Homothorax is first detected at approximately 3 hr of embryogenesis in a broad domain in the central portion of the blastoderm embryo, from approximately 15% to 85% egg length. In addition to a lack of expression at both poles, the ventral-most cells of the embryos, corresponding to the mesoderm primordium, remain unstained. As embryogenesis proceeds, the expression pattern becomes very dynamic. Expression is strongest in the trunk and in isolated regions of the head. Beginning at stage 9, the thoracic segments stain more strongly than the abdominal segments; this difference subsequently increases. By stage 14, expression in the thorax, including the central nervous system, remains strong but has been down-regulated in the abdomen. In the midgut, expression is strongest in the gastric caeca primordia and in a central, broad domain in the endoderm. Expression is absent in the most anterior and posterior regions of the midgut endoderm. By stage 16, strong expression is observed in the Malphigian tubules; expression in the endoderm begins posterior to the first midgut constriction and ends just anterior to the third midgut constriction, with a peak of expression at the second midgut constriction (Rieckhof, 1997).

To determine the spatial pattern of hth expression, in situ hybridization to whole embryos was performed using hth cDNA as a probe. hth expression begins at cellular blastoderm. It is expressed in the region that gives rise to the segmented portion of the embryo and is excluded from the anterior and posterior tips. During gastrulation, hth expression is detected throughout the ectoderm, but is still excluded from the procephalon. Starting at stage 11, high levels of HTH mRNA are detected in the thoracic region, whereas hth expression in the abdominal segments start to decline. Two of the head segments, the mandible and maxilla, express moderate levels of hth, whereas expression is absent from the labium. At the same time, hth expression becomes evident in the developing visceral mesoderm and a very high level of expression appears in the clypeolabrum. At stages 13-14, the levels of hth transcripts remain high in the head and thorax and decline further in the abdomen. hth expression appears also in the rudiments of the Malpighian tubules and the developing central nervous system. During stages 15-17, the ectodermal expression of hth declines whereas expression in the CNS becomes more prominent. The expression of hth in the CNS is graded; it is high in the anterior portion of the VNC and weak in its posterior portion (Kurant, 1998).

Hth protein is not detected prior to the beginning of gastrulation. During gastrulation (stages 6-8), Hth protein is localized to the cytoplasm. At stage 9 (germband extension) Hth starts to accumulate in nuclei in a spatially regulated fashion. Nuclear localization is detected in the ectoderm, and in specific cells within the thoracic portion of the VNC and within the visceral mesoderm (Kurant, 1998).

Proximodistal subdivision of Drosophila legs and wings: The elbow-no ocelli gene complex functions upstream of Hth and Tsh in the formation of the leg primordium

Appendages are thought to have arisen during evolution as outgrowths from the body wall of primitive bilateria. In Drosophila, subsets of body wall cells are set aside as appendage precursors through the action of secreted signaling proteins that direct localized expression of transcription factors. The Drosophila homeodomain protein Distal-less is expressed in the leg primordia and required for formation of legs, but not wings. The homeodomain protein Nubbin is expressed in the wing primordia and required for formation of wings, but not legs. Given that insect legs and wings have a common developmental and evolutionary origin, attempts were made to identify genes that underlie the specification of all appendage primordia. Evidence is presented that the zinc-finger proteins encoded by the elbow and no ocelli genes act in leg and wing primordia to repress body wall-specifying genes and thereby direct appendage formation (Weihe, 2004).

Evidence suggests that the el and noc genes serve as mediators of the function of the Wg and Dpp signaling systems in specification of the appendage field within the imaginal discs. El and Noc are induced by Wg and Dpp and are required to repress the proximally expressed proteins Hth and Tsh. Previous work had identified Dll as a gene required for appendage formation in leg and antenna, and nub as a gene required for wing. This report identifies El and Noc as a pair of zinc-finger proteins that function in both ventral and dorsal appendages. However, there are interesting differences in the way that they do so, when examined in detail (Weihe, 2004).

Dll expression is required for the formation of all leg and antenna elements in the ventral (leg) discs, and until this work Dll was the earliest known marker for the distal region leg disc. Previous work has shown that repression of Hth and Tsh by Dpp and Wg was not required for expression of Dll in the leg, nor could Dll repress Hth and Tsh. Thus an essential mediator of the effects of Wg and Dpp was missing. The current results present evidence that El and Noc serve this function, since their removal leads to ectopic expression of Hth and Tsh. Removal of El and Noc does not cause loss of Dll expression, so it is concluded that Wg and Dpp act independently to induce El and Noc expression and Dll to define the distal region of the leg disc (Weihe, 2004).

The situation differs slightly in the wing. Repression of Tsh is the earliest marker for specification of the distal wing region, preceding the onset of Hth repression or of Nub induction. Loss of Tsh and Hth are required to allow Nub expression. Ectopic expression of Hth and Tsh and loss of Nub is observed in clones lacking El and Noc activity. Thus in the wing, expression of the distal marker Nub cannot be demonstrated to be independent of El and Noc (because ectopic Hth can repress Nub, but not Dll). The vestigial gene is also important for wing development and has been proposed to be a wing specifying gene. However, Vestigial is expressed all along the DV boundary of the wing, both in the wing primordium and in the body wall. This led to the suggestion that while Vestigial is essential for wing development, its expression cannot be taken as a molecular marker for wing identity per se, particularly at early stages. For this reason analysis of the relationship between El, Noc and Vestigial was not performed in this study (Weihe, 2004).

Is the repression of trunk genes needed to specify appendage, as opposed to body wall, in wing and leg discs? In the wing disc the answer appears to be yes; repression of 'trunk genes' like hth is necessary to make the remaining part of the disc competent to form the appendage. However, in the leg the situation is more complex. Coexpression of Dll and Hth does not disrupt proximal-distal axis formation, but leads to homeotic transformation of leg tissue into antennal tissue. Hth is not repressed and limited to proximal areas in the antenna. However, loss of el and noc activities in the leg disc leads to loss of distal leg tissue without any evident transformation into antennal tissue. Thus, El and Noc may regulate the expression of other 'trunk genes', whose restricted expression is required to make the remaining leg and antenna disc competent to form the appendage (Weihe, 2004).

The regional requirements for El and Noc highlight another interesting difference between leg and wing disc development. el noc double mutant cells are excluded from contributing to the tarsal region of the leg but not from contributing to the femur and tibia. Lineage tracing has shown a considerable net flux of cells from the proximal (Tsh-expressing domain) into femur and tibia. While there is no boundary of lineage restriction separating these domains, cells must be able to change from expressing the proximal marker Hth to expressing the distal marker Dll in order to move from one territory to the other. The wing in contrast does not appear to normally exhibit this large net flux of cells from proximal to distal and the el noc double mutant cells are excluded from contributing to the entire wing region. Clonal analysis has suggested that el noc double mutant cells attempt to sort out toward proximal territory, or if that fails, they can be lost from the disc, apparently by sorting out perpendicular to the epithelium. These observations suggest that El and Noc activity may contribute to the production of proximal-distal differences in cell affinities and thereby may help to maintain segregation of these cell populations during development (Weihe, 2004).

The origins of the Drosophila leg revealed by the cis-regulatory architecture of the Distalless gene

Limb development requires the elaboration of a proximodistal (PD) axis, which forms orthogonally to previously defined dorsoventral (DV) and anteroposterior (AP) axes. In arthropods, the PD axis of the adult leg is subdivided into two broad domains, a proximal coxopodite and a distal telopodite. This study shows that the progressive subdivision of the PD axis into these two domains occurs during embryogenesis and is reflected in the cis-regulatory architecture of the Distalless (Dll) gene. Dll protein in the thorax was first detected during embryonic stage 11, and continues to be visualized in this region until the end of embryogenesis. Early Dll expression, governed by the Dll304 enhancer, is in cells that can give rise to both domains of the leg as well as to the entire dorsal (wing) appendage. A few hours after Dll304 is activated, the activity of this enhancer fades, and two later-acting enhancers assume control over Dll expression. The LT enhancer is expressed in cells that will give rise to the entire telopodite, and only the telopodite. By contrast, cells that activate the DKO ("Distalless Keilin Organ") enhancer will give rise to a leg-associated larval sensory structure known as the Keilin's organ (KO). Cells that activate neither LT nor DKO, but had activated Dll304, will give rise to the coxopodite. In addition, the trans-acting signals controlling the LT and DKO enhancers are described; surprisingly, the coxopodite progenitors begin to proliferate ~24 hours earlier than the telopodite progenitors. Together, these findings provide a complete and high-resolution fate map of the Drosophila appendage primordia, linking the primary domains to specific cis-regulatory elements in Dll (McKay, 2009).

To determine how each of the cell fates in the limb primordia is specified, genetic experiments were carried out to identify the regulators of the LT and DKO enhancers. Consistent with LT's dependency on wg and dpp for leg disc expression, LT is activated in the embryo in cells that receive both inputs, as monitored by anti-Wg and anti-PMad staining. To determine whether wg is required for LT activity, a temperature-sensitive allele of wg was used to allow earlier Dll activation. Switching the embryos to the restrictive temperature at stage 11 resulted in the absence of LT activity, despite the presence of Dll protein (probably derived from Dll304 activity. In addition, ectopic activation of the wg pathway [using an activated form of armadillo (arm*)] resulted in more LT-lacZ-expressing cells (McKay, 2009).

Like wg, the dpp pathway is necessary for LT-lacZ expression in leg discs. Paradoxically, dpp signaling represses Dll in the embryo because dpp mutants show an expansion in Dll304-lacZ expression. By contrast, LT-lacZ is not expressed in dpp null embryos. LT-lacZ, but not Dll protein, was also repressed by two dpp pathway repressors, Dad and brk. Conversely, stimulation of the dpp pathway [using an activated form of the Dpp receptor (TkvQD)] resulted in ectopic activation of LT ventrally (McKay, 2009).

Taken together, these data demonstrate that LT is activated by Wg and Dpp in the embryonic limb primordia, just as it (and Dll) is in the leg disc. Similarly, DKO activity also requires Wg and Dpp input (McKay, 2009).

Although LT is activated by wg and dpp in the leg primordia, these signals are also present in each abdominal segment. Consequently, there must be additional factors that restrict LT activity to the thorax. One possibility is that LT is repressed by the abdominal Hox factors, such as Dll304. Alternatively, LT might be regulated by Dll, itself. In Dll null embryos LT-lacZ was initially expressed in a stripe of cells instead of a ring, but then expression decayed. Ectopic expression of Dll resulted in weak ectopic expression of LT-lacZ in the thorax and abdomen. These data suggest that LT activity is restricted to the thorax in part because of the earlier restriction of Dll304 activity to the thorax (McKay, 2009).

The related zinc-finger transcription factors encoded by buttonhead (btd) and Sp1 are also expressed in the limb primordia and are also required for ventral appendage specification. In strong btd hypomorphs, the activity of LT was still detected but the number of cells expressing LT-lacZ was decreased and its pattern was disrupted. LT-lacZ expression was completely eliminated in animals bearing a large deficiency that removes both btd and Sp1. By contrast, Dll304 was activated normally in these animals (data not shown). Importantly, LT-lacZ expression was rescued by expressing btd in these deficiency embryos. By contrast, expressing Dll, tkvQD, or arm* did not rescue LT expression in these deficiency embryos. Ectopic expression of btd resulted in weak ectopic activation of LT-lacZ in cells of the thorax and abdomen. Strikingly, the simultaneous expression of Dll and btd resulted in robust ectopic expression of LT-lacZ in abdominal segments in the equivalent ventrolateral position as the thoracic limb primordia. btd and Dll were not sufficient to activate LT in wg null embryos (data not shown). These data indicate that the thoracic-specific expression of the LT enhancer is controlled by the combined activities of btd and/or Sp1, Dll and the wg and dpp pathways (McKay, 2009).

Although the data suggest that LT is activated by a combination of Wg, Dpp, Btd and Dll, these activators are also present in the precursors of the KO, which activate DKO instead of LT. Because the KO is a sensory structure, the role of members of the achaete-scute complex (ASC) that are expressed in these cells was tested. In embryos hemizygous for a deficiency that removes the achaete-scute complex, LT-lacZ expression was expanded at the expense of the Ct-expressing cells. Consistently, ectopic expression of the ASC gene asense (ase) repressed LT and increased the number of Ct-expressing cells. These data suggest that there is a mutual antagonism between the progenitors of the telopodite and those of the KO. It was also found that DKO-lacZ expression in the leg primordia was lost in Dll or btd null embryos, consistent with the loss of KOs in these mutants. DKO activity was also lost from the limb primordia in embryos deficient for the ASC. These results indicate that DKO is activated by the same genes that promote LT expression but, in addition, requires proneural input from the ASC (McKay, 2009).

One of the most interesting findings from this work is that the temporal control of Dll expression in the limb primordia by three cis-regulatory elements is linked to cell-type specification. The earliest acting element, Dll304, is active throughout the appendage primordia. At the time Dll304 is active, the cells are multipotent and can give rise to any part of the dorsal or ventral appendages, or KO. A few hours later, Dll304 activity fades, and two alternative cis-regulatory elements become active. Together, these two elements allow for the uninterrupted and uniform expression of Dll within the appendage primordia. However, their activation correlates with a higher degree of refinement in cell fate potential: LT, active in only the outer ring of the appendage primordia, is only expressed in the progenitors of the telopodite. By contrast, DKO, active in the cells within the LT ring, is only expressed in the progenitors of the KO. Thus, although the pattern of Dll protein appears unchanged, the control over Dll expression has shifted from singular control by Dll304 to dual control by LT and DKO. Moreover, not only is there a molecular handoff from Dll304 to LT and DKO, the two later enhancers both require the earlier expression of Dll. Thus, the logic of ventral primordia refinement depends on a cascade of Dll regulatory elements in which the later ones depend on the activity of an earlier one (McKay, 2009).

The high-resolution view of the embryonic limb primordia provided in this study allows clarification of some contradictions that currently exist in the literature. Initial expression of Dll in the thorax overlaps entirely with Hth-nExd (referring to nuclear Extradenticle). Subsequently, hth expression is lost from most, but not all, of the Dll-expressing cells of the leg primordia. The first reports describing these changes failed to recognize the persistent overlap between Dll and Hth-nExd in some cells. As a result, and partly because of the analogy with the third instar leg disc, the predominant view of this fate map became that the Dll-positive, Hth-nExd-negative cells of the embryonic primordia gave rise to the telopodite, while the surrounding Hth-positive cells gave rise to the coxopodite. The expression pattern of esg, a gene required for the maintenance of diploidy, was also misinterpreted as being a marker exclusively of proximal leg fates. Counter to these earlier studies, the current experiments unambiguously show that the Dll-positive, Hth-nExd-negative cells in the center of the primordia give rise to the KO, the ring of Dll-positive, Esg-positive, Hth-nExd-positive cells gives rise to the telopodite, and the remaining Esg-positive, Dll-negative cells give rise to the coxopodite (McKay, 2009).

The spurious expression of DKO-lacZ in Dll-non-expressing cells outside the leg primorida complicates the interpretation of several experiments. Attempts to refine DKO activity by changing the size of the cloned fragment proved unsuccessful. Nevertheless, the evidence supports the idea that DKO-positive, Dll-positive cells of the leg primordia give rise to the Keilin's organ, and not the adult appendage (McKay, 2009).

The progenitors of the coxopodite begin to proliferate at approximately 48 hours of development, consistent with previous measurements of leg imaginal disc growth, whereas the progenitors of the telopodite do not resume proliferating for an additional 12 to 24 hours. According to estimates of the cell cycle time in leg discs, this difference in the onset of proliferation results in one to two additional cell divisions in the coxopodite, consistent with images of late second instar leg discs presented in this study. Why might the telopodite and coxopodite begin proliferation at different times? One possibility is that the cells of the coxopodite give rise to the peripodial epithelium that covers the leg imaginal disc, and therefore require additional cell divisions relative to the telopodite. It is also possible that the telopodite is delayed because the neurons of the Keilin's organ serve a pathfinding role for larval-born neurons that innervate the adult limb. Perhaps this pathfinding function requires that the KO and telopodite remain associated with each other through the second instar. Consistently, the leg is the only imaginal disc that has not invaginated as a sac-like structure in newly hatched first instar larvae (McKay, 2009).

A possible explanation for the delay in the onset of telopodite proliferation is the persistent co-expression of hth and Dll in these cells; hth (and tsh) expression is turned off in these cells at about the same time they begin to proliferate. Consistent with this idea, maintaining the expression of hth throughout the primordia blocks the proliferation of the telopodite. Also noteworthy is the finding that the genes no ocelli and elbow have been shown to mediate the ability of Wg and Dpp to repress coxopodite fates. Together with the current findings, it is possible that the activation of these two genes in the LT-expressing progenitors is the trigger that turns off hth and tsh in these cells (McKay, 2009).

The experiments suggest that once LT is activated, and under normal growth conditions, there is a lineage restriction between the telopodite and coxopodite. By contrast, previous lineage-tracing experiments using tsh-Gal4 concluded that the progeny of proximal cells could adopt more distal leg fates. However, tsh is still expressed in the telopodite progenitors far into the second instar, providing an explanation for these results. In contrast to this early restriction, there is no evidence for a later lineage restriction within the telopodite. For example, the progeny of a Dll-positive cell can lose Dll expression and contribute to the dac-only domain (McKay, 2009).

Interestingly, the lineage restriction between coxopodite and telopodite is not defined by the presence or absence of Hth-nExd or Tsh because both progenitor populations express hth and tsh after their fates have been specified. By contrast, when these two domains are specified, the telopodite expresses Dll, while the coxopodite does not, suggesting that Dll may be important for the lineage restriction. However, later in development, some cells in the telopodite lose Dll expression and express dac, but continue to respect the coxopodite-telopodite boundary. Thus, either Dll expression in the telopodite is somehow remembered or the telopodite-coxopodite boundary can be maintained by dac, which is expressed in place of Dll immediately adjacent to the telopodite-coxopodite boundary. Also noteworthy is the finding that clones originating in the coxopodite can contribute to the trochanter, the segment inbetween the proximal and distal components of the adult leg that expresses both Dll and hth in third instar imaginal discs. However, the progeny of such clones do not contribute to fates more distal than the trochanter. Likewise, a clone originating in the telopodite can also contribute to the trochanter, but will not grow more proximally into the coxa. Thus, the lineage restriction uncovered here seems to be determined by distinct combinations of transcription factors expressed in the coxopodite and telopodite progenitors at stage 14. The progeny of cells that express Dll, tsh and hth can populate the telopodite or trochanter, whereas the progeny of cells that express tsh and hth, but not Dll, can populate the coxopodite or trochanter. In light of Minute-positive results, however, the lineage restriction between coxopodite and telopodite does not satisfy the classical definition of a compartment boundary. A similar non-compartment lineage restriction has also been documented along the PD axis of the developing Drosophila wing (McKay, 2009).

