage. At 24 hours APF, AbdB was expressed in A8 and formed a ring. Time-lapse images were taken, and unexpectedly it was found that most of the cells in the AbdB-expressing region underwent a 180° clockwise movement, suggesting that AbdB was not expressed in the A8pp region that moved 360° with A9. To determine the speed of the AbdB-expressing cells, three individual cells were traced in each fly, and the value of the turning angle of the cells (theta_AbdB) was calculated. The findings confirmed that the AbdB-expressing region moved halfway around. Although cells in the AbdB-expressing region moved only 180°, the A8pp (inner ring), which was encircled by the AbdB-expressing region (outer ring), still moved 360°. Furthermore, the imaging data indicated that the movement of the outer ring started 1-2 hours later than that of the A9 region, when the acceleration of the genitalia rotation occurred. These observations raise the possibility that the outer ring movement is related to the acceleration of the genitalia rotation (Kuranaga, 2011).

It was therefore considered that the outer ring movement was restricted in the p35-expressing flies, resulting in an incomplete genitalia rotation of about 180°, which mimics the movement of only the inner ring. To verify this possibility, the movement of the outer ring was examined in the p35-expressing flies (en-GAL4+UAS-p35). Although the inner ring rotated normally, the rotation of the outer ring was impaired in the p35-expressing flies. The turning angles were determined by tracing cells in the p35-expressing flies and it was found that theta_{p35 _inner} increased, while the increase of theta_{p35 _outer} was impaired. These data suggest that the A8 segment is composed of two independently regulated rings, and when apoptosis is inhibited, the inner ring can move only 180° with no outer ring movement, resulting in incomplete genitalia rotation (Kuranaga, 2011).

Thus, to determine whether apoptosis correlates with the outer ring movement, the apoptosis was quantified in A8pa every 10 minutes from 0-8 hours after the start of genitalia rotation. The frequency of apoptosis (R_apoptosis) was normalized to the total number of apoptotic cells in each individual. Pulsatile increases in R_apoptosis were observed, with peaks at 1, 2.5 and 4 hours after the start of genitalia rotation. To verify the participation of R_apoptosis in the initiation of outer ring movement, the acceleration rate of theta_AbdB (a_AbdB) was calculated by measuring V_AbdB as a function of time t, and R_apoptosis was compared with a_AbdB. The starting time of outer ring movement was characterized by the early peaks of a_AbdB. The analysis suggested that the a_AbdB was related to the R_apoptosis, because a_AbdB showed its first two peaks at about 1 and 2.5 hours after genitalia rotation started. To quantify these observations, the correlation was calculated between R_apoptosis and a_AbdB. This analysis confirmed that there was a strong correlation between these parameters, because the correlation between a_AbdB and R_apoptosis is approximately linear during this time. Therefore, these data implied a possible mechanism of apoptosis that facilitates the outer ring movement (Kuranaga, 2011).

To verify this possibility, whether the upregulation of apoptotic signals induces an increase in genitalia rotation speed was meastured. Because the expression of apoptotic genes using an en-GAL4 driver, which is expressed at the embryonic stage, is lethal, the TARGET system was used to control gene expression temporally. Flies were allowed to develop at 18°C until the head of the pupae had just everted, to inhibit gene expression. The pupae were then heat-shocked at 29°C for 12 hours to induce gene expression. Live imaging was performed at 22°C, after the heat shock. At this temperature, the genitalia rotation in the control flies was slower than in control flies bred at 25°C, because a low breeding temperature affects the rate of fly development, including genitalia rotation. Therefore, it was necessary in this experiment to compare the rotation speeds at the same temperature. The expression of reaper (rpr), a pro-apoptotic gene, using the TARGET system, showed that the upregulation of apoptotic signaling significantly increased the timing of acceleration and speed of genitalia rotation. These observations led to the proposal that the outer ring functions like a 'moving walkway' to accelerate the speed of the inner part of the structure, including the A9 genitalia, enabling genitalia to complete rotation within the appropriate developmental time window (Kuranaga, 2011).

According to these observations, it was found that apoptosis drives the movement of cell sheets during the morphogenesis of male terminalia. Further questions remain with regard to how apoptosis contributes to the cell sheet movement. A recent study indicated the possibility that local apoptosis acts as a brake release to regulate genitalia rotation, coupled with left-right determination (Suzanne, 2010). However, it has been reported that the cell shape change by apoptosis enables not only the extrusion of dying cells, but also the reorganization of the actin cytoskeleton in neighboring cells. Therefore, apoptosis could affect the behavior of neighboring cells, to act as a main driving force of the cell-sheet movement. Taken together, apoptosis may generally participate in the morphogenetic process of cell-sheet movement during morphogenesis (Kuranaga, 2011).