Segment-specific neuronal subtype specification by the integration of anteroposterior and temporal cues

The generation of distinct neuronal subtypes at different axial levels relies upon both anteroposterior and temporal cues. However, the integration between these cues is poorly understood. In the Drosophila central nervous system, the segmentally repeated neuroblast 5-6 generates a unique group of neurons, the Apterous (Ap) cluster, only in thoracic segments. Recent studies have identified elaborate genetic pathways acting to control the generation of these neurons. These insights, combined with novel markers, provide a unique opportunity for addressing how anteroposterior and temporal cues are integrated to generate segment-specific neuronal subtypes. Pbx/Meis, Hox, and temporal genes were found to act in three different ways. Posteriorly, Pbx/Meis and posterior Hox genes block lineage progression within an early temporal window, by triggering cell cycle exit. Because Ap neurons are generated late in the thoracic 5-6 lineage, this prevents generation of Ap cluster cells in the abdomen. Thoracically, Pbx/Meis and anterior Hox genes integrate with late temporal genes to specify Ap clusters, via activation of a specific feed-forward loop. In brain segments, 'Ap cluster cells' are present but lack both proper Hox and temporal coding. Only by simultaneously altering Hox and temporal gene activity in all segments can Ap clusters be generated throughout the neuroaxis. This study provides the first detailed analysis of an identified neuroblast lineage along the entire neuroaxis, and confirms the concept that lineal homologs of truncal neuroblasts exist throughout the developing brain. Also this study provides the first insight into how Hox/Pbx/Meis anteroposterior and temporal cues are integrated within a defined lineage, to specify unique neuronal identities only in thoracic segments. This study reveals a surprisingly restricted, yet multifaceted, function of both anteroposterior and temporal cues with respect to lineage control and cell fate specification (Karlsson, 2010).

To understand segment-specific neuronal subtype specification, this study focused on the Drosophila neuroblast 5-6 lineage and the thoracic-specific Ap cluster neurons born at the end of the NB 5-6T lineage. The thoracic appearance of Ap clusters was shown to result from a complex interplay of Hox, Pbx/Meis, and temporal genes that act to modify the NB 5-6 lineage in three distinct ways (see Summary of Hox/Pbx/Meis and temporal control of NB 5-6 development). In line with other studies of anterior-most brain development, it was found that the first brain segment (B1) appears to develop by a different logic. These findings will be discussed in relation to other studies on spatial and temporal control of neuroblast lineages (Karlsson, 2010).

In the developing Drosophila CNS, each abdominal and thoracic hemisegment contains an identifiable set of 30 neuroblasts, which divide asymmetrically in a stem-cell fashion to generate distinct lineages. However, they generate differently sized lineages -- from two to 40 cells, indicating the existence of elaborate and precise mechanisms for controlling lineage progression. Moreover, about one third of these lineages show reproducible anteroposterior differences in size, typically being smaller in abdominal segments when compared to thoracic segments. Thus, neuroblast-specific lineage size control mechanisms are often modified along the anteroposterior axis (Karlsson, 2010).

Previous studies have shown that Hox input plays a key role in modulating segment-specific behaviors of neuroblast lineages. Recent studies have resulted in mechanistic insight into these events. For instance, in the embryonic CNS, Bx-C acts to modify the NB 6-4 lineage, preventing formation of thoracic-specific neurons in the abdominal segments. This is controlled, at least in part, by Bx-C genes suppressing the expression of the Cyclin E cell cycle gene in NB 6-4a. Detailed studies of another neuroblast, NB 7-3, revealed that cell death played an important role in controlling lineage size in this lineage: when cell death is genetically blocked, lineage size increased from four up to 10 cells. Similarly, in postembryonic neuroblasts, both of these mechanisms have been identified. In one class of neuroblasts, denoted type I, an important final step involves nuclear accumulation of the Prospero regulator, a key regulator both of cell cycle and differentiation genes. In 'type II' neuroblasts, grh acts with the Bx-C gene Abd-A to activate cell death genes of the Reaper, Head involution defective, and Grim (RHG) family, and thereby terminates lineage progression by apoptosis of the neuroblast. This set of studies demonstrates that lineage progression, in both embryonic and postembryonic neuroblasts, can be terminated either by neuroblast cell cycle exit or by neuroblast apoptosis. In the abdominal segments, it was found that the absence of Ap clusters results from a truncation of the NB 5-6 lineage, terminating it within the Pdm early temporal window, and therefore Ap cluster cells are never generated. These studies reveal that this truncation results from neuroblast cell cycle exit, controlled by Bx-C, hth, and exd, thereafter followed by apoptosis. In Bx-C/hth/exd mutants, the neuroblast cell cycle exit point is bypassed, and a thoracic sized lineage is generated, indicating that these genes may control both cell cycle exit and apoptosis. However, it is also possible that cell cycle exit is necessary for apoptosis to commence, and that Bx-C/hth/exd in fact only control cell cycle exit. Insight into the precise mechanisms of the cell cycle exit and apoptosis in NB 5-6A may help shed light on this issue (Karlsson, 2010).

Whichever mechanism is used to terminate any given neuroblast lineage -- cell cycle exit or cell death -- the existence in the Drosophila CNS of stereotyped lineages progressing through defined temporal competence windows allows for the generation of segment-specific cell types simply by regulation of cell cycle and/or cell death genes by developmental patterning genes. Specifically, neuronal subtypes born at the end of a specific neuroblast lineage can be generated in a segment-specific fashion 'simply' by segmentally controlling lineage size. This mechanism is different in its logic when compared to a more traditional view, where developmental patterning genes act upon cell fate determinants. But as increasing evidence points to stereotypic temporal changes also in vertebrate neural progenitor cells (Okano, 2009), this mechanism may well turn out to be frequently used to generate segment-specific cell types also in the vertebrate CNS (Karlsson, 2010).

These findings of Hox, Pbx/Meis, and temporal gene input during Ap cluster formation are not surprising -- generation and specification of most neurons and glia will, of course, depend upon some aspect or another of these fundamental cues. Importantly however, the detailed analysis of the NB 5-6T lineage, and of the complex genetic pathways acting to specify Ap cluster neurons, has allowed this study to pin-point critical integration points between anteroposterior and temporal input. Specifically, cas, Antp, hth, and exd mutants show striking effects upon Ap cluster specification, with effects upon expression of many determinants, including the critical determinant col. Whereas Antp plays additional feed-forward roles, and exd was not tested due to its maternal load, it was found that both cas and hth mutants can be rescued by simply re-expressing col. This demonstrates that among a number of possible regulatory roles for cas, hth, Antp, and exd, one critical integration point for these anteroposterior and temporal cues is the activation of the COE/Ebf gene col, and the col-mediated feed-forward loop. Both col and ap play important roles during Drosophila muscle development, acting to control development of different muscle subsets. Their restricted expression in developing muscles has been shown to be under control of both Antp and Bx-C genes. Molecular analysis has revealed that this regulation is direct, as Hox proteins bind to key regulatory elements within the col and ap muscle enhancers. The regulatory elements controlling the CNS expression of col and ap are distinct from the muscle enhancers, and it will be interesting to learn whether Hox, as well as Pbx/Meis and temporal regulatory input, acts directly also upon the col and ap CNS enhancers (Karlsson, 2010).

One particularly surprising finding pertains to the instructive role of Hth levels in NB 5-6T. At low levels, Hth acts in NB 5-6A to block lineage progression, whereas at higher levels, it acts in NB 5-6T to trigger expression of col within the large cas window. It is interesting to note that the hth mRNA and Hth protein expression levels increase rapidly in the entire anterior CNS (T3 and onward). In addition, studies reveal that thoracic and anterior neuroblast lineages in general tend to generate larger lineages and thus remain mitotically active for a longer period than abdominal lineages. On this note, it is tempting to speculate that high levels of Hth may play instructive roles in many anterior neuroblast lineages. In zebrafish, Meis3 acts to modulate Hox gene function, and intriguingly, different Hox genes require different levels of Meis3 expression. In the Drosophila peripheral nervous system, expression levels of the Cut homeodomain protein play instructive roles, acting at different levels to dictate different dendritic branching patterns in different sensory neuron subclasses. Although the underlying mechanisms behind the levels-specific roles of Cut, Meis3 or Hth are unknown, it is tempting to speculate that they may involve alterations in transcription factor binding sites, leading to levels-sensitive binding and gene activation of different target genes (Karlsson, 2010).

The vertebrate members of the Meis family (Meis1/2/3, Prep1/2) are expressed within the CNS, and play key roles in modulating Hox gene function. Intriguingly, studies in both zebrafish and Xenopus reveal that subsequent to their early broad expression, several members are expressed more strongly or exclusively in anterior parts of the CNS, in particular, in the anterior spinal cord and hindbrain. Here, functional studies reveal complex roles of the Meis family with respect to Hox gene function and CNS development. However, in several cases, studies reveal that they are indeed important for specification, or perhaps generation, of cell types found in the anterior spinal cord and/or hindbrain, i.e., anteroposterior intermediate neural cell fates. As more is learned about vertebrate neural lineages, it will be interesting to learn which Meis functions may pertain to postmitotic neuronal subtype specification, and which may pertain to progenitor cell cycle control (Karlsson, 2010).

In anterior segments -- subesophageal (S1-S3) and brain (B1-B3) -- a more complex picture emerges where both the overall lineage size and temporal coding is altered, when compared to the thoracic segments. Specially, whereas all anterior NB 5-6 lineages do contain Cas expressing cells, expression of Grh is weak or absent from many Cas cells. The importance of this weaker Grh expression is apparent from the effects of co-misexpressing grh with Antp -- misexpression of Antp alone is unable to trigger FMRFa expression, whereas co-misexpression with grh potently does so. It is unclear why anterior 5-6 lineages would express lower levels of Grh, since Grh expression is robust in some other anterior lineages (Karlsson, 2010).

In the B1 segment two NB 5-6 equivalents have been identified. However, the finding of two NB 5-6 equivalents is perhaps not surprising, since the B1 segment contains more than twice as many neuroblasts as posterior segments. Due to weaker lbe(K)-lacZ and -Gal4 reporter gene expression, and cell migration, these lineages could not be mapped. However, irrespective of the features of the B1 NB 5-6 lineages, bona fide Ap cluster formation could not be triggered by Antp/grh co-misexpression in B1. Together, these findings suggest that the B1 segment develops using a different modus operandi, a notion that is similar to development of the anterior-most part of the vertebrate neuroaxis, where patterning and segmentation is still debated. On that note, it is noteworthy that although Hox genes play key roles in specifying unique neuronal cell fates in more posterior parts of the vertebrate CNS, and can indeed alter cell fates when misexpressed, the sufficiency of Hox genes to alter neuronal cell fates in the anterior-most CNS has not been reported -- for instance, Hox misexpression has not been reported to trigger motoneuron specification in the vertebrate forebrain. Thus, in line with the current findings that Antp is not sufficient to trigger Ap cluster neuronal fate in the B1 anterior parts, the anterior-most part of both the insect and vertebrate neuroaxis appears to be 'off limits' for Hox genes (Karlsson, 2010).

The Hox, Pbx/Meis, and temporal genes are necessary, and in part sufficient, to dictate Ap cluster neuronal cell fate. However, they only do so within the limited context of NB 5-6 identity. Within each abdominal and thoracic hemisegment, each of the 30 neuroblasts acquires a unique identity, determined by the interplay of segment-polarity and columnar genes. In the periphery, recent studies demonstrate that anteroposterior cues, mediated by Hox and Pbx/Meis genes, are integrated with segment-polarity cues by means of physical interaction and binding to regulatory regions of specific target genes. It is tempting to speculate that similar mechanisms may act inside the CNS as well, and may not only involve anteroposterior and segment-polarity integration, but also extend into columnar and temporal integration (Karlsson, 2010).


The correlation between hth expression and nuclear localized Exd can be observed in leg and antennal imaginal discs: Exd is nuclear in only the peripheral cells of leg and antennal discs and is cytoplasmic in the central portion of these discs. Like nuclear Exd, hth is expressed only in the peripheral cells of these discs. In a few places in imaginal discs, for example, the region of the antennal disc that gives rise to the maxillary palps, Exd is localized to nuclei without detectable expression of hth. Thus, hth expression correlates with nuclear-localized Exd in most (but not all) imaginal disc cells (Rieckhof, 1997).

In the wing disc, hth expression is present in the regions corresponding to notum, wing hinge, and ventral pleura. In the leg discs, expression is in the periphery region, corresponding to the proximal segments of the legs. In the antennal disc, expression is in all but the arista region. In the eye disc, the expression is strong in the anterior region surrounding the eye field, including the regions corresponding to ptilinum, ocellus, and head capsules, and weak in the posterior and lateral margins of the eye disc. Very weak expression is detected in the posterior region composed of mature photoreceptors. Hth is also expressed in all cells of the peripodial membrane in the eye disc. These patterns are very similar to those of nuclear Exd protein. Closer examination shows that the distribution of Hth and nuclear Exd generally coincide. Outside of the Hth expression domain, Exd protein is present in low levels in the cytoplasm (Pai, 1998).

Hth and Exd are expressed in the proximal domain of the leg (the Hth domain) : all the other factors studied (Wingless, Decapentaplegic and Distalless) are expressed in more distal regions (the Dll domain). Dachsund (Dac) is expressed in an intermediate domain, dorsal and lateral to the more distal Dll domain (the Dac domain). What follows is a more complete description of these domains. The expression of several targets of the signaling molecules Wg and Dpp were examined in relation to the hth expression domain. dpp expression in the leg disc at the early third larval instar stage consists of a sector that originates at the center of the disc, extends dorsally to the periphery and shows extensive overlap with Hth. omb, a target of the Dpp-signaling pathway, is expressed in a dorsal sector that, in contrast to dpp, extends dorsally only to abut, but not overlap with, the hth domain. wg expression consists of a ventral sector of cells that extends from the center to the periphery of the disc, whereas H15, an enhancer trap line that requires wg signaling for its activation, is largely not transcribed in the hth domain. The restriction of these Wg and Dpp target genes to non-hth-expressing cells suggests that hth restricts signaling by these two molecules. By the late third larval instar stage, there is a small degree of overlap between hth and omb expression as well as between hth and H15. This expression corresponds to the trochanter domain where gene activation can occur independent of the Wg- and Dpp-signaling pathways. Unlike omb and H15, the Dll and dac genes require input from both the Dpp and Wg signal transduction pathways to be activated in leg discs. Dll encodes a homeodomain protein present in the central portion of leg discs, and its activation requires the highest concentrations of Wg and Dpp. dac encodes a nuclear protein and a putative transcription factor whose expression is repressed by high concentrations, and activated by intermediate concentrations, of Wg and Dpp. By performing triple stains for the dacP-lacZ reporter gene, and Dll and Hth proteins at the early third larval instar stage, it was found that the leg disc is defined by three non-overlapping domains of gene expression. The distal-most domain of the leg disc contains Dll protein (the Dll domain). Dorsal and dorsolateral, but not ventral, to the Dll domain are cells that express dac (the Dac domain). The proximal-most cells of the disc, which surround the dac and Dll domains, express hth (the Hth domain). At the mid 3rd larval instar stage (~96 hours after egg lay, or AEL), the distal-most cells express only Dll and are surrounded by a ring of cells that express both Dll and dac. At this stage, there is also a dorsal patch of cells that express dac but not Dll. hth expression remains limited to the proximal-most cells of the disc and shows no overlap with dac or Dll. By the late 3rd larval instar stage (~120 hours AEL), hth is still not co-expressed with dac or Dll, with the exception of a thin band of cells corresponding to the trochanter domain, where all three genes are co-expressed. Gene expression in the trochanter domain is likely to represent secondary patterning events, because it is not dependent on Wg- or Dpp-signaling. At this stage dac expression also surrounds and partially overlaps the Dll expression domain. It is proposed that the Dll and Dac domains, where hth transcription is off and Exd is cytoplasmic, are Dpp- and/or Wg-responsive domains, as demonstrated by the ability of these cells to respond to these signals by activating the target genes Dll, dac, omb and H15. In contrast, the hth domain, where hth is active and Exd is nuclear, is a Wg- and/or Dpp-non- responsive domain, where these signals are present but cannot activate these targets (Abu-Shaar, 1998).

The developing legs of Drosophila are subdivided into proximal and distal domains by the activity of the homeodomain proteins Homothorax (Hth) and Distal-less (Dll). The expression domains of Dll and Hth are initially reciprocal. In the mature third instar disc, Dll is expressed in a large central domain that corresponds to the presumptive tarsus and distal tibia. Dll is also expressed in a secondary ring. X-gal staining of adult legs carrying a Dll-lacZ reporter gene shows that this ring is located at the proximal edge of the femur, possibly extending slightly into the distal trochanter. The central domain of Dll expression is controlled by Wg and Dpp. The proximal ring arises in third instar and does not depend on Wg or Dpp activity. The leg disc is a continuous single-layered epithelial sheet that forms a series of folds as it grows. The peripheral region of the disc forms the proximal segments. This region is folded back over the central region where Dll is expressed. The domain of Hth expression extends from the peripodial membrane at the top, through the coxa and trochanter segment primordia. The distal-most portion of the Hth domain overlaps the proximal part of the dac-lacZ domain within the proximal ring of Dll expression in the femur. Dll is expressed alone in the central folds of the disc (which correspond to tarsal segment primordia). In proximal tarsus and tibia, Dll and Dac overlap. Dac is expressed alone in the presumptive femur. Because the disc is highly folded, horizontal optical sections make proximal and distal regions of the disc appear to be closely apposed, although they are actually far apart along the PD axis in the plane of the disc epithelium. Hth is expressed in the upper layer and around the lateral sides of the epithelial sac. Dll is expressed in the center of the lower layer. The two expression domains abut, but do not overlap. dac-lacZ is not detectably expressed at this stage, but can be reliably detected in slightly older discs at the transition from second to third instar. These observations suggest that the primary subdivision of the disc is into two domains: a central Dll-expressing domain and a proximal Hth-expressing domain. Wg and Dpp act together to induce Dll and Dac in the center of the leg disc. Wg and Dpp repress Hth and Teashirt, but not through activation of Dll (Wu, 1999).

The expression patterns of Dll and Hth/Exd reflect an early subdivision of the disc into proximal and distal domains. At early stages of disc development, Dll and Hth/Exd are expressed in reciprocal domains which account for all cells of the disc. At this stage, Dac is not yet expressed. What is the relationship between Dll and Hth/Exd expression in the early disc? The Dll domain is defined by Wg and Dpp signaling. The same signals repress nuclear localization of Exd and Hth expression. The reciprocity of Dll and Hth expression suggests a model in which Wg and Dpp act through Dll to repress Hth in the early disc. However, the analysis of marked Dll mutant clones reported here shows that this is not the case. Clones of Dll mutant cells located in the distal region of the leg do not express Hth. This contrasts with recent reports by González-Crespo (1998) and Abu-Shaar (1998) in which evidence is presented for ectopic expression of Exd and Hth in Dll mutant clones. How can the difference in the results between these reports be reconciled? In both studies, the clones were induced in second instar larvae using the same allele of Dll. In the experiments reported here, clones were marked by the absence of Dll protein and by the absence of a neutral beta-gal marker, which permits definitive genotyping of the cells independent of Dll expression. In the other reports, clones were marked only by the absence of Dll. The disc epithelium is highly folded and the proximal Hth-expressing epithelium is very close to the distal Dll-expressing epithelium. Unless cells in the clone are definitively genotyped, it is difficult to distinguish a genuine clone from a patch of the overlying Hth-expressing proximal epithelium that has been pushed downward into the plane of the optical section. Serial optical sections of wild-type discs show that this type of distortion of the disc epithelium can occur in damaged discs as well as in discs that are not obviously damaged. How is Hth repressed by Wg and Dpp? Dac is induced by Wg and Dpp toward the end of second instar. Hth expands distally, to some extent, in Dac mutant discs. These observations suggest that Dac contributes to Hth repression. However, Hth is repressed prior to the onset of Dac expression indicating that Dac cannot be the primary repressor. Whether Wg and Dpp act directly to repress Hth expression or act via another as unidentified repressor remains to be determined (Wu, 1999).

In conclusion, Hth and Dll expression appear to define alternative fates in the second instar disc. Under normal circumstances, there does not appear to be a cell lineage restriction between these populations (i.e. no compartment boundary). These results suggest that cells can cross between these territories if they are able to switch between Hth and Dll expression. This situation appears to be analogous to the DV subdivision of the leg disc (as opposed to the proximal distal subdivision reported here). DV subdivision is stable at the level of gene expression in a cell population, but is not a clonal lineage restriction boundary. Similarly, the separation of proximal and distal cell populations requires Hth function. These results suggest that cells at the interface between these two territories are specialized to allow integration of otherwise immiscible populations of cells (Wu, 1999 and references).

The Drosophila wing imaginal disc gives rise to three main regions along the proximodistal axis of the dorsal mesothoracic segment: the notum, proximal wing, and wing blade. Development of the wing blade requires the Notch and wingless signalling pathways to activate vestigial at the dorsoventral boundary. However, in the proximal wing, Wingless activates a different subset of genes, e.g., homothorax. This raises the question of how the downstream response to Wingless signalling differentiates between proximal and distal fate specification. A temporally dynamic response to Wingless signalling is shown to sequentially elaborate the proximodistal axis. In the second instar, Wingless activates genes involved in proximal wing development; later in the third instar, Wingless acts to direct the differentiation of the distal wing blade. The expression of a novel marker for proximal wing fate, Zn finger homeodomain 2 (zfh-2), is initially activated by Wingless throughout the 'wing primordium,' but later is repressed by the activity of Vestigial and Nubbin, which together define a more distal domain. Thus, activation of a distal developmental program is antagonistic to previously established proximal fate. In addition, Wingless is required early to establish proximal fate, but later when Wingless activates distal differentiation, development of proximal fate becomes independent of Wingless signalling. Since P-element insertions in the zfh-2 gene result in a revertable proximal wing deletion phenotype, it appears that zfh-2 activity is required for correct proximal wing development. These data are consistent with a model in which Wingless first establishes a proximal appendage fate over notum, then the downstream response changes to direct the differentiation of a more distal fate over proximal. Thus, the proximodistal domains are patterned in sequence and show a distal dominance (Whitworth, 2003).

Recent work has indicated that the homeobox gene homothorax (hth) is required for the correct development of the proximal wing by both upregulating Wg expression in the proximal wing and limiting the area of wing blade differentiation. Since loss of Hth function in the proximal wing leads to a dramatic reduction in the level of Wg expression, attempts were made to determine whether Hth is also required for regulation of Zfh-2 expression. In hth- clones, neither the expression pattern nor the level of Zfh-2 is altered compared with neighboring wild type tissue. This is consistent with the observation that late removal of wg does not affect the expression of zfh-2. Similarly, ectopic expression of Hth shows no effect on zfh-2 expression. These data suggest that Hth does not play a role in establishing or regulating the determination of proximal wing fate, since no change in the expression of Zfh-2 was observed. Thus, it appears that the prime functions of Hth in the proximal wing are to maintain Wg expression and define the limits of the wing pouch (Whitworth, 2003).

At the beginning of the second larval instar, the wing imaginal disc expresses markers of proximal fate, hth and tsh, in the entire anlage. During early L2, the expression of wg and zfh-2 is initiated in an anterior-ventral wedge pattern. The data indicate that Wg function is required to activate zfh-2 expression at this stage, since early removal of Wg function leads to a simultaneous loss of zfh-2 expression. As development proceeds, wg and zfh-2 expression rapidly expands filling the whole of the ventral portion of the wing disc by the end of the second instar. Concomitant with the expansion of wg and zfh-2, both hth and tsh become repressed in the ventral portion of the disc. This transition appears to mark the first P-D differentiation of the wing disc into appendage and notum. However, since zfh-2 is expressed in the entire wing anlage at this time, it is believed that the appendage has not differentiated proximal wing and blade. Around the L2-L3 transition, the wing blade markers nub and vgQE are activated by the combined activity of the Wg and N signalling pathways. Nub and Vg, acting together or independently, repress zfh-2 expression in the center of the disc. This marks the second phase of P-D elaboration where the appendage anlage is split into proximal wing and blade. It is noted that, at this time, hth and tsh remain coexpressed in the notum, where zfh-2 is not expressed. The pattern of zfh-2 expression at this stage suggests that it is still influenced by Wg signalling since it remains restricted to areas of high Wg expression. During L3, the division of the wing disc into three distinct domains is maintained and refined as the individual domains undergo their characteristic patterning. At this time, Hth and Wg are upregulated in the proximal wing anlage, where their activities are interdependent, while zfh-2 expression persists but becomes independent of Wg activity (Whitworth, 2003).

Concentric zones, cell migration and neuronal circuits in the Drosophila visual center

The Drosophila optic lobe comprises a wide variety of neurons, which form laminar neuropiles with columnar units and topographic projections from the retina. The Drosophila optic lobe shares many structural characteristics with mammalian visual systems. However, little is known about the developmental mechanisms that produce neuronal diversity and organize the circuits in the primary region of the optic lobe, the medulla. This study describes the key features of the developing medulla and reports novel phenomena that could accelerate understanding of the Drosophila visual system. The identities of medulla neurons are pre-determined in the larval medulla primordium, which is subdivided into concentric zones characterized by the expression of four transcription factors: Drifter, Runt, Homothorax and Brain-specific homeobox (Bsh). The expression pattern of these factors correlates with the order of neuron production. Once the concentric zones are specified, the distribution of medulla neurons changes rapidly. Each type of medulla neuron exhibits an extensive but defined pattern of migration during pupal development. The results of clonal analysis suggest homothorax is required to specify the neuronal type by regulating various targets including Bsh and cell-adhesion molecules such as N-cadherin, while drifter regulates a subset of morphological features of Drifter-positive neurons. Thus, genes that show the concentric zones may form a genetic hierarchy to establish neuronal circuits in the medulla (Hasegawa, 2011).

Concentric genes are expressed in a defined subset of medulla neurons throughout development, suggesting that a part of neuronal identities are pre-determined in the larval medulla primordium. The data suggest that Drf-positive neurons produce nine types of medulla neurons, including lobula projection and medulla intrinsic neurons, while Hth-positive neurons produce at least four types of neurons, including lamina projection and medulla intrinsic neurons. In Hth-positive neurons, Bsh is exclusively expressed in medulla intrinsic Mi1 neurons. A hth mutation caused the neuron to switch type, while a drf mutation affected subsets of morphological features of Drf-positive neurons. Thus, roles of concentric genes may be functionally segregated to form a genetic hierarchy. Apparently, other concentric genes must exist in addition to the four genes reported in this study. Because there are many neurons outside of the Drf domain in the larval medulla, some concentric genes may be expressed in the outer zones. Some transcription factors may have expression patterns that differ from those of concentric genes, and their combined expression may specify restricted subtypes of medulla neurons. For example, apterous (ap) and Cut are widely expressed in medulla neurons. Cut was co-expressed in subsets of Drf-positive neurons, while ap was expressed in all Drf- and Bsh/Hth-positive neurons (Hasegawa, 2011).

Early-born medulla neurons express the inner concentric genes, while late born neurons express the outer ones. Thus, concentric gene expression correlates with neuronal birth order. However, it is still unknown how concentric gene expression is specified. It would be possible to speculate that genes controlling temporal specification of neurons are expressed in NBs to control the concentric gene expression. However, the genes that are known to control neuronal birth order in the embryonic CNS were not expressed in larval medulla NBs. In addition to local temporal mechanisms, such as birth order, global and spatial mechanisms governed by morphogen gradient may also play a role in determining medulla cell type. In addition to birth order or a morphogen gradient, mutual repression among concentric genes may be essential in establishing defined concentric zones. Except for rare occasions, de-repression of other concentric genes was not induced in clones mutant for hth or drf. Additionally, ectopic hth expression did not compromise Drf and Run expression. These results may suggest that unidentified genes act redundantly with these genes to repress expression of other concentric genes and that weak Hth expression in NBs does not play roles in temporal specification of medulla neurons (Hasegawa, 2011).

Various types of cell migration play important roles during vertebrate neurogenesis. Although Drosophila has been a powerful model of neural development, extensive neuronal migrations coupled with layer formation found in this study have not been previously reported. The current findings may establish a model to understand molecular mechanisms that govern brain development via neuronal migrations (Hasegawa, 2011).

It is important to know whether the migration of medulla neurons occurs actively or passively. The distribution of cell bodies in the adult medulla cortex was not random, but organized according to cell type. In particular, the Mi1 neurons identified by Bsh expression migrated outwards and were eventually located in the outermost area of the adult medulla cortex, which was affected in hth mutant clones. The observation that defined localization of cell bodies is under the control of genetic program may not be explained by passive migration. Repression of apoptosis by expressing p35 under the control of elav-Gal4 did not compromise migration of Bsh- and Drf-positive neurons, suggesting that apoptosis is not a driving force of the migration. If neurons migrate actively in an organized manner, what regulates the pattern of migration? In many cases, glial cells play important roles in neuronal migration. Indeed, glial cells and their processes were identified in the medulla cortex. Glial cells or other cell types could provide cues for neuronal migration (Hasegawa, 2011).

The medulla neurons project axons near their targets forming subsets of dendrites in the larval brain; the cell bodies migrated in the presence of preformed neurites during pupal development. During or following cell body migration, additional dendrites were formed along the axonal shafts. Therefore, cell body migration may somehow contribute to circuit formation in the medulla. Indeed, similar strategies have been reported in sensory neurons of C. elegans and cerebellar granule cells in mammals. Thus, cell body migration in the presence of neurites may be a general conserved mechanism of circuit formation. Cell body migration may also allow developing cells to receive inductive cues provided by cells in the vicinity of the medulla cortex. For example, glial cells placed on the surface of the brain may trigger the expression of specific genes (e.g. ChAT) in Mi1 cells that are located in the outermost area of the adult medulla cortex (Hasegawa, 2011).

In adults, Mi1 neurons have arborization sites at M1 and M5, which coincide with the L1 lamina neuron terminals. In Golgi studies, Mi1 neurons were found in all parts of the retinotopic field. Indeed, the number of Bsh expressing medulla neurons was about 800, a figure similar to the number of ommatidial units. Therefore, the Mi1 neurons identified by Bsh expression are most probably columnar neurons with direct inputs from L1 neurons. Because L1 is known to have inputs from R1-6, which processes motion detection, Mi1 may participate in the motion detection circuit (Hasegawa, 2011).

If the genetic codes that specify each type of neuron are found, it may encourage the functional study of defined neurons. In the medulla, bsh-Gal4 is solely expressed by Mi1 neurons. Although the expression of Bsh is also observed in L4/5 lamina neurons, intersectional strategies such as split Gal4 may enable the activity of Mi1 to be specifically manipulated by inducing expression of neurogenetic tools like shibirets. This could provide insight into high-resolution functional neurobiology in the Drosophila visual system (Hasegawa, 2011).

Development of the mammalian central nervous system reiteratively establishes cell identity, directs cell migration and assembles neuronal layers, processes similar to the patterns observed during medulla development. In the cerebral cortex, neurons are generated within the ventricular or subventricular zones and migrate outwards, leaving their birthplace along the radial glial fibers. Later-born neurons migrate radially into the cortical plate, past the deep layer neurons and become the upper layers. The layers of the cortex are thus created inside-out. In the developing spinal cord, neuronal types are specified according to morphogen gradients. Within each domain along the dorsoventral axis, neuronal and glial types are specified according to their birth order. The spinal cord neurons then migrate extensively along the radial, tangential and rostrocaudal axes. Therefore, the initial organization of spinal cord neurons is disrupted in the mature system (Hasegawa, 2011).

The medulla shares intriguing similarities with the mammalian central nervous system. For example, the concentric zones established in the larval medulla resemble the dorsoventral subdivisions of the spinal cord. Extensive migrations of medulla neurons disrupt concentric zones, as observed in the spinal cord. However, this study found that the locations of cell bodies were organized according to neuronal type, a distribution that may be similar to the cortical organization of the cerebral cortex. Thus, the development of the medulla may share characteristics with various forms of neurogenesis found in the mammalian central nervous system. A comprehensive study of important features of neurogenesis will now be possible using the Drosophila visual center and powerful tools of Drosophila genetics. Unveiling all aspects of development in the medulla will not only shed light into the functional neurobiology of the visual system, but also elucidate the developmental neurobiology of vertebrates and invertebrates (Hasegawa, 2011).

Temporal patterning of Drosophila medulla neuroblasts controls neural fates

In the Drosophila optic lobes, the medulla processes visual information coming from inner photoreceptors R7 and R8 and from lamina neurons. It contains approximately 40,000 neurons belonging to more than 70 different types. This study describes how precise temporal patterning of neural progenitors generates these different neural types. Five transcription factors - Homothorax, Eyeless, Sloppy paired, Dichaete and Tailless - are sequentially expressed in a temporal cascade in each of the medulla neuroblasts as they age. Loss of Eyeless, Sloppy paired or Dichaete blocks further progression of the temporal sequence. Evidence is provided that this temporal sequence in neuroblasts, together with Notch-dependent binary fate choice, controls the diversification of the neuronal progeny. Although a temporal sequence of transcription factors had been identified in Drosophila embryonic neuroblasts, this work illustrates the generality of this strategy, with different sequences of transcription factors being used in different contexts (Li, 2013).

In the developing medulla, the wave of conversion of neuroepithelium into neuroblasts makes it possible to visualize neuroblasts at different temporal stages in one snapshot, with newly generated neuroblasts on the lateral edge and the oldest neuroblasts on the medial edge of the expanding crescent shaped neuroblast region. An antibody screen was conducted for transcription factors expressed in the developing medulla and five transcription factors, Hth, Ey, Slp1, D and Tll, were identified that are expressed in five consecutive stripes in neuroblasts of increasing ages, with Hth expressed in newly differentiated neuroblasts, and Tll in the oldest neuroblasts. This suggests that these transcription factors are sequentially expressed in medulla neuroblasts as they age. Neighbouring transcription factor stripes show partial overlap in neuroblasts with the exception of the D and Tll stripes, which abut each other. Previous studies have reported that Hth and Ey were expressed in medulla neuroblasts, but they had not been implicated in controlling neuroblast temporal identities. Hth and Tll also show expression in the neuroepithelium (Li, 2013).

To address whether each neuroblast sequentially expresses the five transcription factors, their expression was examined in the neuroblast progeny. Hth, Ey and Slp1 are expressed in three different layers of neurons that correlate with birth order, that is, Hth in the first-born neurons of each lineage in the deepest layers; Ey or Slp1 in correspondingly more superficial layers, closer to the neuroblasts. This suggests that they are born sequentially in each lineage. D is expressed in two distinct populations of neurons. The more superficial population inherit D from D+ neuroblasts. D+ neurons in deeper layers (corresponding to the Hth and Ey layers) turn on D expression independently and will be discussed later. Single neuroblast clones were generated, and the expression of the transcription factors was examined in the neuroblast and its progeny. Single neuroblast clones in which the neuroblast is at the Ey+ stage include Ey+ GMCs/neurons as well as Hth+ neurons. This indicates that Ey+ neuroblasts have transited through the Hth+ stage and generated Hth+ neurons. Clones in which the neuroblast is at the D+ stage contain Slp1+ GMCs and Ey+ neurons, suggesting that D+ neuroblasts have already transited through the Slp+ and Ey+ stages. This supports the model that each medulla neuroblast sequentially expresses Hth, Ey, Slp1 and D as it ages, and sequentially produces neurons that inherit and maintain expression of the transcription factor (Li, 2013).

slp1 and slp2 are two homologous genes arranged in tandem and function redundantly in embryonic and eye development. Slp2 is expressed in the same set of medulla neuroblasts as Slp1. Slp1 and Slp2 are referred to collectively as Slp (Li, 2013).

Tll is expressed in the oldest medulla neuroblasts. The oldest Tll+ neuroblasts show nuclear localization of Prospero (Pros), suggesting that they undergo Pros-dependent cell-cycle exit at the end of their life, as in larval nerve cord and central brain neuroblasts. Tll+ neuroblasts and their progeny express glial cells missing (gcm), and the progeny gradually turn off Tll and turn on Repo, a glial-specific marker. These cells migrate towards deeper neuronal layers and take their final position as glial cells around the medulla neuropil. Thus, Tll+ neuroblasts correspond to previously identified glioblasts between the optic lobe and central brain that express gcm and generate medulla neuropil glia. Clones in which the neuroblast is at the Tll+ stage contain Hth+ neurons and Ey+ neurons, among others, confirming that Tll+ neuroblasts represent the final temporal stage of medulla neuroblasts rather than a separate population of glioblasts. Therefore, these data clearly show that medulla neuroblasts sequentially express five transcription factors as they age. The four earlier temporal stages generate neurons that inherit and maintain the temporal transcription factor present at their birth, although a subset of neurons born during the Ey, Slp or D neuroblast stages lose expression of the neuroblast transcription factor. At the final temporal stage, neuroblasts switch to glioblasts and then exit the cell cycle (Li, 2013).

Whether cross-regulation among transcription factors of the neuroblast temporal sequence contributes to the transition from one transcription factor to the next was examined. Loss of hth or its cofactor, extradenticle (exd), does not affect the expression of Ey and subsequent progression of the neuroblast temporal sequence (Li, 2013).

ey-null mutant clones were generated using a bacterial artificial chromosome (BAC) rescue construct recombined on a chromosome containing a Flip recombinase target (FRT) site in an eyJ5.71 null background. eyJ5.71 homozygous mutant larvae were also tested. In both cases, Slp expression is lost in neuroblasts, along with neuronal progeny produced by Slp+ neuroblasts, marked by the transcription factor Twin of eyeless (Toy, see below). However, neuroblast division is not affected, and Hth remains expressed in only the youngest neuroblasts and first-born neurons. Targeted ey RNA interference (RNAi) using a Vsx-Gal4 driver that is expressed in the central region of the neuroepithelium and neuroblasts gives the same phenotype. This suggests that Ey is required to turn on the next transcription factor, Slp, but is not required to repress Hth (Li, 2013).

In clones of a deficiency mutation, slpS37A, that deletes both slp1 and slp2, neuroblasts normally transit from Hth+ to Ey+, but older neuroblasts maintain the expression of Ey and do not progress to express D or Tll, suggesting that Slp is required to repress ey and activate D (Li, 2013).

Similarly, in D mutant clones, neuroblasts are also blocked at the Slp+ stage, and do not turn on Tll, indicating that D is required to repress slp and activate tll. Finally, in tll mutant clones, D expression is not expanded into oldest neuroblasts, suggesting that tll is not required for neuroblasts to turn off D. Thus, in the medulla neuroblast temporal sequence, ey, slp and D are each required for turning on the next transcription factor. slp and D are also required for turning off the preceding transcription factor (Li, 2013).

Gain-of-function phenotypes of each gene were studied. However, misexpression of Hth, Ey, Slp1 or Slp2, or D in all neuroblasts or in large neuroblast clones is not sufficient to activate the next transcription factor or repress the previous transcription factor in neuroblasts. Only misexpressing tll in all neuroblasts is sufficient to repress D expression (Li, 2013).