Distinct genetic requirements for BX-C mediated specification of abdominal denticles

Hox genes encode transcription factors playing important role in segment specific morphogenesis along the anterior posterior axis. Most work in the Hox field aimed at understanding the basis for specialised Hox functions, while little attention was given to Hox common function. In Drosophila, genes of the Bithorax complex [Ultrabithorax (Ubx), abdominalA (abdA) and AbdominalB (AbdB)] all promote abdominal identity. While Ubx and AbdA share extensive sequence conservation, AbdB is highly divergent, questioning how it can perform similar functions than Ubx and AbdA. This study investigated the genetic requirement for the specification of abdominal-type denticles by Ubx, AbdA and AbdB. The impact of ectopic expression of Hox proteins in embryos deprived for Exd as well as for Wingless or Hedgehog signaling involved in intrasegmental patterning was analyzed. Results indicated that Ubx and AbdA do not require Exd, Wg and Hh activity for specifying abdominal-type denticles, while AbdB does. These results support that distinct regulatory mechanisms underlie Ubx/AbdA and AbdB mediated specification of abdominal-type denticles, highlighting distinct strategies for achieving a similar biological output. This suggests that common function performed by distinct paralogue Hox proteins may also rely on newly acquired property, instead of conserved/ancestral properties (Sambrani, 2013b).

This study relies on a gain of function strategy, scoring phenotypes in the thorax, where none of these three BX-C proteins are expressed, but where Exd, Wg, and Hh are expressed. This allows circumventing the difficulty resulting from the incapacity to unambiguously identify posterior most abdominal segments, where AbdB acts, and avoid complications in interpreting results that would arise from cross regulation between BX-C genes in the abdomen. However the approach also questions whether the conclusion of this study applies for BX-C proteins activity in their endogenous expression domains. Regarding Exd requirements, maternal and zygotic loss results in abdominal segment fusion, where segments are fused by pairs: A1/A2-3/A4-5/A6-7/A8. Although denticle belts are highly disorganized, denticles are clearly of abdominal type in anterior abdominal segments. When present in A8, the belt of denticles is very much reduced, and in most embryos is absent. This indicates that Ubx and AbdA do not require Exd for the specification of abdominal-type denticles in their endogenous expression domain, while AbdB does. The complete segment fusion resulting from loss of Wg and Hh signaling make it impossible to unambiguously identify the A8 segments, and therefore does not allow addressing if AbdB also requires Wg and Hh signaling in its endogenous expression domain. Denticles are found in continuous lawn, that encompasses most abdominal segments. Therefore denticles in the Ubx and AbdA expression domains are clearly of abdominal types, indicating that Ubx and AbdA do not require Wg and Hh signaling for the specification of abdominal-type denticles. Thus, whenever possible, resident BX-C Hox protein activity in loss of exd, wg and hh function are consistent with the conclusion raised in the gain of function approach, further supporting that Ubx/AbdA and AbdB use distinct regulatory mechanisms for achieving a common function (Sambrani, 2013b).

Such a conclusion was recently reached by studying the molecular mechanisms underlying repression of the limb-promoting gene Dll by Ubx and AbdB. It was shown that the cofactor requirement and intrinsic protein domain requirement for Ubx versus AbdB repression of Dll was distinct. Ubx represses Dll by binding DNA cooperatively with the Exd and Hth cofactors, which relies on the UbdA domain, a domain specific to Ubx and AbdA and located C-terminal to the HD (Sambrani, 2013a). Surprisingly, Ubx DNA binding is dispensable, probably due to cooperative binding to DNA with Exd and Hth, DNA binding proteins that likely compensate for Ubx loss of DNA binding. By contrast, AbdB represses Dll without the help of the Exd and Hth, and DNA binding of AbdB is strictly required for repression. It was further established that in specifying posterior spiracles and regulating empty spiracles expression, Exd/Hth antagonize AbdB activity, showing that the AbdB/Exd partnership depends on the biological context. Mechanisms at the origin of cooperativity/antagonism are still to be discovered (Sambrani, 2013b).

The present study corroborates the conclusion reached by the analysis of Dll repression by Ubx and AbdB and extends it in several ways: first by using a distinct Hox biological activity as functional readout; second by including in the analysis the AbdA Hox protein; and third by examining additional genetic requirements (Wg and Hh signaling). The work therefore provides further support for the view that distinct molecular strategies underlie an apparent unicity in BXC protein controlled biological function (Sambrani, 2013b).

Given the observation that Ubx and AbdA are very similar, sharing a highly conserved HD as well as additional protein domains such as the HX and UbdA motifs, while AbdB lacks these domains and has a highly divergent HD, it is not surprising that the genetic requirements are similar for Ubx/AbdA and distinct for AbdB. More unexpected was the finding that Ubx and AbdA do not require Exd for specifying abdominal-type denticles, while AbdB does. This indeed contrasts with the known and previously described Exd requirement for Ubx in A1 segment identity specification and Dll repression, and also contrasts with the dispensability of Exd for A8 segment identity specification, posterior spiracle specification and Dll repression (Sambrani, 2013a). This highlights that requirement of Exd for Hox activity depends on the specific function examined, rather than being a general and universal requirement (Sambrani, 2013b).

A salient difference between the central Ubx/AbdA and posterior AbdB Hox proteins is the mode of Hox DNA binding. Posterior paralogue Hox proteins have usually a stronger affinity for DNA when binding as monomer than central class Hox proteins. This difference mainly stems from the ability of posterior but not central class Hox proteins to make extensive contacts with the DNA backbone. These differences provide a frame to understand the requirement of Exd/Pbx cofactor for central class Hox proteins, which upon interaction with Hox proteins raises their DNA binding affinity. In the case of specification of abdominal-type denticles, the contribution of Exd is likely different, as required for AbdB and not Ubx/AbdA activity. This suggests that Exd may be involved in regulating the activity, rather than DNA binding, a function previously suggested in the regulation of Deformed Hox protein function (Sambrani, 2013).