In summary, cross-regulation among transcription factors is required for at least some of the transitions. No cross-regulation was observed between hth and ey. Because ey is already expressed at low levels in the neuroepithelium and in Hth+ neuroblasts, an as yet unidentified factor might gradually upregulate ey and repress hth to achieve the first transition. As tll is sufficient but not required to repress D expression, additional factors must act redundantly with Tll to repress D (Li, 2013).

The temporal sequence of neuroblasts described above could specify at least four neuron types plus glia (in fact more than ten neuron types plus glia considering that neuroblasts divide several times at each stage with overlaps between neighbouring temporal transcription factors). As this is not sufficient to generate the 70 medulla neuron types, it was asked whether another process increases diversity in the progeny neurons born from a neuroblast at a specific temporal stage. Apterous (Ap) is known to mark about half of the 70 medulla neuron types. In the larval medulla, Ap is expressed in a salt-and-pepper manner in subsets of neurons born from all temporal stages. In the progeny from Hth+ neuroblasts, all neurons seem to maintain Hth, with a subset also expressing Ap. However, only half of the neurons born from neuroblasts at other transcription factor stages maintain expression of the neuroblast transcription factor. For instance, in the progeny of Ey+ neuroblasts, Ey+ neurons are intermingled with about an equal number of Ey neurons that instead express Ap. Neuroblast clones contain intermingled Ey+ and Ap+ neurons. This is also true for the progeny of Slp+ neuroblasts: Slp1+ neurons are intermingled with Slp1 Ap+ neurons. In the progeny of D+ neuroblasts, D and Ap are co-expressed in the same neurons, and they are intermingled with neurons that express neither D nor Ap. Neurons in deeper neuronal layers (corresponding to the Ey+ and Hth+ neuron layers) also express D independently, and these neurons are Ap. The expression of Ap is stable from larval to adult stages (Li, 2013).

The intermingling of Ap+ and Ap neurons raised the possibility that asymmetric division of GMCs gives rise to one Ap+ and one Ap neuron. Two-cell clones were generated to visualize the two daughters of a GMC. In every case, one neuron is Ap+ and the other is Ap-, suggesting that asymmetric division of GMCs diversifies medulla neuron fates by controlling Ap expression (Li, 2013).

Asymmetric division of GMCs in Drosophila involves Notch (N)-dependent binary fate choice. In the developing medulla, the N pathway is involved in the transition from neuroepithelium to neuroblast, and loss of Su(H), the transcriptional effector of N signalling, leads to faster progression of neurogenesis and neuroblast formation. However, Su(H) mutant neuroblasts still follow the same transcription factor sequence and generate GMCs and neuronal progeny, allowing analysis of the effect of loss of N function on GMC progeny diversification. Notably, neurons completely lose Ap expression in Su(H) mutant clones. All mutant neurons born during the Hth+ stage still express Hth, but not Ap, suggesting that the NON daughters of Hth+ GMCs are the neurons expressing both Ap and Hth. In contrast to wild-type clones, all Su(H) mutant neurons born during the Ey+ neuroblast stage express Ey and none express Ap. Similarly, all mutant neurons born during the Slp+ neuroblast stage express Slp1 but lose Ap. These data suggest that, for Ey+ or Slp+ GMCs, the NOFF daughter maintains the neuroblast transcription factor expression, whereas the NON daughter loses this expression but expresses Ap. In the wild-type progeny born during the D+ neuroblast stage, Ap+ neurons co-express D. Both D and Ap are lost in Su(H) mutant clones in the D+ neuroblast progeny, confirming that D is transmitted to the Ap+ NON daughter of D+ GMCs. By contrast, the D+ Ap neurons in the deeper layers (corresponding to the NOFF progeny born during the Ey+ and Hth+ neuroblast stages, see above) are expanded in Su(H) mutant clones at the expense of Ap+ neurons. Therefore, the deeper layer of D expression is turned on independently in the NOFF daughters of Hth+ and Ey+ GMCs (Li, 2013).

Finally, in wild type, a considerable amount of apoptotic cells were observed dispersed among neurons, suggesting that one daughter of certain GMCs undergoes apoptosis in some of the lineages. Together these data suggest that Notch-dependent asymmetric division of GMCs further diversifies neuronal identities generated by the temporal sequence of transcription factors (Li, 2013).

How does the neuroblast transcription factor temporal sequence, together with the Notch-dependent binary fate choice, control neuronal identities in the medulla? Transcription factor markers specifically expressed in subsets of medulla neurons, but not in neuroblasts, were examined including Brain-specific homeobox (Bsh) and Drifter (Dfr), as well as other transcription factors identified in the antibody screen, for example, Lim3 and Toy. Bsh is required and sufficient for the Mi1 cell fate, and Dfr is required for the morphogenesis of nine types of medulla neurons, including Mi10, Tm3, TmY3, Tm27 and Tm27Y (Hasegawa, 2011). Investigation were carried out to identify at which neuroblast temporal stage these neurons were born by examining co-expression with the inherited neuroblast transcription factors. Then whether the neuroblast transcription factors regulate expression of these markers and neuron fates was investigated. The results for each neuroblast stage are described below (Li, 2013).

Bsh is expressed in a subset of Hth+ neurons, suggesting that Bsh is in the NON daughter of Hth+ GMCs. Indeed, Bsh expression is lost in both Su(H) and hth mutant clones. Thus, both Notch activity and Hth are required for specifying the Mi1 fate, consistent with the previous report that Hth is required for the Mi1 fate. Ectopic expression of Hth in older neuroblasts is also sufficient to generate ectopic Bsh+ neurons, although the phenotype becomes less pronounced in later parts of the lineage. These data suggest that Hth is necessary and sufficient to specify early born neurons, but the competence to do so in response to sustained expression of Hth decreases over time. This is similar to embryonic CNS neuroblasts, where ectopic Hb is only able to specify early born neurons during a specific time window (Li, 2013).

Lim3 is expressed in all Ap progeny of both Hth+ and Ey+ neuroblasts. Toy and Dfr are expressed in subsets of neurons born from Ey+ neuroblasts, as indicated by their expression in the Ey+ neuron progeny layer. The most superficial row of Ey+ Ap neurons express Toy (and Lim3), suggesting that they are the NOFF progeny of the last-born Ey+ GMCs. Dfr is co-expressed with Ap in two or three rows of neurons that are intermingled with Ey+ neurons, suggesting that they are the NON progeny from Ey+ GMCs. In addition to these Ap+ Dfr+ neurons, Dfr is also expressed in some later-born neurons that are Ap but express another transcription factor: Dachshund (Dac), in specific sub-regions of the medulla crescent (Li, 2013).

Whether Ey in neuroblasts regulates Dfr expression in neurons was tested. As expected, Dfr-expressing neurons are lost in ey-null mutant clones, suggesting that they require Ey activity in neuroblasts, even though Ey is not maintained in Ap+ Dfr+ neurons. Furthermore, in slp mutant clones in which neuroblasts remain blocked in the Ey+ state, the Ap+ Dfr+ neuron population is expanded into later-born neurons, suggesting that the transition from Ey+ to Slp+ in neuroblasts is required for shutting off the production of Ap+ Dfr+ neurons. In addition, Ap+ Dfr+ neurons are lost in Su(H) mutant clones. Thus, Ey expression in neuroblasts and the Notch pathway together control the generation of Ap+ Dfr+ neurons (Li, 2013).

In addition to its expression with Ey in the NOFF progeny of the last-born Ey+ GMCs, Toy is also expressed in Ap+ (NON) neurons in more superficial layers generated by Slp+ and D+ neuroblasts. Consistently, in Su(H) mutant clones, an expansion of Toy+ Ey+ neurons is seen in the Ey progeny layer, followed by loss of Toy in the Slp and D progeny layer (Li, 2013).

Tests were performed to see whether Slp is required for the neuroblasts to switch from generating Toy+ Ap neurons, progeny of Ey+ neuroblasts, to generating Toy+ Ap+ neurons. Indeed, in slp mutant clones, the Toy+ Ap+ neurons largely disappear, whereas Toy+ Ap neurons expand (Li, 2013).

WAp and Toy expression was examined in specific adult neurons. OrtC1-gal4 primarily labels Tm20 and Tm5 plus a few TmY10 neurons, and these neurons express both Ap and Toy. To examine whether Slp is required for the specification of these neuron types, wild-type or slp mutant clones were generated using the mosaic analysis with a repressible cell marker (MARCM) technique by heat-shocking for 1 h at early larval stage, and the number of OrtC1-gal4-marked neurons in the adult medulla was examined. In wild-type clones, OrtC1-gal4 marks ~100 neurons per medulla. By contrast, very few neurons are marked by OrtC1-gal4 in slp mutant clones. Slp is unlikely to directly regulate the Ort promoter because Slp expression is not maintained in Ap+ Toy+ neurons. Furthermore, the expression level of OrtC1-gal4 in lamina L3 neurons is not affected by slp mutation. These data suggest that loss of Slp expression in neuroblasts strongly affects the generation of Tm20 and Tm5 neurons (Li, 2013).

In summary, these data show that the sequential expression of transcription factors in medulla neuroblasts controls the birth-order-dependent expression of different neuronal transcription factor markers, and thus the sequential generation of different neuron types (Li, 2013).

Although a temporal transcription factor sequence that patterns Drosophila nerve cord neuroblasts was reported more than a decade ago, it was not clear whether the same or a similar transcription factor sequence patterns neural progenitors in other contexts. The current identification of a novel temporal transcription factor sequence patterning the Drosophila medulla suggests that temporal patterning of neural progenitors is a common theme for generating neuronal diversity, and that different transcription factor sequences might be recruited in different contexts (Li, 2013).

There are both similarities and differences between the two neuroblast temporal sequences. In the Hb-Kr-Pdm-Cas-Grh sequence, ectopically expressing one gene is sufficient to activate the next gene, and repress the previous gene, but these cross-regulations are not necessary for the transitions, with the exception of Castor. In the Hth-Ey-Slp-D-Tll sequence, removal of Ey, Slp or D does disrupt cross-regulations necessary for temporal transitions (except the Hth-Ey transition). However, in most cases these cross-regulations are not sufficient to ensure temporal transitions, suggesting that additional timing mechanisms or factors are required (Li, 2013).

For simplicity, the medulla neuroblasts are represented as transiting through five transcription factor stages, whereas in fact the number of stages is clearly larger than five. First, neuroblasts divide more than once while expressing a given temporal transcription factor, and each GMC can have different sub-temporal identities. Furthermore, there is considerable overlap between subsequent temporal neuroblast transcription factors: neuroblasts expressing two transcription factors are likely to generate different neuron types from neuroblasts expressing either one alone (Li, 2013).

Although the complete lineage of medulla neuroblasts is still being investigated, this study shows how a novel temporal sequence of transcription factors is required to generate sequentially the diverse neurons that compose the medulla. The requirement for transcription factor sequences in the medulla and in embryonic neuroblasts suggests that this is a general mechanism for the generation of neuronal diversity. Interestingly, the mammalian orthologue of Slp1, FOXG1, acts in cortical progenitors to suppress early born cortical cell fates. Thus, transcription-factor-dependent temporal patterning of neural progenitors might be a common theme in both vertebrate and invertebrate systems (Li, 2013).

A temporal mechanism that produces neuronal diversity in the Drosophila visual center

The brain consists of various types of neurons that are generated from neural stem cells; however, the mechanisms underlying neuronal diversity remain uncertain. A recent study demonstrated that the medulla, the largest component of the Drosophila optic lobe, is a suitable model system for brain development because it shares structural features with the mammalian brain and consists of a moderate number and various types of neurons. The concentric zones in the medulla primordium that are characterized by the expression of four transcription factors, including Homothorax (Hth), Brain-specific homeobox (Bsh), Runt (Run) and Drifter (Drf/Vvl), correspond to types of medulla neurons. This study examined the mechanisms that temporally determine the neuronal types in the medulla primordium. For this purpose, transcription factors were sought that are transiently expressed in a subset of medulla neuroblasts (NBs, neuronal stem cell-like neural precursor cells) and identified five candidates [Hth, Klumpfuss (Klu), Eyeless (Ey), Sloppy paired (Slp) and Dichaete (D)]. The results of genetic experiments at least explain the temporal transition of the transcription factor expression in NBs in the order of Ey, Slp and D. The results also suggest that expression of Hth, Klu and Ey in NBs trigger the production of Hth/Bsh-, Run- and Drf-positive neurons, respectively. These results suggest that medulla neuron types are specified in a birth order-dependent manner by the action of temporal transcription factors that are sequential ly expressed in NBs (Suzuki, 2013).

In the embryonic central nervous system, the heterochronic transcription factors suchas Hb, Kr, Pdm, Cas and Grh are expressed in NBs to regulate the temporal specification of neuronal identity. They regulate each other to achieve sequential changes in their expression in NBs without cell-extrinsic factors. However, expression of the embryonic heterochronic genes was not detected in the medulla NBs.Instead this study found that Hth, Klu, Ey, Slp and D are transiently and sequentially expressed in medulla NBs. The expression of Hth and Klu was observed in lateral NBs, while that of Ey/Slp and D was observed in intermediate and medial NBs, respectively. These observations suggest that the expression of heterochronic transcription factors changes sequentially as each NB ages, as observed in the development of the embryonic central nervous system (Suzuki, 2013).

This study demonstrates that at least three of the temporal factors Ey, Slp and D regulate each other to form a genetic cascade that ensures the transition from Ey expression to D expression in the medulla NBs. Ey expression in NBs activates Slp, while Slp inactivates Ey expression. Similarly, Slp expression in NBs activates D expression, while D inactivates Slp expression. In fact, the expression of Slp is not strong in newer NBs in which Ey is strongly expressed, but is up regulated in older NBs in which Ey is weakly expressed in the wildtype medulla. A similar relationship is found between Slp and D, supporting the idea that Ey, Slp and D regulate each other's expression to control the transition from Ey-expression to D-expression. In the embryonic central nervous system, similar interaction is mainly observed between adjacent genes of the cascade hb-Kr-pdm-cas-grh, and this concept may also be applied to the medulla primordium. The expression pattern and function of Ey, Slp and D suggest that they are adjacent to each other in the cascade of transcription factor expression in medulla NBs (Suzuki, 2013).

However, no such relationship was found between Hth, Klu and the other temporal factors.The sequential expression of Hth and Klu could be regulated by an unidentified mechanism that is totally different from the genetic cascade that controls the transition through Ey-Slp-D. Or, there might be unidentified temporal factors that are expressed in lateral NBs which act upstream of Hth and Klu to regulate their expression. It is necessary to identify additional transcription factors that are transiently expressed in medulla NBs (Suzuki, 2013).

The expression of concentric transcription factors in the medulla neurons correlates with the temporal sequence of neuron production from the medulla NBs (Hasegawa, 2011). In the larval medulla primordium, the neurons are located in the order of Hth/Bsh-, Run- and Drf-positive cells from inside to outside, and these domains are adjacent to each other (Hasegawa, 2011). Given that NBs generate neurons toward the center of the developing medulla, Hth/Bsh-positive neurons are produced at first, and then Run-positive and Drf-positive neurons. Thus Hth/Bsh, Run and Drf were used as markers to examine roles of Hth, Klu, Ey, Slp and D expressed in NBs in specifying types of medulla neurons. The continuous expression of Hth and Ey from NBs to neurons and the results of clonal analyses that visualize the progeny of NBs expressing each one of the temporal transcription factors suggest that the temporal windows of NBs expressing Hth, Klu and Ey approximately correspond to the production of Hth/Bsh-, Run- and Drf- positive neurons, respectively. Indeed, the results of the genetic study suggest that Hth and Ey are necessary and sufficient to induce the production of Hth/Bsh- and Drf-positive neurons,respectively (Hasegawa, 2011, 2013). Ectopic Klu expression at least induces the produc tion of Run-positive neurons (Suzuki, 2013).

Slp and D expression in NBs may correspond to the temporal windows that produce medulla neurons in the outer domains of the concentric zones, which are most likely produced after the production of Drf-positive neurons. The results at least suggest that Slp is necessary and sufficient and D is sufficient to repress the production of Drf-positive neurons. Identification of additional markers that are expressed in the outer concentric zones compared to the Drf-positive domain would be needed to elucidate the roles of Slp and D in specification of medulla neuron types (Suzuki, 2013).

D mutant clones did not produce any significant phenotype except for derepression of Slp expression in NBs. Drf expression in neurons was not affected either. Since D is a Sox family transcription factor, SoxN, another Sox family transcription factor, is a potential candidate molecule that acts together with D in the medulla NBs. However, its expression was found in neuroepithelia cells and lateral NBs that overlap with Hth-positive cells but not with D-positive cells. All the potential heterochronic transcription factors examined in this study are expressed in three to five cell rows of NBs. Nevertheless, one NB has been observed to produce one Bsh- positive and one Run-positive neuron (Hasegawa, 2011). Therefore, the expression pattern of the heterochronic transcription factors is not sufficient to explain the stable production of one Bsh-positive and one Run-positive neuron from a single NB.The combinatorial action of multiple temporal factors expressed in NBs may play important roles in the specification of Bsh- and Run- positive neurons (Suzuki, 2013).

Another possible mechanism that guarantees the production of a limited number of the same neuronal type from multiple rows of NBs expressing a temporal transcription factor could be a mutual repression between concentric transcription factors expressed in medulla neurons. For example, Hth/Bsh, Run and Drf may repress each other to restrict the number of neurons that express either of these transcription factors. However, expression of Run and Drf was not essentially affected in hth mutant clones and in clones expressing Hth (Hasegawa, 2011). Similarly, expression of Hth and Drf was not essentially affected in clones expressing run RNAi under the control of AyGal4, in which Run expression is eliminated. Hth and Run expression was not affected in drf mutant clones (Hasegawa, 2011). These results suggest that Hth/Bsh, Run and Drf do not essentially regulate each other during the formation of concentric zones in the medulla (Suzuki, 2013).

During embryonic development, the heterochronic genes that are expressed in NBs (hb-Kr-pdm-cas-grh) are maintained and act in GMCs to specify neuronal type. Similarly, Hth and Ey are continuously expressed from NBs to neurons, suggesting that their expression may also be inherited through GMCs (Hasegawa, 2011). However, this type of regulatory mechanism may be somewhat modified in the case of Klu, Slp and D (Suzuki, 2013).

Klu is expressed in NBs and GMCs, but not in neurons. Slp and D are predominantly detected in NBs and neurons visualized by Dpn and Elav, respectively. Occasionally, however, expression of D was found in putative GMCs, which are situated between NBs and neurons. Additionally, both D-positive and D-negative cells were found among Miranda-positive GMCs. Slp expression was not found in Miranda-positive GMCs. Finally, D is expressed in medulla neurons forming a concentric zone in addition to its expression in medial NBs. However, D expression was abolished in slp mutant NBs but remained in the mutant neurons, suggesting that D expression in medulla neurons is not inherited from the NBs. These results suggest that Slp and D expression are not maintained from NBs to neurons and that not all the temporal transcription factors expressed in NBs are inherited through GMCs. However, it is possible to speculate that Klu, Slp and D regulate expression of unidentified transcription factors in NBs that are inherited from NBs to neurons through GMCs (Suzuki, 2013).

Integration of temporal and spatial patterning generates neural diversity

In the Drosophila optic lobes, 800 retinotopically organized columns in the medulla act as functional units for processing visual information. The medulla contains over 80 types of neuron, which belong to two classes: uni-columnar neurons have a stoichiometry of one per column, while multi-columnar neurons contact multiple columns. This study shows that combinatorial inputs from temporal and spatial axes generate this neuronal diversity: all neuroblasts switch fates over time to produce different neurons; the neuroepithelium that generates neuroblasts is also subdivided into six compartments by the expression of specific factors (see The OPC neuroepithelium is patterned along its dorsal-ventral axis). Uni-columnar neurons are produced in all spatial compartments independently of spatial input; they innervate the neuropil where they are generated. Multi-columnar neurons are generated in smaller numbers in restricted compartments and require spatial input; the majority of their cell bodies subsequently move to cover the entire medulla. The selective integration of spatial inputs by a fixed temporal neuroblast cascade thus acts as a powerful mechanism for generating neural diversity, regulating stoichiometry and the formation of retinotopy (Erclik, 2017).

The optic lobes, composed of the lamina, medulla and the lobula complex, are the visual processing centres of the Drosophila brain. The lamina and medulla receive input from photoreceptors in the compound eye, process information and relay it to the lobula complex and central brain. The medulla, composed of ~40,000 cells, is the largest compartment in the optic lobe and is responsible for processing both motion and colour information. It receives direct synaptic input from the two colour-detecting photoreceptors, R7 and R8. It also receives input from five types of lamina neuron that are contacted directly or indirectly by the outer photoreceptors involved in motion detection (Erclik, 2017).

Associated with each of the ~800 sets of R7/R8 and lamina neuron projections are 800 medulla columns defined as fixed cassettes of cells that process information from one point in space. Columns represent the functional units in the medulla and propagate the retinotopic map established in the compound eye. Each column is contributed to by more than 80 neuronal types, which can be categorized into two broad classes. Uni-columnar neurons have arborizations principally limited to one medulla column and there are thus 800 cells of each uni-columnar type. Multi-columnar neurons possess wider arborizations, spreading over multiple columns. They compare information covering larger receptor fields. Although they are fewer in number, their arborizations cover the entire visual field (Erclik, 2017).

The medulla develops from a crescent-shaped neuroepithelium, the outer proliferation centre (OPC). During the third larval instar, the OPC neuroepithelium is converted into lamina on its lateral side and into medulla neuroblasts on its medial side. A wave of neurogenesis moves through the neuroepithelial cells, transforming them into neuroblasts; the youngest neuroblasts are closest to the neuroepithelium while the oldest are adjacent to the central brain. Neuroblasts divide asymmetrically multiple times to regenerate themselves and produce a ganglion mother cell that divides once more to generate medulla neurons. Recent studies have shown that six transcription factors are expressed sequentially in neuroblasts as they age: neuroblasts first express Homothorax (Hth), then Klumpfuss (Klu), Eyeless (Ey), Sloppy-paired 1 (Slp1), Dichaete (D) and Tailless (Tll). This temporal series is reminiscent of the Hb --> Kr --> Pdm --> Cas --> Grh series observed in Drosophila ventral nerve cord neuroblasts that generates neuronal diversity in the embryo. Indeed, distinct neurons are generated by medulla neuroblasts in each temporal window. Further neuronal diversification occurs through Notch-based asymmetric division of ganglion mother cells. In total, over 20 neuronal types can theoretically be generated using combinations of temporal factors and Notch patterning mechanisms. However, little is known about how the OPC specifies the additional ~60 neuronal cell types that constitute the medulla (Erclik, 2017).

To understand the logic underlying medulla development, late larval brains were stained with 215 antibodies generated against transcription factors and 35 genes were identifiied that are expressed in subsets of medulla progenitors and neurons. The OPC neuroepithelial crescent can be subdivided along the dorsal-ventral axis by the mutually exclusive expression of three homeodomain-containing transcription factors: Vsx1 is expressed in the central OPC (cOPC), Rx in the dorsal and ventral posterior arms of the crescent (pOPC), and Optix in the two intervening 'main arms' (mOPC). These three proteins are regionally expressed as early as the embryonic optic anlage and together mark the entire OPC neuroepithelium with sharp, non-overlapping boundaries. Indeed, these three regions grow as classic compartments: lineage trace experiments show that cells permanently marked in the early larva in one OPC region do not intermingle at later stages with cells from adjacent compartments. Of note, Vsx1 is expressed in cOPC progenitor cells and is maintained in a subset of their neuronal progeny whereas Optix and Rx are not expressed in post-mitotic medulla neurons. The OPC can be further subdivided into dorsal (D) and ventral (V) halves: a lineage trace with hedgehog-Gal4 (hh-Gal4) marks only the ventral half of the OPC, bisecting the cOPC compartment. As hedgehog is not expressed in the larval OPC, this dorsal-ventral boundary is set up in the embryo. Thus, six compartments (ventral cOPC, mOPC and pOPC and their dorsal counterparts) exist in the OPC. The pOPC compartment can be further subdivided by the expression of the wingless and dpp signalling genes. Cells in the wingless domain behave in a very distinct manner from the rest of the OPC, and have been described elsewhere (Erclik, 2017).

The Hth --> Klu --> Ey --> Slp1 --> D --> Tll temporal progression is not affected by the compartmentalization of the OPC epithelium; the same neuroblast progression throughout the entire OPC. Thus, in the developing medulla, neuroblasts expressing the same temporal factors are generated by developmentally distinct epithelial compartments (Erclik, 2017).

To test whether the intersection of the dorsal-ventral and temporal neuroblast axes leads to the production of distinct neural cell types, focus was placed on the progeny of Hth neuroblasts, which maintain Hth expression. In the late third instar, Hth neurons are found in a crescent that mirrors the OPC (see Distinct neuronal cell types are generated along the dorsal-ventral axis of the OPC). The NotchON (NON) progeny of Hth+ ganglion mother cells express Bsh and Ap, and they are distributed throughout the entire medulla crescent. In contrast, the NotchOFF (NOFF) progeny, which are BshHth+ neurons, express different combinations of transcription factors, and can be subdivided into three domains along the dorsal-ventral axis: (1) in the cOPC, NOFFHth+ neurons express Vsx1, Seven-Up (Svp) and Lim3; (2) in the pOPC, these neurons also express Svp and Lim3, but not Vsx1; (3) in the ventral pOPC exclusively, these neurons additionally express Teashirt (Tsh). NOFFHth+ cells are not observed in the mOPC. Rather, Cleaved-Caspase-3+ cells are intermingled with Bsh+ neurons. When cell death is prevented, Bsh+Hth+ cells become intermingled with neurons that express the NOFF marker Lim3, confirming that the NOFFHth+ progeny undergo apoptosis in the mOPC (Erclik, 2017).

It was therefore possible to distinguish three regional populations of Hth neurons (plus one that is eliminated by apoptosis) and a fourth population that is generated throughout the OPC. The neuronal identity of each of these populations was identified, as follows. (1) Bsh is a specific marker of Mi1 uni-columnar interneurons that are generated in all regions of the OPC. (2) To determine the identity of Hth+NOFF cOPC-derived neurons, Hth+ single cell flip-out clones were generated (using hth-Gal4) in the adult medulla. The only Hth+ neurons that are also Vsx1+Svp+ are Pm3 multi-columnar local neurons. (3) For Hth+NOFF pOPC-derived neurons, 27b-Gal4 was used; it drives expression in larval pOPC Hth+NOFF neurons and is maintained to adulthood. Flip-out clones with 27b-Gal4 mark Pm1 and Pm2 neurons, as well as Hth- Tm1 uni-columnar neurons that come from a different temporal window. Both Pm1 and Pm2 neurons (but not Tm1) express Hth and Svp. Pm1 neurons also express Tsh, which only labels larval ventral pOPC neurons (Erclik, 2017).

Thus, in addition to uni-columnar Mi1 neurons generated throughout the OPC, Hth neuroblasts generate three region-specific neuronal types: multi-columnar Pm3 neurons in the cOPC; multi-columnar Pm1 neurons in the ventral pOPC; and multi-columnar Pm2 neurons in the dorsal pOPC (Erclik, 2017).

To determine the contribution of the temporal and spatial factors to the generation of the different neuronal fates, the factors were mutated them and whether neuronal identity was lost was examined. To test the temporal axis, hth was mutated. As previously reported, Bsh expression is lost in hth mutant clones. Loss of hth in clones also leads to the loss of the Pm3 marker Svp without affecting expression of Vsx1, indicating that Vsx1 is not sufficient to activate Svp and can only do so in the context of an Hth+ neuroblast. Hth is also required for the specification of Pm1 and Pm2 in the pOPC as Svp and Tsh expression is lost in hth mutant larval clones. Ectopic expression of Hth in older neuroblasts is not able to expand Pm1, 2 or 3 fates (on the basis of the expression of Svp) into later born neurons, although it is able to expand Bsh expression. Thus, temporal input is necessary for the specification of all Hth+ neuronal fates but only sufficient for the generation of Mi1 neurons (see Temporal and spatial inputs are required for neuronal specification in the medulla. ) (Erclik, 2017).

Next, whether regional inputs are necessary and/or sufficient to specify neuronal fates in the progeny of Hth+ neuroblasts was determined. In Vsx1 RNA interference (RNAi) clones, Svp expression is lost in the cOPC but Bsh is unaffected. Additionally, Hth+Lim3+ cells are absent, suggesting that NOFF cells undergo apoptosis in these clones. Conversely, ectopic expression of Vsx1 leads to the expression of Svp in mOPC Hth+ neurons but does not affect Bsh expression. Therefore, Vsx1 is both necessary and sufficient for the specification of Pm3 fates in the larva. However, unlike the temporal factor Hth, Vsx1 does not affect the generation of Mi1 neurons (Erclik, 2017).

In Rx whole mutant larvae and in mutant clones, Svp+Lim3+Hth+ larval neurons (that is, Pm1 and Pm2 neurons) in the pOPC are lost. Additionally, the Pm1 marker Tsh is lost in ventral pOPC Hth+ cells. Consistent with the Vsx1 mutant data, larval Bsh expression is not affected by the loss of Rx. In adults, the Pm1/Pm2 markers (Svp, Tsh and 27b-Gal4) are lost in the medulla (Erclik, 2017).

Ectopic expression of Rx leads to the activation of Svp in mOPC Hth+ neurons, but does not affect the expression of Bsh. It also leads to the activation of Tsh, but only in the ventral half of mOPC Hth+ neurons, suggesting that a ventral factor acts together with Rx to specify ventral fates. Taken together, the above data show that Rx is both necessary and sufficient for the specification of Pm1/2 neurons but (like Vsx1) does not affect the generation of Mi1 neurons (Erclik, 2017).

Finally, the role of the mOPC marker Optix in neuronal specification was examined. In Optix mutant clones, Svp is ectopically expressed in the mOPC, but Bsh expression is not affected. Of note, these ectopic Svp+ neurons fail to express the region-specific Pm markers Vsx1 or Tsh (in ventral clones), which suggests that they assume a generic Pm fate. Conversely, ectopic expression of Optix leads to the loss of Svp expressing neurons in both the cOPC and pOPC but does not affect Bsh. These NOFF neurons die by apoptosis as no Lim3+ neurons are found intermingled with Bsh+Hth+NON neurons. When apoptosis is prevented in mOPC-derived neurons, Svp is not derepressed in the persisting Hth+NOFF neurons, which suggests that Optix both represses Svp expression and promotes cell death in Hth+NOFF neurons (Erclik, 2017).

The above data demonstrate that input from both the temporal and regional axes is required to specify neuronal fates. The temporal factor Hth is required for both Mi1 and Pm1/2/3 specification. The spatial genes are not required for the specification of NON Mi1 neurons, consistent with the observation that Mi1 is generated in all OPC compartments. The spatial genes, however, are both necessary and sufficient for the activation (Vsx1 and Rx) or repression (Optix) of the NOFF Pm1/2/3 neurons. Thus, Hth+ neuroblasts generate two types of progeny: NOFF neurons that are sensitive to spatial input (Pm1/2/3) and NON neurons that are refractory to spatial input (Mi1). Vsx1 expression in the cOPC is only maintained in Hth+NOFF neurons, suggesting that spatial information may be 'erased' in Mi1, thus allowing the same neural type to be produced throughout the OPC (Erclik, 2017).

Do spatial genes regulate each other in the neuroepithelium? In Vsx1 mutant clones, Optix (but not Rx) is derepressed in the cOPC epithelium. Conversely, ectopic Vsx1 is sufficient to repress Optix in the mOPC and Rx in the pOPC. Similarly, Optix, but not Vsx1, is derepressed in Rx mutant clones in the pOPC epithelium and ectopic Rx is sufficient to repress Optix in the mOPC (but not Vsx1 in the cOPC). In Optix mutant clones, neither Vsx1 nor Rx are derepressed in the mOPC epithelium, but ectopic Optix is sufficient to repress both Vsx1 in the cOPC and Rx in the pOPC. The observation that Optix is not necessary to suppress Vsx1 or Rx in the mOPC neuroepithelium is surprising because Svp is activated in a subset of Hth+ neurons in the mOPC in Optix mutant clones. Nevertheless, when cell death in the mOPC is abolished, the ectopic undead NOFF neurons express Lim3 but not Svp, which confirms that Optix represses Svp expression in mOPC neurons. Taken together, these results support a model in which Optix is sufficient to repress Vsx1 and Rx, to promote the death of Hth+NOFF neurons and to repress Pm1/2/3 fates (see Spatial genes cross-regulate each other in the OPC neuroepithelium). Vsx1 and Rx act to promote Pm3 (Vsx1) or Pm1/2 (Rx) fates but can only do so in the absence of Optix (Erclik, 2017).

These results suggest that multi-columnar neurons are generated at specific locations in the medulla crescent. However, since these neurons are required to process visual information from the entire retina in the adult medulla, how does the doral-ventral position of neuronal birth in the larval crescent correlate with their final position in the adult? Lineage-tracing experiments were performed with Vsx1-Gal4 to permanently mark neurons generated in the cOPC and with Optix-Gal4 for mOPC neurons, and the position of the cell bodies of these neurons was analyzed. In larvae, neurons from the cOPC or from the mOPC remain located in the same dorsal-ventral position where they were born. However, in adults, both populations have moved to populate the entire medulla cortex along the dorsal-ventral axis (see Neuronal movement during medulla development is restricted to multi-columnar cell types). The kinetics of cell movement during development was analyzed by following cOPC neurons. Neurons born in the cOPC remain tightly clustered until 20 h after puparium formation (P20), after which point the cell bodies spread throughout the medulla cortex. By P30 the neurons are distributed over the entire dorsal-ventral axis of the medulla cortex. In the adult, most neurons derived from the cOPC neuroepithelium are located throughout the cortex although there is an enrichment of neurons in the central region of the cortex (Erclik, 2017).

To determine whether these observed movements involve the entire neuron or just the cell body, the initial targeting of cOPC or mOPC-derived neurons in larvae was examined before the onset of cell movement. In larvae, both populations send processes that target the entire dorsal-ventral axis of the medulla neuropi. Therefore, medulla neurons first send projections to reach their target columns throughout the entire medulla. Later, remodelling of the medulla results in extensive movement of cell bodies along the dorsal-ventral axis, leading to their even distribution in the cortex (Erclik, 2017).

What is the underlying logic behind why some neurons move while others do not? Markers were studied for the Mi1 (Bsh), Pm2 (Hth+Svp+), Pm1 (Hth+Svp+Tsh+), and Pm3 (Vsx1+Svp+Hth+) populations of neurons through pupal stages and up to the adult. Mi1 neurons are generated evenly throughout the larval OPC and remain regularly distributed across the dorsal-ventral axis at all stages. The lineage-tracing experiment was repeated with Vsx1-Gal4 to follow Mi1 neurons produced by the cOPC. These Mi1 neurons remain exclusively in the centre of the adult medulla cortex, demonstrating that they do not move. In contrast, Pm3 neurons remain tightly clustered in the central region until P20, at which point they move to occupy the entire cortex (Erclik, 2017).

However, not all multi-columnar neurons have cell bodies that move to occupy the entire medulla cortex. Unlike Mi1 and Pm3, adult Pm1 and Pm2 cell bodies are not located in the adult medulla cortex but instead in the medulla rim, at the edges of the cortex. Pm1 and Pm2 markers remain clustered at the ventral (Pm1) or dorsal (Pm2) posterior edges of the medulla cortex throughout all pupal stages. In the adult, both populations occupy the medulla rim from where they send long horizontal projections that reach the entire dorsal-ventral axis of the medulla neuropil. The pOPC may be a specialized region where many of the medulla rim cell types are generated. Even though most of cOPC-derived neurons move during development, a cOPC-derived multi-columnar neuron (TmY14) was identified that sends processes targeting the entire dorsal-ventral length of the medulla neuropil but whose cell bodies remain in the central medulla cortex in the adult (Erclik, 2017).

Thus, the four populations of Hth neurons follow different kinetics: Mi1 neurons are born throughout the OPC and do not move; Pm3 neurons are born centrally and then move to distribute throughout the entire cortex; and Pm1/Pm2 neurons are born at the ventral or dorsal posterior edges of the OPC and occupy the medulla rim in adults (Erclik, 2017).

It is noted that uni-columnar Mi1 neurons, whose cell bodies do not move, reside in the distal cortex whereas multi-columnar Pm3 neurons, which move, reside in the proximal cortex. The hypothesis was thus tested that neurons whose cell bodies are located distally in the medulla cortex represent uni-columnar neurons generated homogeneously throughout the OPC that do not move. In contrast, proximal neurons, which are fewer in number and are generated in specific subregions of the medulla OPC, would be multi-columnar and move to their final position (Erclik, 2017).

It was first confirmed that neurons that move have their cell bodies predominantly in the proximal medulla cortex. The cell body position of neurons born ventrally that have moved dorsally was analyzed using the hh-Gal4 lineage trace: in the adult, the cell bodies found dorsally are mostly in the proximal medulla cortex, whereas the cell bodies in the ventral region are evenly distributed throughout the distal-proximal axis of the ventral cortex. They probably represent both distal uni-columnar neurons that did not move as well as proximal multi-columnar neurons that remained in the ventral region (Erclik, 2017).

Next the pattern of movement of Tm2 uni-columnar neurons from the ventral and dorsal halves of the OPC was analyzed using the hh-based lineage-trace. The cell bodies of Tm2 neurons are located throughout the dorsal-ventral axis in the adult medulla cortex but are co-labelled with the hh lineage marker only in the ventral half. Thus, like Mi1, Tm2 uni-columnar neurons do not move. Furthermore, uni-columnar Tm1 neurons, labelled by 27b-Gal4, are born throughout the dorsal-ventral axis of the OPC crescent with distal cell bodies in the adult, suggesting that they also remain where they were born (Erclik, 2017).

Conversely, it was asked whether neurons that are specified in only one region, such as the Vsx+ neurons of the cOPC, are multi-columnar in morphology. By sparsely labelling cOPC-derived neurons using the Vsx1-Gal4 driver, 13 distinct cell types were characterized that retain Vsx1 expression in the adult medulla. Strikingly, all are multi-columnar in morphology, further supporting the model that it is the multi-columnar neurons that move during pupal development (Erclik, 2017).