In summary, this work together with the study of Dll repression by BX-C proteins highlights that distinct regulatory mechanisms and molecular strategies underlies common Hox protein functions. Thus while sequence divergence following gene duplication promotes functional divergence, it also generates novel gene regulatory mechanisms and molecular strategies that yet promotes a common biological output (Sambrani, 2013).

Abdominal-B neurons control Drosophila virgin female receptivity

Female sexual receptivity offers an excellent model for complex behavioral decisions. The female must parse her own reproductive state, the external environment, and male sensory cues to decide whether to copulate. In the fly Drosophila melanogaster, virgin female receptivity has received relatively little attention, and its neural circuitry and individual behavioral components remain unmapped. Using a genome-wide neuronal RNAi screen, this study identified a subpopulation of neurons responsible for pausing, a novel behavioral aspect of virgin female receptivity characterized in this study. Abdominal-B (Abd-B), a homeobox transcription factor, was shown to be required in developing neurons for high levels of virgin female receptivity. Silencing adult Abd-B neurons significantly decreased receptivity. Two components of receptivity were characterized that are elicited in sexually mature females by male courtship: pausing and vaginal plate opening. Silencing Abd-B neurons decreased pausing but did not affect vaginal plate opening, demonstrating that these two components of female sexual behavior are functionally separable. Synthetic activation of Abd-B neurons increased pausing, but male courtship song alone was not sufficient to elicit this behavior. These results provide an entry point to the neural circuit controlling virgin female receptivity. The female integrates multiple sensory cues from the male to execute discrete motor programs prior to copulation. Abd-B neurons control pausing, a key aspect of female sexual receptivity, in response to male courtship (Bussell, 2014).

Female receptivity is a complex behavior comprising multiple motor programs and requiring the integration of sensory cues across several modalities. Nevertheless, Drosophila mating behavior is innate, and receptivity is likely controlled by hardwired neural circuits. This study identified seven candidate genetic markers of receptivity neurons by using a neuronal RNAi screen. The data suggest a central role for one of these, the transcription factor Abd-B, in forming a neural circuit that functions in receptivity (Bussell, 2014).

This study has refined the behavioral components of receptivity beyond mere copulation acceptance. Vaginal plate opening occurs throughout courtship and depends on sexual maturity. The historically noted slowing down of receptive females is attributed to punctuated bouts of pausing during courtship rather than decreased walking speed. Pausing behavior is specific to female receptivity: it is decreased in both unreceptive females and in mature virgin females not being actively courted by a male. The increased level of pausing associated with receptivity requires the integration of multiple sensory inputs, including song, from a courting male. Abd-BLDN neuronal activity is both necessary for this pausing response and sufficient to induce it, thus establishing direct function of these neurons within the receptivity circuit (Bussell, 2014).

How do Abd-B neurons control pausing? The Abd-BLDN neurons important for receptivity are not themselves motor neurons, and females with silenced Abd-BLDN neurons are not generally deficient in movement or posture. This suggests that Abd-BLDN neurons play a role downstream of the sensation of individual male courtship sensory inputs but upstream of motor output. The abdominal ganglion is emerging as a potential locus coordinating female-specific behavior, and Abd-BLDN neurons there are well-positioned to interact with other neurons involved in female behavior, including the postmating response. These neurons could therefore potentially function to integrate male courtship cues and internal inputs and promote pausing (Bussell, 2014).

Silencing Abd-BLDN neurons affects pausing, but not vaginal plate opening, which demonstrates that it is possible to uncouple these two aspects of receptivity. However, activation of Abd-BLDN neurons affects both pausing and the movement of the vaginal plates. It is therefore possible that Abd-BLDN neurons, or subsets within them, function in both of these aspects of receptivity. There are likely to be additional circuit components involved in plate opening that might be able to act redundantly in the absence of Abd-BLDN neurons, and the involvement of additional neurons in the control of the vaginal plates is consistent with the fact that Abd-BLDN activation does not induce periodic vaginal plate opening but rather locks the plates in the open position. How the receptivity circuitry coordinates vaginal plate opening with pausing and male copulation attempts remains unknown. Abd-BLDN neurons provide an important entry point to dissect the two female motor programs. It was observed that vaginal plate opening occurs both while the female is moving and while she is stationary. Female movement has been shown to provide feedback to the male during courtship, and it could be that pausing provides an important connection between the sexes within the context of the courtship duet (Bussell, 2014).