Finally, MARCM clones were generated in the OPC neuroepithelium and visualized using cell-type-specific Gal4 drivers in the adult medulla. Two classes of adult clone distribution were observed: clones in which neurons are tightly clustered, and clones in which neurons are dispersed. Consistent with the model, the clustered clones are those labelled with uni-columnar neuronal drivers, whereas the dispersed clones are those labelled with a multi-columnar driver (Erclik, 2017).

Taken together, these data demonstrate that neurons that do not move are uni-columnar (with cell bodies in the distal cortex), whereas most multi-columnar neurons (with cell bodies in the proximal cortex) move (Erclik, 2017).

This study shows that combinatorial inputs from the temporal and spatial axes act together to promote neural diversity in the medulla. Previous work has shown that a temporal series of transcription factors expressed in medulla neuroblasts allows for a diversification of the cell types generated by the neuroblasts as they age. This study now shows that input from the dorsal-ventral axis leads to further diversification of the neurons made by neuroblasts; at a given temporal stage, neuroblasts produce the same uni-columnar neuronal type globally as well as smaller numbers of multi-columnar cell types regionally. This situation is reminiscent of the mode of neurogenesis in the Drosophila ventral nerve cord, in which each neuroblast also expresses a (different) temporal series of transcription factors that specifies multiple neuronal types in the lineage. Spatial cues from segment polarity, dorsal-ventral and Hox genes then intersect to impart unique identities to each of the lineages. However, neuroblasts from the different segments give rise to distinct lineages to accommodate the specific function of each segment. In contrast, in the medulla, the entire OPC contributes to framing the repeating units that form the retinotopic map. It is therefore likely that each neuroblast produces a common set of neurons that connect to each pair of incoming R7 and R8 cells, or L1-L5 lamina neurons. This serves to produce 800 medulla columns with a 1:1 stoichiometry of medulla neurons to photoreceptors. The medulla neurons that are produced by neuroblasts throughout the dorsal-ventral axis of the OPC are thus uni-columnar The production of the same neuronal type along the entire OPC could be achieved by selectively 'erasing' spatial information in uni-columnar neurons, as observed in Mi1 neurons (Erclik, 2017).

Regional differences in the OPC confer further spatial identities to neuroblasts with the same temporal identity, and lead to specific differences in the lineages produced in the compartments along the dorsal-ventral axis of the medulla. These differences produce smaller numbers of multi-columnar neurons whose stoichiometry is much lower than 1:1. The majority of these neurons move during development to be uniformly distributed in the adult medulla cortex. This combination of regional and global neuronal specification in the medulla presents a powerful mechanism to produce the proper diversity and stoichiometry of neuronal types and generate the retinotopic map (Erclik, 2017).

Yki/YAP, Sd/TEAD and Hth/MEIS control tissue specification in the Drosophila eye disc epithelium

During animal development, accurate control of tissue specification and growth are critical to generate organisms of reproducible shape and size. The eye-antennal disc epithelium of Drosophila is a powerful model system to identify the signaling pathway and transcription factors that mediate and coordinate these processes. This study shows that the Yorkie (Yki) pathway plays a major role in tissue specification within the developing fly eye disc, in squamous peripodial portion of the epithelium (PE), at a time when organ primordia and regional identity domains are specified. RNAi-mediated inactivation of Yki, or its partner Scalloped (Sd), or increased activity of the upstream negative regulators of Yki cause a dramatic reorganization of the eye disc fate map leading to specification of the entire disc epithelium into retina. On the contrary, constitutive expression of Yki suppresses eye formation in a Sd-dependent fashion. It was also showm that knockdown of the transcription factor Homothorax (Hth), known to partner Yki in some developmental contexts, also induces an ectopic retina domain, that Yki and Scalloped regulate Hth expression, and that the gain-of-function activity of Yki is partially dependent on Hth. These results support a critical role for Yki - and its partners Sd and Hth - in shaping the fate map of the eye epithelium independently of its universal role as a regulator of proliferation and survival (Zhang, 2011).

The process of regional specification in the eye-antennal disc of Drosophila involves a number of signaling pathways that regulate the expression of identity-defining transcriptional regulators, often referred to as selector factors. This study shows that the Hippo signaling pathway and the transcription factors Yki, Sd, and Hth play critical roles in the regional specification of this disc. Specifically, all three factors are required for the establishment of the peripodial cell layer of the eye disc. Yki function is required in the PE to maintain tissue identity and appears to works in concert with Sd and Hth. Moreover, negative regulation of Yki through the Hippo/Warts genetic pathway is required in the disc proper (DP) to modulate Yki activity such that it promotes proliferation and survival of retina progenitor cells without interfering with their specification. The specification role of Yki and its partners in the eye epithelium is central to proper disc development, occurs in the early stages of regional specification within this disc, and appears to be distinct from its more general role in proliferation and survival (Zhang, 2011).

Yki and Sd have been shown to form a complex and regulate the anti-apoptotic gene Diap1 in the wing and eye discs of Drosophila. In the eye, Yki is also strongly required for proliferation and survival, whereas Sd makes a lesser contribution to these processes (Zhang, 2011).

Does Yki function in a complex with Sd in the context of regional specification within the eye disc and how? The evidence presented in this study shows that (1) the RNAi-induced knock-down of either gene induces essentially identical PE-to-DP transformations and (2) sd-RNAi can suppress the gain-of-function, anti-retina effect of Yki over-expression. These findings, together with the biochemical evidence of protein-protein interactions, suggest that Yki and Sd may indeed control transcription together in a complex during PE specification (Zhang, 2011).

Surprisingly, a comparison of loss-of-function analyses in mosaic discs versus RNAi-mediated disc-wide knock-downs has uncovered a discrepancy in the induced phenotypes. Specifically, loss-of-function clones of either yki or sd do not show signs of cell fate transformation. This is the case for traditional flip-FRT loss-of-function clones generated using mutant alleles as well as for knock-down MARCM clones induced by RNAi expression. Differences in reagents cannot explain this discrepancy, because the same yki-RNAi or sd-RNAi lines were used in disc-wide versus clonal analyses. Hence, this discrepancy must depend on the two different loss-of-function approaches employed. The two approaches differ significantly in two ways: (1) the amount of mutant tissue induced, (2) and the timing of loss-of-gene function (Zhang, 2011).

Thus far, two major functions of Hth have been uncovered in the DP cell layer: 1) Hth, together with Tsh, suppresses the expression of the late retinal determination (RD) genes eya and so, thus slowing down the conversion of early eye progenitors (Ey, Tsh and Hth positive) into more mature (Ey, Tsh, Eya and So positive) eye precursor cells, and 2) Hth together with Tsh and Yki enhances proliferation by inducing expression of the proliferation-promoting microRNA Bantam. The combined effect of these two activities results in the generation of an abundant pool of eye progenitor cells. Protein-protein interactions among these factors have been documented in vitro and/or in vivo, leading to the proposal that two complexes (inclusive of Hth/Tsh and Hth/Yki/Tsh) may perform these major functions (Zhang, 2011).

The data presented in this study uncover another critical function of Hth in the eye disc. In the PE cell layer, fated to give rise to portions of the head cuticle, Hth prevents conversion of this tissue into retina. As in the eye progenitors within the DP, this anti-retina activity of Hth involves suppression of late-RD factors (Eya, So, Dac). However, unlike its role in eye progenitors, it also entails down-regulation of the early factors Ey and Tsh. Thus, Hth suppresses late but not early RD factors in early eye disc progenitors in conjunction with Tsh, whereas it suppresses retina formation at the level if the early factors in the PE through a process that appears to involve Yki and Sd, but not Tsh (whose expression is normally absent from the PE). In agreement with this context dependent role of Hth, hth-RNAi reverses the anti-retina effect of Yki over-expression, at least in part, by relieving Yki suppression of the early retinal determination factor Ey, and expression of exogenous Tsh within the PE cell layer leads to the formation of an ectopic retina (Zhang, 2011).

Lastly, this study has shown that Hth expression is under the genetic, positive control of Yki and Sd, and that neither yki-RNAi nor sd-RNAi can suppress the gain-of-function activity of Hth. These findings, together with the reported association of the Hth and Yki proteins in S2 cells and on the regulatory region of Bantam, suggest a potentially more complex relationship between Hth and Yki in the development of the PE. The observation that Yki and Sd control Hth expression does not preclude the possibility that the Hth protein also functions in a complex with these factors. Indeed, a related example is offered by the retinal determination factor Eya, which first induces and then partners with the Sine oculis to regulate retina development. It is proposed therefore that Hth is one of the direct or indirect targets of a Yki/Sd complex in the PE. Thereafter, association with Hth may modify Yki/Sd activity eventually resulting in the transcriptional silencing of critical retinal determination genes, such as Tsh (Zhang, 2011).

The Hippo/Yki signaling pathway is a general regulator of cell proliferation and survival in metazoans. In a handful of cases, the Hippo pathway has been shown to regulate processes other than proliferation or cell death, such as the maintenance of the undifferentiated state of progenitor cell types in the neural tube and in the gut and, in two cases, aspects of neuronal differentiation. In one, the pathway controls the choice of opsin gene expressed in the R8 cells of the adult fly eye; in the other, it is required for maintenance of the dendritic harbors of body-wall sensory neurons in the Drosophila larva. While transcriptional regulation by Yki is thought to play a role in these non-proliferation-related processes, it is difficult to draw parallels between these cases and what is seen in the eye disc (Zhang, 2011).

Two more recent reports implicate Yki in processes related to tissue specification. In one case, a transient, expanding burst of Yki activity within rows of cells at the border of an already established wing field results in a marginal expansion of the wing primordium, ultimately ensuring proper wing size. In a second example, the YAP/Yki and TEAD4/Sd proteins specify the trophoectoderm as distinct from the inner cell mass (ICM) in mammalian embryos. It is thought that the circumferential cell-cell contacts experienced by ICM cells triggers Hpo activity and cytoplasmic retention of YAP, whereas the less extensive cell-cell contacts experienced by outer cells do not, thus allowing nuclear accumulation of YAP (Zhang, 2011).

Based on similarities with the latter example, it is tempting to hypothesize that less extensive cell-cell contacts in the squamous cell layer, than in the columnar one, would promote a more efficient nuclear localization of Yki in the PE, than in the DP cells. At moderate activity levels in the DP, Yki would then promote proliferation and survival without interfering with retina specification, whereas at higher activity levels in the PE, Yki would be able to not only promote cell proliferation and survival but also PE-identity by suppressing retina formation. This scenario would be consistent with the observed need for down-regulation of Yki activity by Warts in the DP and the stronger expression of Diap1-2-lacZ in the PE. However, the observation that the two cell layers differ significantly in the availability of factors found in Yki-based complexes suggests that 'Yki-complex composition' plays a critical role in the PE versus DP/retina distinction in the developing fly eye (Zhang, 2011).

Yki association with specific co-factors would, therefore, modify the output of the Hpo-Yki pathway to bring about different outcomes in distinct regions of the eye epithelium. A Yki/Hth/Tsh complex would ensure maintenance and expansion of the retina progenitors pool, whereas a Yki/Sd, and possibly Hth, complex would contribute to regional specification within the eye disc by ensuring formation of PE-derived head structures. That Yki/YAP-based complexes can include a variety of different co-factors is supported by other recent examples, including the above mentioned role of Yki, Hth and Tsh in promoting eye progenitors' proliferation in the fly, and its interaction with IRS1 to promote proliferation of neural precursors or with Smad1 to enhance BMP-mediated suppression of neuronal differentiation of embryonic stem cells in the mouse (Zhang, 2011).

In the L2 eye disc, the Sd protein is believed to be available throughout, but does not appear to contribute greatly to Yki-induced proliferation in either cell layer. On the contrary, in the PE, it behaves as a critical co-factor in the control of tissue identity. Unlike Sd, Tsh expression has been shown to be restricted specifically to the DP cell layer. Thus, Yki must perform its PE-promoting tasks in transcriptional complexes that do not include Tsh. Interestingly, misexpression of Tsh within the PE has been shown to induce retina development in this tissue. Whether Tsh does so, at least in part, by diverting Yki activity away from 'anti-retina-fate' functions remains to be determined. Hth, also present broadly in both cells layer at the L2 stage, would play a more general role contributing to Yki's function in both proliferation and tissue specification (Zhang, 2011).

One possible scenario is that the tissue specification functions of Yki in the PE are critically dependent on its association with Sd, and that the availability of Tsh specifically in the DP interferes with the ability of Sd and Yki to associate or work together on fate-determining tasks, thereby preventing any interference with retina formation (Zhang, 2011).

As shown by the paucity of examples, this analysis is still in the early days of deciphering how Yki and its partners regulate cell fate. Nonetheless, this role is apparently separate from its contribution to cell proliferation and survival, and likely involves a number of distinct molecular mechanisms including co-factor specificity and differing levels of Yki activity (Zhang, 2011).

Genetic dissection of photoreceptor subtype specification by the Drosophila melanogaster zinc finger proteins Elbow and No ocelli

The elbow/no ocelli (elb/noc) complex of Drosophila melanogaster encodes two paralogs of the evolutionarily conserved NET family of zinc finger proteins. These transcriptional repressors share a conserved domain structure, including a single atypical C2H2 zinc finger. In flies, Elb and Noc are important for the development of legs, eyes and tracheae. Vertebrate NET proteins play an important role in the developing nervous system, and mutations in the homolog ZNF703 human promote luminal breast cancer. However, their interaction with transcriptional regulators is incompletely understood. This study shows that loss of both Elb and Noc causes mis-specification of polarization-sensitive photoreceptors in the 'dorsal rim area' (DRA) of the fly retina. This phenotype is identical to the loss of the homeodomain transcription factor Homothorax (Hth)/dMeis. Development of DRA ommatidia and expression of Hth are induced by the Wingless/Wnt pathway. The current data suggest that Elb/Noc genetically interact with Hth, and two conserved domains crucial for this function were identified. Furthermore, Elb/Noc specifically interact with the transcription factor Orthodenticle (Otd)/Otx, a crucial regulator of rhodopsin gene transcription. Interestingly, different Elb/Noc domains are required to antagonize Otd functions in transcriptional activation, versus transcriptional repression. It is proposed that similar interactions between vertebrate NET proteins and Meis and Otx factors might play a role in development and disease (Wernet, 2014; PubMed: 24625735).

The transcription factors Homothorax (Hth) and Extradenticle (Exd) have been well characterized as co-factors for Hox genes. Hth/Exd can also act as co-factors for non-Hox transcription factors, like for Engrailed. This study showed that loss of both Elb and Noc phenocopies the loss of Hth at the dorsal rim of the retina. All markers of DRA ommatidia are lost in elb,noc double mutants: Rh3 expression and Sens repression in DRA R8, as well as the DRA-specific inner photoreceptor rhabdomere morphology in DRA R7 and DRA R8. The data shows that Elb/noc act downstream of Hth in the specification of DRA cell fates. Elb and Noc are expressed strongly in DRA R7 and R8. This expression is expanded to all R7 and R8 by ectopic Hth (but never into outer photoreceptors R1-6), while Hth expression is not affected in elb,noc double mutants. One possibility is that Elb/Noc serve as cofactors for Hth/Exd, since Hth loses its potential to induce the DRA fate in a double mutant retina. The vertebrate homologs of Elb and Noc function as repressors of transcription (Nakamura, 2008). Therefore, aspects of the Hth/Exd and Elb/Noc loss-of-function phenotypes could be due to a direct failure of their complex to repress common target genes. For instance, the de-repression of the R8 marker Sens by dominant-negative hthHM, as well as in elb,noc double mutants could be explained by loss of a repressor complex containing all four proteins. Interestingly, functional antagonism between the Hox/Hth/Exd complex and Sens have been described in the Drosophila embryo. However, in this case the factors were shown to compete for overlapping binding sites in the promoter of the common target gene rhomboid. Gene expression profiling data revealed that the Hox gene Abd-B also directly represses Sens in the embryo using Hth/Exd as cofactors. Elb and Noc might therefore provide a missing link for transcriptional repression of Sens by Hth/Exd (Wernet, 2014).

Much work on NET family proteins has focused on functional characterization of their evolutionarily conserved domains. The C-terminus of NET proteins is required for nuclear localization (Pereira-Castro, 2013; Runko, 2004), as well as for self-association of the zebrafish ortholog Nlz1, although neither self-association nor heterodimerization with Nlz2 was found to be necessary for wild type function (Runko ). 'buttonhead box' , a conserved 7-10 amino acid motif which was not investigated in this study, may be required for transcriptional activation (Athanikar, 1997). Deletion of the 'buttonhead box' in zebrafish Nlz proteins transformed them into dominant-negatives, an effect that was proposed to be due to reduced affinity to co-repressor Groucho and histone de-acetylases. Interestingly, deletion of N-terminal sequences, including the Sp/SPLALLA motif also leads to dominant negative proteins. These data are consistent with findings that a protein with a mutated Sp/SPLALLA motif has a dominant-negative effect on DRA specification. The Sp motif was proposed to mediate transcriptional repression by directly binding to cofactors. It should be noted that both N-terminal Sp/SPLALLA deletion and the VP16 fusions have the same dominant-negative effect for zebrafish Nlz1. While this is consistent with a pure repressor function of the zebrafish protein, the differences between Sp/SPLALLA mutation and VP16-fusion (as well as the observation of a phenotype for the Engrailed fusion) reported in this study hint towards a more complex role of Elb and Noc in transcriptional regulation (Wernet, 2014).

This study has shown that mutation of the conserved zinc finger of Elbow also transforms this protein into a dominant-negative. Usually, multiple zinc fingers are required for DNA binding, suggesting that the NET family zinc finger is a protein-protein interaction domain. Deletion of the zinc finger from zebrafish Nlz proteins leads to a loss of nuclear localization, and the Nlz1 zinc finger is necessary for transcriptional repression. Although the possibility that Elb and Noc bind DNA through their zinc finger cannot be excluded, it is likely that mutation of the zinc finger either leads to an inactive complex by sequestration of another co-repressor, or that such complex could be trapped in the cytoplasm. Given that mutation of either Sp/SPLALLA motif or zinc finger both lead to a dominant-negative effect raises the possibility that protein binding to both motifs could be necessary for in vivo function, possibly through the formation of higher order transcriptional complexes (Wernet, 2014).

Loss of both elb and noc does not result in Rhodopsin phenotypes outside the DRA. However, over-expression of different forms of Elb or Noc recapitulates all Rhodopsin phenotypes observed in otdUVI mutants. This phenotype might therefore arise from forcing a direct interaction between over-expressed Elb protein and Otd. Little is known about the regulatory relationship between Elb/Noc and Otd. However, the overlapping expression patterns and similar phenotypes for certain alleles of otd named ocelliless, and for no ocelli (noc) at the anterior pole of the fly embryo, as well as their common requirement in the morphogenesis of ocelli suggests that these proteins also interact positively outside of the retina. The antagonism that was observed might therefore be a dominant-negative effect resulting from sequestration of the Otd protein by over-expressed Elb. Alternatively, different combinations of transcriptional cofactors present between tissues (for instance DRA versus non-DRA R8 cells) might decide whether Elb and Noc act in concert with Otd, or as antagonists (Wernet, 2014).