Abd-B is required in neurons during development for females to become highly receptive to male courtship. How does the Abd-B protein affect the receptivity circuitry? Abd-BLDN > Abd-B RNAi experiments show that Abd-BLDN- Gal4 labels the neurons in which Abd-B functions during development to affect receptivity. However, it is possible that these developmental Abd-BLDN neurons are not identical to the adult Abd-BLDN neurons that function in receptivity. In developing neuroblasts, Abd-B can have different, even opposing, functions, promoting cell death or promoting a particular cell fate or repressing it, depending on neuroblast identity and context. In Abd-BLDN > Abd-B RNAi experiments, no obvious changes were observed in either the number or projections of Abd-BLDN neurons in the adult, but this does not exclude the possibility of subtle anatomical changes or changes in cell identity. Finally, it is noted that modularity in the control of complex innate behavior has been found across a variety of species and systems. From flies to mice, both aggression and mating are controlled by eliciting different modules in a sexually dimorphic way. Thus, female fly receptivity fits into a larger pattern of sex-specific control of innate behavioral components (Bussell, 2014).

Effects of Mutation or Deletion

The proteins responsible for m and r activities were ectopically expressed in fly embryos. The resultant larval cuticular transformations are consistent with the genetically defined role of each protein during normal embryogenesis. Both ABD-B proteins activate ectopic expression of transcripts encoding the m protein, but the levels of Antennapedia, Ultrabithorax and abdominal-A transcripts are differentially repressed (Kuziora, 1993).

To determine when the homeotic genes are required for specific developmental events Ultrabithorax, abdominal-A and Abdominal-B proteins were expressed at different times during development using the GAL4 targeting technique. Early transient homeotic gene expression has no lasting effects on the differentiation of the larval epidermis, but it switches the fate of other cell types irreversibly (e.g. the spiracle primordia). One cell type in the peripheral nervous system makes sequential, independent responses to homeotic gene expression. There is also an in vivo competition between the bithorax complex proteins for the regulation of their down-stream targets (Castelli-Gair, 1994).

The metameric organization of the Drosophila melanogaster tail is obscured by developmental events that partially suppress or fuse some of its regions. engrailed patterns in different bithorax complex mutants ( Abd-B morphogenetic (m) and regulatory (r) mutants) demonstrate that Abd-B acts to suppress embryonic ventral epidermal structures on the posterior side of A8 to A9 (Kuhn, 1995).

The tumorous-head-3 (tuh-3) mutation has been associated with the insertion of mobile element Delta 88 at +200 on the bithorax complex (BX-C) DNA map, 5' of all Abdominal-B (Abd-B) transcripts. Different phenotypes of tuh-3 are regulated by the tumorous-head-1 (tuh-1) maternal effect locus. In the presence of the recessive tuh-1h maternal effect, tuh-3 offspring produce homeotic abdominal and genital tissue in the head. In the presence of the dominant tuh-1g maternal effect, tuh-3 offspring have normal heads but now show genital defects. One other mutant, I127B, produces flies with identical defects to that of tuh-3 in the presence of both maternal effects. Molecular analysis of I127B reveals the insertion of mobile element 297 in the Abd-B gene, approximately 25 kb downstream of the Delta 88 insertion in tuh-3. No other abnormalities are detected. Reexamination of the tuh-3 strain reveals a 297 insertion in an identical region to that of I127B, in addition to the Delta 88 insertion. Recombinants of tuh-3, carrying 297 only, produce homeotic head defects and genital defects in the presence of the tuh-1h and tuh-1g maternal effects, respectively. Recombinants of tuh-3, carrying Delta 88 only, fail to produce any defects in the presence of either maternal effect. Based upon these results, it is proposed that it is the 297 insertion in the Abd-B gene, not Delta 88, that is responsible for the tuh-3 mutation (Mack, 1997).

The genital disc of Drosophila, which gives rise to the genitalia and analia of adult flies, is formed by cells from different embryonic segments. To study the organization of this disc, the expressions of segment polarity and homeotic genes were investigated. The organization of the embryonic genital primordium and the requirement of the engrailed and invected genes in the adult terminalia were also analysed. The three primordia, the female and male genitalia plus the analia, are composed of an anterior and a posterior compartment. In some aspects, each of the three primordia resemble other discs: the expression of genes such as wingless and decapentaplegic in each anterior compartment is similar to that seen in leg discs; the absence of engrailed and invected causes duplications of anterior regions, as occurs in wing discs. The absence of lineage restrictions in some regions of the terminalia and the expression of segment polarity genes in the embryonic genital disc suggest that this model of compartmental organization evolves, at least in part, as the disc grows. The expression of homeotic genes suggests a parasegmental organization of the genital disc, although these genes may also change their expression patterns during larval development (Casares, 1997).

Mutations in Abd-B transform female genitalia into abdomen, suggesting that the activity of Abd-B is a prerequisite for the specification of the terminalia by the sex-determing genes. abd-A is expressed only in female genital discs, in the region corresponding to the female genital primordium, particularly in the prospective internal female genitalia. abd-A expression is coincident with engrailed in the central region of the female genital primordium engrailed band. Abd-B transcripts are located in the genital disc. The Abd-B protein is present in the male and female primordia in both male and female discs, leaving unstained the region where the analia map. Abd-B expression is coincident with en bands 1 and 2. In female discs, Abd-B m transcript is present only in the female genital primordium: transcript levels are strong in the prospective external genitalia and faint in the prospective internal genitalia. In the male disc, only the repressed male primordium is labelled. Abd-B r transcript is expressed in the repressed male primordium of female discs and the male genetal primordium of male discs. caudal is located in the analia primordium of the genital disc, overlapping with the third engrailed band. However, caudal and enoverlap in only a few, dorsally located, epidermal nuclei of stage 14 embryos. This overlap is not seen in the ventrally located embryonic genital disc where caudal expression is observed in its posterior region. This suggests that en expression in anal primordium of mature genital discs appears during larval development. The perianal ring corresponds to the terminal band of en, and the co-expression of en and cad is maintained from the third instar disc until the adult stage (Casares, 1997)

The genital disc consists of three primordia: moving from anterior to posterior they are the female genital primordium, the male genital primordium and the anal primordia. Only one of the two genital primordia develops, depending on the individual's sex, whereas the anal primordium develops in both sexes. It is proposed here that the genital disc, which is of ventral origin, is organized in a manner similar to the antennal and leg discs: the expression domains of decapentaplegic and wingless are mostly complementary and abut engrailed expression. An analysis was made of the roles of the genes hedgehog, patched, dpp and wg in the development of the three primordia that form the genital disc. The morphogenetic alterations produced by ectopic expression of hh mimic a lack of ptc function. Both genetic conditions cause derepression of dpp and wg. Ectopic expression of either of these genes causes non-autonomous duplications and/or reductions of genital and anal structures. Some of these alterations are explained by the mutual repression of wg and dpp. In the development of the genital disc, the functional relationships between these genes seem to be analogous to those described for leg and antennal discs: dpp and wg are induced in the anterior compartment by Hh protein blocking the repressive effect of Ptc, and the mutual repression of dpp and wg restrict one another to their respective domains. It may be concluded that dpp and wg act as general organizers for development of the genital disc (Sanchez, 1997).

The gene doublesex controls which genital primordium of the genital disc will grow and which will be repressed. The female genital primordium develops from A8 and the male genital primordium develops from A9. Therefore, the gene doublesex must act in concert with another regulatory gene(s) to determine the genital primordium that develops in each sex. A possible candidate for this additional regulatory element is the homeotic gene Abd-B, since this gene is reponsible for specification of posterior segments. Under normal conditions, the female genital primordium can develop either into genitalia or remain in the repressed state, producing no adult structures. In mutant conditions for Abdominal-B m transcript, it can develop into an abdominal tergite plus sternite. Similarly, under normal conditions, the male genital primordium can develop into either normal genitalia or remain in the repressed state, forming no adult strucures. In Abd-B r transcript mutants it develops into rudimentary genitalia (Sanchez, 1997 and references).

The development of the genital primordia is based on two processes: cell proliferation and sexual differentiation. Cell proliferation refers to the capacity of each genital primordium to grow or to be kept in the repressed state. Sexual differentiation refers to the type of adult structure formed by each genital primordium. It is proposed that the control of cell proliferation in the male and female genitalia requires the concerted action of Abd-B and doublesex, either directly or indirectly, through the expression of the genes dpp and wingless. Thus, in female genital discs, the repressed male primordium does not express dpp whereas the repressed female primordium of the male genital discs expresses a reduced level of dpp. This reduced level seems to be insufficient to stimulate cell growth. In contrast, when strong dpp levels are obtained in the repressed female primordium of male discs, repressed female primordia overproliferate in mutants for patched or costal-2, as well as in the discs where uniform ectopic expreession of hedgehog is produced. The genes dpp and wg, however, do not participate in the sexual differentiation process, which depends on sexual cytodifferentiation genes. Thus the growth of repressed female primordia of the patched mutant male discs would give rise to no adult female genital structures since the genetic sex is male (Sanchez, 1997 and references).

The Drosophila kismet gene is related to chromatin-remodeling factors and is required for both segmentation and segment identity

The Drosophila trithorax group gene kismet (kis) was identified in a screen for extragenic suppressors of Polycomb (Pc) and subsequently shown to play important roles in both segmentation and the determination of body segment identities. One of the two major proteins encoded by kis (Kis-L) is related to members of the SWI2/SNF2 and CHD families of ATP-dependent chromatin-remodeling factors. To clarify the role of Kis-L in gene expression, its distribution on larval salivary gland polytene chromosomes was examined. Kis-L is associated with virtually all sites of transcriptionally active chromatin in a pattern that largely overlaps that of RNA Polymerase II (Pol II). The levels of elongating Pol II and the elongation factors SPT6 and CHD1 are dramatically reduced on polytene chromosomes from kis mutant larvae. By contrast, the loss of Kis-L function does not affect the binding of PC to chromatin or the recruitment of Pol II to promoters. These data suggest that Kis-L facilitates an early step in transcriptional elongation by Pol II (Srinivasan, 2005).

The Drosophila kismet gene was identified in a screen for dominant suppressors of Polycomb, a repressor of homeotic genes. kismet mutations suppress the Polycomb mutant phenotype by blocking the ectopic transcription of homeotic genes. Loss of zygotic kismet function causes homeotic transformations similar to those associated with loss-of-function mutations in the homeotic genes Sex combs reduced and Abdominal-B. kismet is also required for proper larval body segmentation. Loss of maternal kismet function causes segmentation defects similar to those caused by mutations in the pair-rule gene even-skipped. The kismet gene encodes several large nuclear proteins that are ubiquitously expressed along the anteriorposterior axis. The Kismet proteins contain a domain conserved in the trithorax group protein Brahma and related chromatin-remodeling factors, providing further evidence that alterations in chromatin structure are required to maintain the spatially restricted patterns of homeotic gene transcription (Daubresse, 1999).