In the retina, Otd acts in a 'coherent feedforward loop' with Spalt to directly activate transcription of rh3 and rh5. As a consequence, Rh3 and Rh5 are lost in otd mutants. Furthermore, Otd activates transcription of the repressor Dve, forming an 'incoherent feedforward loop', resulting in repression of rh3 and rh5 in outer photoreceptors. Since rh6 is activated by a distinct factor, Pph13, loss of Otd leads to a specific de-repression of rh6 into outer photoreceptors. This study shows that different domains of Elb specifically interfere with different aspects of Otd function in these feedforward loops. Mutation of the Groucho-binding motif FKPY only abolishes the ability of over-expressed Elbow protein to antagonize Otd function in repressing rh6 in outer photoreceptors, while mutation of the Sp/SPLALLA motif specifically antagonizes Otd function in activating both rh3 and rh5, without affecting repression of rh6 in outer photoreceptors (mediated by induction of Dve). Furthermore, while the Elb zinc finger is also required for antagonizing the function of Otd in outer photoreceptors, it is also necessary for antagonizing activation of rh3 by Otd, but not rh5. Hence, these two activator functions of Otd could be separated by mutating the zinc finger (Wernet, 2014).

The different Rhodopsin phenotypes caused by loss of Otd can be mapped to different protein domains. The current data therefore reveal specific genetic interactions between the protein domains of Elb/Noc and Otd. Such interactions could be direct or be mediated through additional proteins. For instance, the Otd C-terminus mediates the repression of rh6 in outer photoreceptors, making it a possible interaction domain for Groucho binding to the Elb/Noc FKPY motif. The N-terminus of Otd is necessary for most activation potential on rh3, while activation of rh5 predominantly maps to the C-terminus. This correlates well with the Rhodopsin-specific phenotypes seen after mutation of Sp/SPLALLA (affecting rh3 and rh5), or the zinc finger (affecting rh3 and rh6) motifs. Finally, the results using VP16- and en[R]-fusions of Noc show that potentially direct transcriptional effects on rhodopsin genes can only be induced in R8 cells. Both fusion proteins specifically regulate expression of rh5, while all other rhodopsins remain unaffected. Elb and Noc are both expressed strongly in R8 cells outside of the DRA where they may contribute the repression of Rh5. The absence of a non-DRA R8 rhodopsin phenotype in elb,noc double mutants, as well as the R8-specific action of VP16:noc could therefore be due to the existence of redundant, R8-specific factors required for Elb/Noc function there, but not for DRA specification. These factors remain unknown, since it was found that expression of elb and noc is not altered in homozygous mutants affecting p/y cell fate decisions in R8 cells (melt and wts (Wernet, 2014).

Mutations in the human Elb/Noc homolog ZNF703 promote metastasis (Slorach, 2011). This study has shown that over-expression of both human NET family proteins UAS-ZNF503 and UAS-ZNF703 in the Drosophila retina result in weak co-expression of Rh5 and Rh6, resembling over-expression of a VP16:noc protein. It is therefore possible that the genetic interaction of NET family proteins with Otd/Otx proteins is evolutionarily conserved, especially since a central domain of Otd was previously shown to mediate mutual exclusion of Rh5 and Rh6 (McDonald, 2010). This study presents a new role for Drosophila NET proteins in retinal patterning. Both zebrafish homologs of Elb/Noc, Nlz1 and Nlz2 are also required for optic fissure closure during eye development (Brown, 2009). Furthermore, expression of the Elb/Noc mouse homologue znf503 suggests that NET family genes are involved in the development of mammalian limbs(McGlinn, 2008). Given previous reports from Drosophila on the proximo-distal specification of leg segments, it appears that NET family members act in similar processes across species. This raises the possibility that NET proteins serve as evolutionarily conserved modules that have been re-utilized for analogous processes during evolution. Based on the current data, their conserved domain structure might be crucial for interacting with transcription factor networks involving conserved families of factors like Otx or Meis. Given their medical relevance in breast cancer, a better understanding of the role NET proteins play in the transcriptional control of tissue patterning will be of great importance (Wernet, 2014).

Functional dissection of the splice variants of the Drosophila gene homothorax (hth)

TALE-homeodomain family member Homothorax interacts with a second TALE-homeodomain protein Extradenticle to facilitate Exd entrance to the nucleus. The many different functions described for Hth rely on the complexity of the locus, from which six different isoforms arise. The isoforms can be grouped into full-length and short versions, which contain either one or the two conserved domains of the protein (homeodomain and Exd-interacting domain). This study used molecular and genetic tools to analyze the levels of expression, the distribution and the function of the isoforms during embryonic development. The results clearly show that the isoforms display distinct levels of expression and are differentially distributed in the embryo. This detailed study also shows that during normal embryonic development not all the Hth isoforms translocate Exd into the nucleus, suggesting that both the proteins can also function separately. The full-length Hth protein activates transcription of exd, augmenting the levels of exd mRNA in the cell. The higher levels of Exd protein in those cells facilitate its entrance to the nucleus. This work demonstrates that hth is a complex gene that should not be considered as a functional unit. The roles of the different isoforms probably rely on their distinct protein domains and conformations and, at the end, on interactions with particular partners (Corsetti, 2013).

Effects of Mutation or Deletion

Loss of homothorax function results in severe head defects, including a failure of head involution, and in the transformation of the thoracic and abdominal segments into a more posterior identity. For example, in homozygotes of one allele, the denticle belts present in the thoracic segments have an abdominal-like morphology, and the first abdominal segment is transformed into an identity that resembles the fifth abdominal segment. Similar posterior-directed transformations are seen in other combinations of hth alleles. In the strongest allelic combination, segmental fusions are observed in addition to these transformation (Rieckhof, 1997).

Mutations in homothorax (hth) have pleiotropic effects on embryonic development. Cuticle preparations of loss-of-function alleles of hth reveal defects in segmentation and head involution. The head skeleton is reduced or absent. Thoracic segments appeared deranged and the thoracic denticle belts are eliminated. Abdominal denticle belts appear more dispersed and less differentiated than normal and exhibit a weak engrailed-like phenotype. In addition, the width of the denticle belts in anterior abdominal segments appears reduced and similar to that of the seventh or eighth abdominal segment. Two distinct phenotypes are evident in the ventral nerve cord (VNC) of mutant embryos: substantial widening of the VNC in thoracic segments and abnormal scaffold of CNS axons throughout the VNC. The longitudinal pathways are absent or reduced in all thoracic and abdominal segments. The anterior commissure is present, but the posterior commissure is often reduced. The spacing between the anterior and posterior commissures is also reduced, most notably in thoracic segments. Another phenotype observed in the CNS of hth mutant embryos is the outgrowth of multiple nerve roots on each side of the CNS, as compared to two nerve roots in wild-type embryos (Kurant, 1998).

Mutations in homothorax (also known as dorsotonals) seem to alter the identity of the abdominal chordotonal neurons, which depend on Abd-A for their normal development. However, these mutations do not alter the expression of the abd-A gene, suggesting that hth may be involved in modulating abd-A activity. In wild-type embryos, the LCh5 neurons are located invariably in the lateral PNS cluster of abdominal segments A1-A7. In contrast, these neurons are situated in a more dorsal position in (respectively) either 25% or 36% of the abdominal segment in the PNS of embryos homozygous for hth H321 (n=91) or hth J186 (n=56). The affected Ch neurons remain associated with the dorsal PNS cluster, or occasionally, are positioned between the dorsal and lateral PNS clusters. The orientation of the affected neurons is also abnormal. Whenever the affected LCh5 neurons remain associated with the dorsal PNS cluster, their dendrites point ventrally or posteriorly instead of dorsally. The 'dorsal chordotonals' phenotype can be detected in all the abdominal segments in varying frequencies. In weak alleles, it is observed more frequently in the posterior abdominal segments (A5-A7). Stronger alleles affect all the abdominal segments in similar frequencies. Weak hth alleles do not affect any PNS neurons other than the LCh5 neurons. Strong hypomorphic mutations in hth affect not only the position and orientation of the LCh5 neurons, but also cause a reduction in their number. Only three dorsal Ch neurons are observed in nearly 100% of abdominal segments of mutants for strong alleles. Most of the affected neurons remain associated with the dorsal PNS cluster; their dendrites point ventrally. In spite of their abnormal location and orientation, the affected Ch neurons appear fully differentiated, as judged by their overall morphology and the presence of normal-looking scolopales at the tips of their dendrites. The precursors of the LCh5 neurons are born in a normal dorso-lateral position in hth mutant embryos. In the dorsal cluster one dorsal ES neuron and 2-3 Cut-negative MD neurons are lost. The ventral Ch neurons are only rarely lost in strong mutants (Kurant, 1998).

A similar phenotype was observed in embryos homozygous for mutations in the homeotic selector gene abd-A. In the absence of abd-A activity, the LCh5 neurons are transformed into DCh3 neurons, and as such they remain associated with the dorsal PNS cluster and their dendrites pointed ventrally. Since the PNS phenotype associated with loss of hth function suggests a homeotic transformation of LCh5 neurons towards the identity of DCh3 or A8-LCh3 neurons, which do not depend on abd-A for their development, the expression pattern of the Abd-A protein was examined in hth mutant embryos. Abd-A is normally expressed in the ectoderm of abdominal segments from PS7 to the anterior region of PS13. In addition, Abd-A is expressed in the LCh5 neurons of segments A1-A7 and in the VNC in segments A2-A7. The spatial distribution of the Abd-A protein is not altered in the ectoderm or CNS of embryos homozygous for the hth K1-8 allele as compared to wild-type embryos, although a slight reduction in the level of the protein is observed. It is concluded that hth may be required for the activity of Abd-A, rather than its expression. A similar dorsal chordotonal phenotype is found in extradenticle mutants (Kurant, 1998)

Why do the LCh5 neurons remain dorsal in the absence of hth activity? Although the process of Ch organ migration and rotation is not understood, the system can be divided conceptually into two components: the neuronal cells and their environment (or the receiving and signaling components of the pathway, respectively). Two scenarios can be envisioned that are not mutual exclusive. One is that hth affects the homeotic identity of the LCh5 neurons themselves. The other possibility is that hth affects the environment in which these neurons form and migrate. In midgut development abd-A andUbx, which are expressed in neighboring parasegments of the visceral mesoderm, regulate dpp and wingless expression, which affects the underlying endoderm. It is possible that the influence of HTH and EXD on Abd-A activity in the ectoderm affects signaling molecules such as Wingless and DPP, which in turn affect the localization of the Ch neurons. For example, the dpp gene controls tracheal cell migration along the dorso-ventral axis of the embryo. Support for this idea comes from phenotypic analysis of hth mutations in adult flies. Loss of hth activity in eye imaginal discs results in ectopic eye formation in the ventral head tissue, whereas ectopic expression of hth suppressed normal eye development (Pai, 1998). These phenotypes are consistent with a role for Hth protein in activating Wingless and/or repressing Dpp signaling. (Kurant, 1998).

This work describes the structure of the hth locus, the characterization at the molecular level of a collection of mutant alleles of hth, and discusses the correlation between the identified structural defects and their consequent phenotypes. The hth locus spans more than 100 kb and contains 14 exons. Several of the exon-intron boundaries within the homeodomain and the MH domain-coding regions are conserved between Drosophila and C. elegans. The analysis of hth mutations demonstrates that the homeodomain of Hth is not required for nuclear localization of Exd and that the MH domain-containing first 240 residues are sufficient for nuclear localization of both Exd and Hth. Mutations that alter or delete the homeodomain cause only partial homeotic transformations in the PNS, whereas mutations affecting the MH domain cause distinct and more severe PNS phenotypes. These observations suggest that driving nuclear localization of Exd is the main role of Hth in patterning the embryonic PNS. They also suggest that homeodomain-defective Hth protein retains some of its transcription-regulating functions by binding DNA via its interaction with Exd (Kurant, 2001).

The Extradenticle (Exd) protein in Drosophila acts as a cofactor to homeotic proteins. Its nuclear localization is regulated. The Drosophila homothorax (hth) gene is a homolog of the mouse Meis1 proto-oncogene, which has a homeobox related to that of exd. Comparison with Meis1 finds two regions of high homology: a novel MH domain and the homeodomain. In imaginal discs, hth expression coincides with nuclear Exd. hth and exd also have virtually identical, mutant clonal phenotypes in adults. These results suggest that hth and exd function in the same pathway. hth acts upstream of exd and is required and sufficient for Exd protein nuclear localization (Pai, 1998).

Mutant hth clones on ventral head tissue (from antenna to postorbital bristles) result in ectopic eye formation, some of which develop at the tips of tubular outgrowths. However, hth mutant clones do not show morphological phenotypes in either their dorsal head structures or within their compound eyes. When clones cross the eye border, the shape of the eye can become distorted. In the eye-antenna discs of late third instar larvae bearing hth mutant clones, ectopic photoreceptor differentiation and local overgrowth can be detected. Consistent with the adult phenotypes, these ectopic photoreceptors are found only in the ventral margin of the eye-antenna disc. Clones in the dorsal margin of the eye disc do not lead to ectopic photoreceptor development. Mutant clones on the second or third antennal segments result in transformation to leg-like structures, with the larger clones giving a clear claw structure, indicative of a distal leg. Mutant clones on the coxa, femur, or tibia often cause fusion of these leg segments, whereas those on the tarsal segments are morphologically normal. The structures affected by hth mutant clones are limited to the proximal antenna and leg segments, consistent with the expression of hth in the proximal region but not in the distal region of the antenna and leg discs. Clones on the mesonotum and abdomen do not have significant morphological phenotypes (Pai, 1998).

It is concluded that hth and exd are both negative regulators of eye development; their mutant clones caused ectopic eye formation. Targeted expression of hth, but not of exd, in the eye disc abolishes eye development completely. It is suggested that hth acts with exd to delimit the eye field and prevent inappropriate eye development (Pai, 1998).

The Drosophila wing imaginal disc gives rise to three body parts along the proximo-distal (P-D) axis: the wing blade, the wing hinge and the mesonotum. The more distal portion of the hinge is continuous with the wing blade, but contains three identifiable structures: the costa (Co), the radius (Ra) and the allula (Al). A second, more proximal part of the hinge (or axillary region), is morphologically demarcated from the rest of the wing and consists of several sclerites (Scl), which are mostly devoid of trichomes, and the axillary cord (aCrd). The tegula (Te), although positioned just anterior to the sclerites, fate maps in the wing disc to a distinct and more dorso-proximal region than these hinge structures, and therefore is not considered a part of the hinge. Correspondingly, the distalmost portion of third instar wing discs is referred to as the wing pouch, which will give rise to the wing blade. Surrounding the wing pouch is a region that will give rise to the hinge and, more proximally, there is a large dorsal territory that will give rise to the mesonotum (mnt) and a thin ventral region that gives rise to the pleura (pl) (Casares, 2000).

Several genes are known to be expressed in the wing pouch including vestigial (vg), scalloped (sd), nubbin (nub) and Distal-less (Dll), which encode transcription factors, and four-jointed (fj), which encodes a putative secreted factor. Development of the wing blade initiates along part of the dorsal/ventral (D/V) compartment boundary and requires input from both the Notch and wingless (wg) signal transduction pathways. wg is expressed along the D/V compartment boundary within the wing blade and in two concentric rings that surround the wing blade region. The rings of wg expression have been fate mapped to the adult hinge and, using a wg-lacZ reporter gene, they map within the hinge as follows: the outer wg ring (OR) maps to the proximal hinge, and the inner wg ring (IR) stains structures in the distal hinge, including the medial costa (mCo), distal radius (dRa) and part of the allula (Al). hth is also highly expressed in the wing hinge region of third instar wing discs, straddling both wg rings. Using a hth-lacZ reporter gene, hth expression maps to the same structures in the adult hinge as does wg. In late third instar wing discs, teashirt (tsh), which encodes a Zn-finger transcription factor, is strongly expressed in cells that are more proximal than hth-expressing cells, although low levels of tsh and hth overlap in the proximal hinge region. Consistent with this expression pattern, tsh-expressing cells fate map in the adult to the axillary sclerites and pleura (Casares, 2000).

In order to examine the role that hth plays in the wing disc the consequences of both removing hth activity and ectopically expressing hth during development were examined. To remove hth activity hth minus clones were generated by mitotic recombination. In hth - clones in the adult that are within the hinge region, hinge structures are severely disrupted or absent. Specifically, the radius, axillary cord, sclerites, proximal and medial costa and allula do not form in the absence of hth. In contrast, the tegula and distal costa are formed in the absence of hth. Similar phenotypes have been observed in the absence of extradenticle (exd) function, consistent with the role that hth plays in the nuclear localization of the exd gene product. Ectopic expression of hth in the wing pouch, via the Gal4 method, reduces the wing to a rudiment. Driving hth expression with the 1096- Gal4 driver line, which is expressed primarily in the dorsal compartment of the wing disc, results in winglets that, on the dorsal surface, have three types of tissue: (1) an apparent extension of distal hinge tissue that is similar to the radius (by the density and size of trichomes), (2) an unpigmented transparent cuticle that may be sclerital tissue, and (3) a small amount of D/V boundary tissue. This phenotype is interpreted as resulting from a repression of wing development and a partial transformation of wing into radius and sclerite tissues. Together with the loss-of-function phenotypes, these data suggest that hth is required for hinge development and, in some contexts, is sufficient to specify hinge structures (Casares, 2000).

Additional experiments presented here suggest that tsh collaborates with hth to interfere with Notch's ability to activate wg at the D/V boundary. During wild-type wing disc development, both hth and tsh are coexpressed in all non-wing blade cells and, at least in the posterior compartment, the D/V boundary expresses vg but not wg. Consistent with these wild-type expression patterns, the combination of Hth plus Tsh is sufficient to completely block wg expression at the D/V boundary in the wing blade. In contrast, vg is still expressed at the D/V boundary in the presence of both Hth and Tsh. It is suggested that the repression of wg by Hth and Tsh represents a normal function of these two proximally expressed transcription factors. The results further suggest that hth is necessary for this repression, because wg is derepressed in hth minus clones that straddle the D/V boundary (Casares, 2000).

In summary, these experiments demonstrate that hth plays at least two roles in wing development. (1) hth is required to limit where, along the D/V boundary, the wing blade will form. It is suggested that hth carries out this function at least in part by interfering with Notch's ability to activate wg. In addition, it is possible that hth also interferes with Wg's ability to activate the vg quadrant enhancer. These results further suggest that, in wild-type wing discs, hth works together with tsh to block wing blade development. (2) hth is required for the identity of the proximal wing (the hinge), because in the absence of hth function, the hinge cannot form. It is of interest that both of these functions have parallels in leg development, where hth is also required for proximal appendage identities, and also interferes with the activities of signaling pathways (Casares, 2000).

The proximal distal axis of the Drosophila leg is patterned by expression of a number of transcription factors in discrete domains along the axis. The homeodomain protein Homothorax and the zinc-finger protein Teashirt are broadly coexpressed in the presumptive body wall and proximal leg segments. Homothorax has been implicated in forming a boundary between proximal and distal segments of the leg. Evidence is presented that Teashirt is required for the formation of proximal leg segments, but Tsh has no role in boundary formation (Wu, 2000).