The genetic interactions between kis and Pc provided the first clue that kis plays an important role in the determination of body segment identity. kis mutations suppress the adult Pc phenotype by preventing the ectopic transcription of homeotic genes. Thus, kis is a member of the trithorax group of homeotic gene activators. Mosaic analyses reveal that loss of kis function causes homeotic transformations, including the transformation of first leg to second leg and the fifth abdominal segment to a more anterior identity. These phenotypes are identical to those associated with loss-of-function Scr and Abd-B mutations, respectively. Taken together, these findings suggest that kis acts antagonistically to Pc to activate the transcription of both Scr and Abd-B. It is intriguing that kis mutations alter the fate of only the fifth abdominal segment, since the identities of the fifth through ninth abdominal segments are determined by a single homeotic gene, Abd-B (Daubresse, 1999).

Variations in the levels of Abd-B protein result in the differences between these abdominal segments, with Abd-B expression being lowest in the fifth abdominal segment. Parasegment-specific cis-regulatory regions, termed infra-abdominal (iab) regions control Abd-B expression. Each iab region is named for the segment that it affects (iab-5 through iab-9). Mutations in both iab-5 and kis affect the identity of only the fifth abdominal segment, suggesting that the Kis protein may interact specifically with the iab-5 cis-regulatory element of Abd-B (Daubresse, 1999).

kis probably interacts not only with Scr and Abd-B, but with other homeotic genes as well. For example, the isolation of kis mutations as enhancers of loss-of-function Deformed (Dfd) mutations suggests that kis is probably also required to activate transcription of this ANTC homeotic gene. Furthermore, kis duplications strongly enhance the transformation of wing to haltere in Pc heterozygotes, a phenotype caused by the ectopic transcription of Ubx in the wing imaginal disc. However, kis mutations do not cause haltere-to-wing transformations due to decreased Ubx transcription. A possible explanation for the lack of homeotic transformations in kis clones in segments other than the prothoracic and fifth abdominal segment is that the mutations used in these studies are not null alleles. kis¹ is a strong loss-of-function mutation. It has not been characterized at the molecular level, however, and may not completely eliminate kis function. It is also possible that sufficient levels of Kis protein persist in homozygous mutant tissue following mitotic recombination to support normal development. Further genetic studies, including the analysis of conditional kis alleles, will be necessary to distinguish between these possibilities (Daubresse, 1999).

Germline clonal analysis has revealed an unanticipated role for kis in segmentation. Embryos from mosaic kis^S females exhibit a deletion or alteration of every other segment, while mutant embryos from mothers bearing germline clones of the stronger kis¹ allele usually develop only half of the normal number of segments. This variation in phenotypic severity is closely correlated with the extent to which en expression is disrupted. The phenotypes associated with loss of maternal kis function resemble those caused by mutations in pair-rule segmentation genes that cause the deletion of the odd-numbered parasegments. kis thus appears to be necessary for the expression (or function) of one or more pair-rule genes. Recent genetic studies have suggested that kis may also be involved in the Notch signaling pathway. Thus it appears that kis plays roles in addition to the regulation of homeotic genes (Daubresse, 1999).

What pair-rule genes might require kis for their activity? Based on the kis mutant phenotype, perhaps the best candidates are eve and hairy (h), both of which are required for the formation of odd-numbered parasegments. Unlike eve, h and most other segmentation genes, kis is uniformly expressed in the early embryo. This raises the possibility that Kis functions as an essential cofactor or modifier of Eve or other pair-rule proteins. It is also possible that loss of kis function might result in pair-rule genes being transcribed outside of their normal expression domains. Additional work will be necessary to determine the molecular basis of the segmentation defects resulting from loss of maternal kis function (Daubresse, 1999).

Abdominal-B is essential for proper sexually dimorphic development of the Drosophila gonad

Sexual dimorphism requires the integration of positional information in the embryo with the sex determination pathway. Homeotic genes are a major source of positional information responsible for patterning along the anterior–posterior axis in embryonic development, and are likely to play a critical role in sexual dimorphism. The role of homeotic genes in the sexually dimorphic development of the gonad has been investigated in Drosophila. Abdominal-B (ABD-B) is expressed in a sexually dimorphic manner in the embryonic gonad. Furthermore, Abd-B is necessary and sufficient for specification of a sexually dimorphic cell type, the male-specific somatic gonadal precursors (msSGPs). In Abd-B mutants, the msSGPs are not specified and male gonads now resemble female gonads with respect to these cells. Ectopic expression of Abd-B is sufficient to induce formation of extra msSGPs in additional segments of the embryo. Abd-B works together with abdominal-A to pattern the non-sexually dimorphic somatic gonad in both sexes, while Abd-B alone specifies the msSGPs. These results indicate that Abd-B acts at multiple levels to regulate gonad development and that Abd-B class homeotic genes are conserved factors in establishing gonad sexual dimorphism in diverse species (DeFalco, 2004).

The homeotic genes initially work to specify the distinct types of somatic cells that will contribute to the gonad. Abd-B is necessary for the specification of msSGPs in PS13, and is sufficient to induce msSGP clusters in ectopic positions. Thus, Abd-B appears to restrict msSGP development to PS13. Consistent with this idea, the anterior limit of Abd-B expression is initially in PS13, and only later extends into more anterior regions (DeFalco, 2004).

In a similar manner, abd-A is required for the specification of SGPs in PS10-12. abd-A acts to promote SGP development by blocking srp and fat body development in these PS. abd-A is also sufficient to induce ectopic SGPs when expressed in more anterior regions. Thus, the first stage where the homeotic genes act in patterning the somatic gonad is in restricting SGP and msSGP development to their proper PS (DeFalco, 2004).

The homeotic genes next act to pattern distinct identities within the somatic gonad. abd-A alone specifies anterior SGP identity, a combination of abd-A and Abd-B specifies posterior SGP identity, and Abd-B alone is required to specify msSGP identity. This role for the homeotic genes is greatly facilitated by the fact that the cells of the somatic gonad are originally specified in four different PS of the embryo, allowing these cells to acquire unique homeotic gene expression profiles, or Hox codes, that will determine A-P identities. These Hox codes are maintained as the SGPs and msSGPs move anteriorly and coalesce with the germ cells to form a gonad in PS10, as clearly evidenced by the maintenance of Abd-B expression in the msSGPs and posterior SGPs in the coalesced gonad (DeFalco, 2004).

The precursors for the dorsal vessel, the Drosophila heart, are similarly specified in separate PS (4-13), allowing distinct identities to be patterned along the A-P axis by Ultrabithorax, abd-A, and Abd-B. This is also true in other tissues, such as the visceral mesoderm and fat body. Thus, it is a common theme that organ precursors are specified in a spatially segregated manner, allowing the cells to acquire distinct identities that are preserved during organogenesis (DeFalco, 2004).

The last stage where homeotic genes act is in the development of sexual dimorphism in the gonad. The unique identity of the msSGPs, provided in part by Abd-B, allows these cells to behave differently in males and females. In males these cells join the posterior of the coalescing gonad, while they are removed by programmed cell death in the female. Furthermore, the anterior SGPs also behave differently in males vs. females, indicating that the unique SGP identity conferred by abd-A also allows cells to respond differently to distinct sexual identities. How cell identity in the gonad, regulated by the homeotic genes, interacts with the sex determination pathway to produce distinct outputs is a fascinating area for future study (DeFalco, 2004).

There appears to be a common regulatory link between cell types specified by Abd-B and sex-specific regulation by the sex determining gene dsx. Abd-B is critical for specifying msSGP identity, and dsx is critical for causing these cells to behave differently in males and females. The head involution defective (hid) gene is essential for female-specific programmed cell death of the msSGPs, and is a candidate for being differentially regulated by Abd-B and dsx in the two sexes (DeFalco, 2004).

A similar relationship between Abd-B and dsx has been observed in several other examples. It has been shown that these genes interact to control the pattern of sex-specific pigmentation in the Drosophila abdomen, and that bric à brac (bab) integrates positional and sexual inputs in this tissue. The combination of Abd-B and female identity allows bab to act in blocking pigment formation, whereas in males, Abd-B can repress bab in order to allow pigment formation to occur (DeFalco, 2004).

Abd-B and dsx also cooperate in sex-specific development of the genital disc, which gives rise to the non-gonadal structures that must eventually join with the gonad to form the functional adult reproductive system. Abd-B and dsx act through the signaling molecules Wingless and Decapentaplegic to pattern the genital disc, and through the FGF ligand Branchless to regulate mesodermal cell migration into the disc. The expression of a key regulator of genital disc development, dachshund, has been shown to be affected by both Abd-B and dsx (DeFalco, 2004).

Thus, Abd-B and dsx are used in combination to pattern several independent tissues during development. Other cell-type-specific factors must be involved, since these tissues exhibit distinct responses to Abd-B and dsx. However, Abd-B and dsx clearly form a common regulatory network used multiple times in development to create sexual dimorphism (DeFalco, 2004).

Data from studies on Caenorhabditis elegans and mice suggest that regional identities conferred by homeotic genes are required for the proper development and sexual dimorphism of the gonad in these species. An Abd-B homolog in C. elegans, egl-5, is expressed in the somatic gonad and is required for SGP development. Furthermore, in a certain percent of egl-5 mutant males it appears as if the somatic gonad takes on a hermaphrodite-like morphology. This sex-specific phenotype may be analogous to what is seen in Drosophila, in which Abd-B mutant male gonads take on a partial female phenotype (as characterized by an absence of msSGPs). Due to a great deal of gene expansion in the mammalian homeotic complex resulting in potential gene redundancy or overlapping function, it may prove difficult to find a single mouse gene with a similar phenotype to Abd-B or egl-5. However, Hoxa10 male knockout mice exhibit blocks in spermatogenesis, while the female gonad can produce functional eggs, demonstrating a sexually dimorphic role for posterior Hox genes in mouse gonad development (DeFalco, 2004).

In addition, studies of the Polycomb (Pc) group of homeotic regulators are also consistent with a role for homeotic genes in establishing sexual dimorphism. C. elegans Pc homologs mes-2, mes-3, and mes-6 have been shown to regulate homeotic gene expression, in particular egl-5 and mab-5, the latter of which is necessary for sexually dimorphic male V-ray sense organs. Knockouts of the mouse Pc homolog M33 have altered expression of Hox genes resulting in sterility and male-to-female sex reversal (DeFalco, 2004).

These results indicate that the regulation of homeotic gene expression is important for gonad development and sexual dimorphism in diverse organisms. Although methods of initial sex determination have widely diverged among animal species, many lines of evidence strongly suggest that mechanisms to promote sexual dimorphism in the gonad are conserved. Positional information provided by the homeotic genes is likely to be a key conserved element in creating sexual dimorphism (DeFalco, 2004).

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).

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).

Differential activity of Drosophila Hox genes induces myosin expression and can maintain compartment boundaries

Compartments are units of cell lineage that subdivide territories with different developmental potential. In Drosophila, the wing and haltere discs are subdivided into anterior and posterior (A/P) compartments, which require the activity of Hedgehog, and into dorsal and ventral (D/V) compartments, needing Notch signaling. There is enrichment in actomyosin proteins at the compartment boundaries, suggesting a role for these proteins in their maintenance. Compartments also develop in the mouse hindbrain rhombomeres, which are characterized by the expression of different Hox genes, a group of genes specifying different structures along their main axis of bilaterians. This study shows that the Drosophila Hox gene Ultrabithorax can maintain the A/P and D/V compartment boundaries when Hedgehog or Notch signaling is compromised, and that the interaction of cells with and without Ultrabithorax expression induces high levels of non-muscle myosin II. In the absence of Ultrabithorax there is occasional mixing of cells from different segments. A similar role in cell segregation was shown for the Abdominal-B Hox gene. The results suggest that the juxtaposition of cells with different Hox gene expression leads to their sorting out, probably through the accumulation of non-muscle myosin II at the boundary of the different cell territories. The increase in myosin expression seems to be a general mechanism used by Hox genes or signaling pathways to maintain the segregation of different groups of cells (Curt, 2013).

The sorting out of cells with distinct Hox activity in Drosophila has been reported before and in the case of the Hox gene Deformed a possible function in cell segregation has been assigned to such activity. This study has observed some cases that show that Ubx is needed to maintain segregation of cells from different segments during pupation. It is possible that Drosophila Hox genes may have a function in cell segregation during this pupal stage, where cells from different discs and histoblast nests fuse to develop the adult cuticle. The mechanism of segregation seems to rely on the confrontation of cells with different Hox function and not on the absolute levels of Hox expression. This implies that Hox activity in neighboring cells may be checked through proteins at the cell membrane whose expression or levels must be controlled by Hox genes. In the embryo, the Hox gene Abd-B has been shown to regulate molecules like cadherins, and such proteins may mediate segregation between adjacent cells with distinct Hox input (Curt, 2013).

In vertebrates, cells from different rhombomeres are also almost completely prevented from freely mixing. As was shown in this study for Drosophila, it has been proposed that the tension provided by the activity of actomyosin molecules, controlled by Hox genes, could prevent mixing of cells in the vertebrate's rhombomeres. Hox-directed cell segregation, therefore, prevents cells with different Hox code to intermingle, and therefore the appearance of homeotic transformations. This function of Hox genes may be an old one in evolution, required in animals in which development of different body regions is not coupled to the mechanisms of segmentation. In Drosophila, this role of Hox genes may not be needed in cells that are physically separated during most of development (as in imaginal discs and histoblasts from different segments) or superseded by the activity of proteins like Engrailed and Hedgehog, but the maintenance of different affinities by Hox genes and signaling pathways through myosin accumulation may be a general mechanism to segregate cell populations in different species (Curt, 2013).

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).

Retinal determination genes coordinate neuroepithelial specification and neurogenesis modes in the Drosophila optic lobe

Differences in neuroepithelial patterning and neurogenesis modes contribute to area-specific diversifications of neural circuits. In the Drosophila visual system, two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers, generate neuron subtypes for four ganglia in several ways. Whereas neuroepithelial cells in the medial OPC directly convert into neuroblasts, in an IPC subdomain they generate migratory progenitors by epithelial-mesenchymal transition that mature into neuroblasts in a second proliferative zone. The molecular mechanisms that regulate the identity of these neuroepithelia, including their neurogenesis modes, remain poorly understood. Analysis of Polycomblike revealed that loss of Polycomb group-mediated repression of the Hox gene Abdominal-B (Abd-B) causes the transformation of OPC to IPC neuroepithelial identity. This suggests that the neuroepithelial default state is IPC-like, whereas OPC identity is derived. Ectopic Abd-B blocks expression of the highly conserved retinal determination gene network members Eyes absent (Eya), Sine oculis (So) and Homothorax (Hth). These factors are essential for OPC specification and neurogenesis control. Finally, eya and so are also sufficient to confer OPC-like identity, and, in parallel with hth, the OPC-specific neurogenesis mode on the IPC (Apitz, 2016).

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