The leg disc consists of a single epithelial sheet in which the presumptive distal segments are specified in the center and the presumptive proximal segments are specified in the periphery. Cross-sections show that proximal segments, which express Hth and Tsh, fold back over the distal segments, which express Dll and Dac. Hth and Tsh expression is limited to the proximal region of the disc through repression by the combined activities of Wg and Dpp. Although the Hth and Tsh expression domains overlap through much of the proximal region, Hth expression extends more distally than Tsh. This is visible as a band of Hth expression that does not overlap Tsh in a basal optical section. This band coincides with the outer ring of Dll expression. The Tsh domain overlaps the proximal edge of the Dll ring by one or two cells. Tsh expressing cells are also found beneath the disc epithelium. Their location suggests that these may be adepithelial cells. Hth functions as a repressor to modulate Tsh expression. More distally located hth mutant clones lose Tsh expression. Loss of Tsh expression in hth correlates with ectopic expression of Dachshund. hth mutant clones cause ectopic expression of Dac close to the endogenous Dac domain, but do not do so in more proximal regions. The differential effect on Dac expression of hth clones located at different positions along the PD axis has been attributed to a role of Hth as a repressor of Wg and Dpp signaling. Thus the paradoxical loss of Tsh in more distal hth clones can be explained as an indirect effect of Hth on Dac expression. Dac can repress both Tsh and Hth when overexpressed. Thus the different distal limits of the Hth and Tsh expression domains presumably reflect a difference in their sensitivity to repression by Dac. The observation that Tsh levels increase in proximal hth clones suggests that Hth serves as a repressor of Tsh. Thus Hth modulates Tsh expression levels in the proximal leg in two ways. Hth may act directly to reduce Tsh expression levels in the proximal leg, and indirectly via repression of Dac to define the distal limit of Tsh expression (Wu, 2000).

To assess the role of Tsh in development of the proximal leg the phenotypes of adult viable mutant alleles of tsh were examined. tshGAL4 is a weak allele caused by insertion of the GAL4 enhancer-trap P-element. The trochanter is strongly reduced in legs of flies homozygous for tshGAL4. The coxa is reduced and lacks most of the bristles and sense organs found in wild-type. The femur is short, but contains the normal complement of proximal sense organs (including the sc11 group of campaniform sensillae that is normally located at the joint between femur and trochanter. Although the trochanter is reduced, joints can still be seen between coxa, trochanter and femur segments. One or two sensilla trichodea are generally found at the articulation between the reduced trochanter and coxa segments (there are normally two groups of 5-7 sensilla trichodea at this position in wild-type). The tibia and tarsal segments appear to be normal in tsh mutants. In a stronger mutant combination, tshGAL4/tshHD1 the trochanter is no longer detectable as a discrete segment and the coxa appears to articulate directly with the femur. Both coxa and femur are reduced in size. No sense organs can be recognized on the coxa and trochanter rudiment, but the sc11 group of campaniform sensillae was reliably found on the proximal femur where it articulates with the coxa. The shortening of the femur in the strong tsh mutant combination suggests that defects in the trochanter and coxa may have non-autonomous effects on femur development. This may reflect the finding that many of the cells that contribute to femur development originate in the Tsh-expression domain at earlier stages of development and are displaced distally as the disc grows (Wu, 2000).

Reducing Tsh activity produces a phenotype quite distinct from that of removing Hth activity. Tsh is required for the development of trochanter and coxa but does not appear to have a role in segment boundary formation. Hth and its partner Extradenticle are required to prevent fusion of coxa and trochanter with the femur. To better understand the basis for the defects in tsh mutant legs, Hth, Dll and Dac expression were studied in tshGAL4/tshHD1 and tshGAL4/tshGAL4 mutant discs. In wild-type discs Hth and Dac expression overlap in the proximal ring of Dll expression. Hth function in this ring is required for the affinity boundary between proximal and distal regions of the leg. This basic relationship holds in the tshGAL4/tshHD1leg disc. Hth, Dll and Dac expression overlap, and the affinity boundary between proximal and distal leg segments appears to be intact. The principal difference in these discs is expansion of the Dll domain into the proximal, Hth-expressing region. The spatial relationship between Hth and Dac is normal. Ectopic Dll expression is not sufficient to repress Hth but does appear to reduce the size of the coxa and trochanter and to cause problems that result in loss of pattern elements from the remaining portions of these segments. Even slight reductions in Tsh activity causes loss of sensory bristles from the coxa. In contrast, small clones of hth mutant cells are capable of differentiating bristles (Wu, 2000).

Taken together, these observations suggest that Tsh and Hth have distinct functions in the proximal leg. Hth limits the proximal extent of Dac expression, and is required for the affinity boundary between trochanter and femur. Tsh limits the proximal extent of Dll expression and is required for proper growth and differentiation of proximal segments, but does not appear to have a role in PD boundary formation (Wu, 2000).

The morphological diversification of appendages represents a crucial aspect of animal body plan evolution. The arthropod antenna and leg are homologous appendages, thought to have arisen via duplication and divergence of an ancestral structure. To gain insight into how variations between the antenna and the leg may have arisen, the epistatic relationships among three major proximodistal patterning genes, Distal-less, dachshund and homothorax, have been compared in the antenna and leg of Drosophila. Drosophila appendages are subdivided into different proximodistal domains specified by specific genes, and limb-specific interactions between genes and the functions of these genes are crucial for antenna-leg differences. In particular, in the leg, but not in the antenna, mutually antagonistic interactions exist between the proximal and medial domains, as well as between medial and distal domains. The lack of such antagonism in the antenna leads to extensive coexpression of Distal-less and homothorax, which in turn is essential for differentiation of antennal morphology. Furthermore, a fundamental difference between the two appendages is the presence in the leg and absence in the antenna of a functional medial domain specified by dachshund. These results lead to a proposal that the acquisition of particular proximodistal subdomains and the evolution of their interactions has been essential for the diversification of limb morphology (Dong, 2001).

Each segment in the Drosophila leg is considered to be homologous to part or all of a segment in the antenna. The correspondences are based on reproducible homeotic transformations that can occur between parts of the two limbs. Such correlation enables a comparison of the expression domains of Dll, dac and hth between the antenna and the leg. The relative wild-type expression of these three important PD patterning genes of the leg differs in the antenna, indicating that their PD axes are differentially subdivided (Dong, 2001).

For example, at late third instar, Dll expression extends more proximally in the antenna into regions homologous to the leg trochanter. In addition, dac is expressed at lower levels and is expressed in fewer segments in the antenna than in the leg. The dac expression domain in the antenna lies completely within the Dll expression domain. In contrast, the dac and Dll domains in the leg are exclusive when dac expression is activated and remain largely non-overlapping at late third instar. hth is expressed only proximally in the leg, but is expressed throughout the antenna disc until early larval stages when it is lost from distal cells. Because Dpp and Wg, which regulate Dll, dac and hth in the leg, are similarly expressed in the antenna, it is thought unlikely that the differences in Dll, dac and hth expression could be accounted for by variations in Dpp and Wg expression. Instead, it is hypothesized that the differences are due to limb type-specific interactions between Dll, dac and hth. The results of experiments described here confirm that this is the case (Dong, 2001).

Gradients of the morphogens, Wg and Dpp, initiate the PD organization of the Drosophila leg by activating Dll and repressing dac distally and by repressing hth in the distal and medial leg. This creates three domains, distal, medial and proximal, that are specified by the expression Dll, dac and hth, respectively. The expression of dac is derepressed in clones of Dll-null cells in the presumptive distal region of the leg disc. The reciprocal is observed in dac null clones, where Dll expression expands into the medial domain. Mutually repressive interactions between the distal and medial domains therefore are required to keep these domains distinct from one another (Dong, 2001).

The interactions between proximal and medial domains were analyzed. dac is only rarely derepressed in hth-null clones, and ectopic expression of Hth is insufficient to downregulate dac expression in the medial leg. Thus, proximal-to-medial antagonism does not occur via hth. However, ectopic expression of a second proximal leg gene, tsh, can repress dac, and dac expression expands proximally in tsh hypomorphic leg discs. Proximal-to-medial antagonism therefore does occur in the Drosophila leg. Derepression of tsh expression in the dac-null clones has not been observed, but derepression of hth in dac-null clones has been observed. It is therefore concluded that mutually antagonistic interactions between the proximal and medial domains occur via the repression of dac by Tsh and repression of hth by Dac (Dong, 2001).

If the antenna is homologous to the leg, one might expect to find many genetic parallels, particularly with respect to the three major PD patterning genes of the leg, Dll, dac and hth. As in the leg, Dll and hth are required to specify the distal and proximal domains of the antenna. However, dac has a different function in the antenna. No deletions of antennal segments are observed in dac-null flies. In addition, the genetic relationships between Dll, dac and hth are different in the developing antenna. Specifically, the extensive overlap in expression of these three genes in the antenna indicates that domains are not kept separated as they are in the leg. The normal expression domain of dac in the antenna lies completely within an area of hth and Dll coexpression, making it unlikely that dac represses either gene. Nonetheless, because Dll and hth appear to have slightly lower levels of expression where dac is normally expressed, a test was performed to see whether either Hth or Dll levels would be elevated if dac were removed. No detectable changes in the levels of either Dll or Hth were observed in clones of cells that lack Dac. Therefore unlike the situation in the leg, Dac is insufficient to antagonize the expression of either Dll or hth in the antenna. Taken together, these data indicate that mutual antagonism is not a universal feature of appendage development (Dong, 2001).

The antennal regulation of dac by Dll also differs from that of the leg. The regulation of dac by Dll in the antenna varies depending on the proximodistal location. Dll can be a dac repressor or activator, or exert no effect on dac. Dac expression is not activated in Dll-null clones in the presumptive arista, whereas Dll-null clones in the presumptive base of the arista (segments a4 and a5) exhibit non-cell-autonomous dac activation, and Dll-null clones in the presumptive third antennal segment (a3), where dac is normally expressed, result in loss of dac. These data indicate that the regulation of dac by Dll in the antenna is different from that in the leg. They also indicate that the normal antennal expression of dac both requires Dll and has PD regional specificities. Because both Dll and Hth are required for antennal identity and are coexpressed with dac, Hth may also be required for the antennal expression of dac. Consistent with this view, ectopic expression of either Dll in antennal cells expressing Hth or of Hth in antennal cells expressing Dll can activate dac, as can ectopic coexpression of Dll and Hth in the wing disc. Furthermore, antennal dac expression, is not efficiently repressed by ectopic Hth (Dong, 2001).

Unlike Dll-null clones, both Dll hypomorphs and hth-null clones exhibit antenna-to-leg transformations. Examination of Dll hypomorphs and hth-null clones therefore reveals their homeotic functions. One such function may be the repression of leg dac. Leg expression of dac encompasses more segments and occurs at higher levels compared with the antenna. As in Dll hypomorphic leg discs, in Dll hypomorphic antenna discs, dac expression expands distally. hth-null clones exhibit derepression of dac in a1, a2 and a4 and elevation of Dac levels in a3. It is therefore proposed that the derepression of dac in Dll hypomorphs and in hth-null clones may represent leg-specific dac expression. Conclusive evidence for this awaits identification of dac enhancer elements and analysis of their regulatory inputs. Nonetheless, taken together, these data support the view that the regulation of leg and antennal dac expression occurs via distinct mechanisms and that the homeotic functions of Dll and hth are mediated not only through activation of antenna-specific genes such as spalt, but also through the active repression of leg development (Dong, 2001).

Appendages are subdivided by mutually antagonistic domains. Gradients of the morphogens Dpp and Wg initiate the PD organization of the Drosophila leg by activating Dll and repressing dac and hth distally, and by allowing the activation of dac while repressing hth medially. This creates three domains, distal, medial and proximal, that are specified respectively by expression of Dll, dac and hth. Further refinement and maintenance of the borders between domains requires mutually antagonistic interactions between proximal and medial domains as well as between medial and distal domains. Specifically, Dll and dac are mutually repressive. Also, mutually repressive interactions between the proximal and medial domains do exist via Tsh repression of dac and Dac repression of hth. Thus, pattern formation in the leg requires mutually antagonistic interactions among all three domains in order to refine and maintain borders that initially were set up by morphogens (Dong, 2001).

In contrast to the situation in the Drosophila leg, Dll, dac and hth are expressed in largely overlapping patterns in the antenna. This suggests that there is not mutual antagonism between Dll and hth in the antenna. Furthermore, that the entire antennal expression domain of dac lies within an area of Dll and hth coexpression indicates that Dac was unlikely to repress the antennal expression of either Dll or hth. Analysis of dac mutants confirms that Dac does not antagonize either proximal or distal development in the antenna but it does so in the leg. Therefore mutual antagonism is not a universal feature of appendage development (Dong, 2001).

Interestingly, in more basal insects like the cricket, Acheta domesticus, Dll and n-Exd expression are exclusive in the antenna. Since n-Exd is normally coincident with hth expression, it is inferred that Dll and Hth expression are exclusive in the cricket antenna. If exclusion reflects mutual antagonism, this in turn could indicate that mutual antagonism between proximal and distal domains is lost in the antenna within the insect lineage during the course of dipteran evolution (Dong, 2001).

It is noted that the absence of antagonism of any single PD domain towards another leads to overlap of otherwise exclusively expressed transcription factors. This, in turn, may permit the coexpressed factors to execute additional functions. Indeed, while Hth is required for proximal patterning of both antenna and leg, and Dll is required for distal patterning of both antenna and leg, their coexpression leads to the differentiation of antenna-specific cell fates. Thus, expression of distinct combinations of transcription factors such as Dll, Dac and n-Exd/Hth both in specific domains along the PD axis and between appendage types is likely to activate and repress particular suites of target genes, thereby contributing to differences in appendage morphologies (Dong, 2001).

The ability of Dll, Dac and n-Exd/Hth to repress the expression of one another undoubtedly is context-dependent. However, the only known factor involved in context specification is the Hox protein Antp. In the presence of Antp in the antenna, Dll and Hth are no longer coexpressed. Conversely, when Antp is removed from the leg, hth is derepressed in cells expressing Dll. Thus Antp appears to play a role in some aspects of domain antagonism. It remains unclear whether Antp directly modulates interactions among Dll, Dac and n-Exd/Hth or whether there are other molecules that intervene (Dong, 2001).

n-Exd/hth and Dll, and their homologs are expressed respectively in the proximal and distal domains in the appendages of animals as diverse as arthropods and vertebrates, and are required for the proximal and distal development in many Drosophila appendages. It is therefore suggested that the existence of both proximal and distal domains in appendages pre-dates the evolution of the arthropods. However, with the available information, it cannot be said whether these domains in the ancestral appendage were distinct, as they are in the modern Drosophila leg, or overlapping, as they are in the Drosophila antenna. It is speculated that n-Exd and hth, and their vertebrate homologs, the Pbx and Meis genes, were ancestrally expressed in the body wall because they are in modern animals and that as limbs evolved, they were originally expressed throughout the entire outgrowth. Subsequent antagonism by distal factors such as Dll could have allowed for the evolution of additional domains within different appendages (Dong, 2001).

This comparison of the Drosophila antenna and leg leads to the conclusion that a fundamental difference between these homologous appendages is the presence of a functional medial domain in the leg, specified by dac. The antenna has fewer segments, with dac expressed at relatively low levels and in only one of the segments, whereas dac is expressed in at least four leg segments. Loss of dac results in medial deletions in the leg but not in the antenna. Repression of proximal and distal genes by dac is not observed in the antenna, as it is in the leg. Consequently, the antennal expression of n-Exd/hth and Dll are not separated in the antenna by a medial domain that expresses dac. For these reasons, it is proposed that the acquisition of a medial domain, possibly through the use of dac, may have been a distinct step in appendage evolution. Consistent with this, increasing the territory and levels of dac expression in the antenna leads to repression of hth and Dll and to the differentiation of medial leg structures (Dong, 2001).

Two scenarios by which the existing Drosophila domain organizations may have arisen can be envisioned, given primitive appendages that had only proximal and distal domains. One possibility is that the medial domains were initially acquired by both the antenna and leg, but lost from the antenna sometime prior to the evolution of Drosophila. A second possibility is that the medial domain is an innovation of only the leg and may never have existed in the antenna. The expression of dac in the legs and its absence in the antennae of other arthropods may provide support for the latter scenario. Comparison of the relative domains of expression and the functions of Dll, dac and hth in other organisms will undoubtedly lead to further insights into how distinct PD domains were acquired and became patterned during the course of appendage evolution (Dong, 2001).

Homothorax (Hth) is a homeobox-containing protein that plays multiple roles in the development of the embryo and the adult fly. Hth binds to the homeotic cofactor Extradenticle (Exd) and translocates it to the nucleus. Its function within the nucleus is less clear. It was shown, mainly by in vitro studies, that Hth can bind DNA as a part of ternary Hth/Exd/HOX complexes, but little is known about the transcription regulating function of Hth-containing complexes in the context of the developing fly. Genetic evidence is presented, from in vivo studies, for the transcriptional-activating function of Hth. The Hth protein was forced to act as a transcriptional repressor by fusing it to the Engrailed (En) repression domain, or as a transcriptional activator, by fusing it to the VP16 activation domain, without perturbing its ability to translocate Exd to the nucleus. Expression of the repressing form of Hth in otherwise wild-type imaginal discs phenocopies hth loss of function. Thus, the repressing form works as an antimorph, suggesting that normally Hth is required to activate the transcription of downstream target genes. This conclusion was further supported by the observation that the activating form of Hth causes typical hth gain-of-function phenotypes and can rescue hth loss-of-function phenotypes. Similar results were obtained with XMeis3, the Xenopus homolog of Hth, extending the known functional similarity between the two proteins. Competition experiments demonstrate that the repressing forms of Hth or XMeis3 worked as true antimorphs competing with the transcriptional activity of the native form of Hth. The phenotypic consequences of Hth antimorph activity are described in derivatives of the wing, labial and genital discs. Some of the described phenotypes, for example, a proboscis-to-leg transformation, have not been previously associated with alterations in Hth activity. Observing the ability of Hth antimorphs to interfere with different developmental pathways may uncover new targets of Hth. The Hth antimorph described in this work presents a new means by which the transcriptional activity of the endogenous Hth protein can be blocked in an inducible fashion in any desired cells or tissues without interfering with nuclear localization of Exd (Inbal, 2001).

Hth activity can have opposite effects on organ development in different contexts. For example, Hth ectopic expression in the developing eye leads to a reduction in size or a complete loss of this organ, implying that Hth is a negative regulator of eye development. Conversely, in the antenna Hth is required for the formation of the organ, functioning as an antennal determining gene. It is possible that in the context of the developing eye, Hth induces the transcription of a repressor of eye development. In contrast, in the developing antenna Hth may activate the transcription of genes that promote antennal development. For example, spalt is thought to be a downstream target of the combined action of Hth and Distal-less (DLL) in this pathway (Inbal, 2001).

In the leg, Hth is required for proper proximodistal axis formation. Normally, Hth expression is limited to the proximal segments of the developing leg disc and Dll is expressed in the presumptive distal leg. The expression domains of Hth and Dll are mutually exclusive. When Hth is ectopically expressed in the Dll domain or along the anteroposterior (AP) border, distal leg structures fail to form, suggesting that Hth interferes with Dll function. When the expression of En-Hth1-430 (the repressive form of Hth) was induced along the AP border, all leg segments were affected. Proximally, fusion of coxa, trochanter and proximal femur was evident; a phenotype that was also observed when hth mutant clones were generated in the developing proximal leg region. All tarsal segments were missing, and the remaining structures, which appeared to be mostly of tibial identity, were extremely deformed. The phenotypes caused by either En-Hth1-430 or hth loss of function in the proximal leg are the same, and are therefore in accordance with the assumption that Hth normally induces transcription. Intriguingly however, in the distal region the ectopic expression of either normal Hth or En-Hth1-430 led to a loss of distal leg structures. This result can be interpreted in several ways. First, it is possible that in the specific context of the developing distal leg, ectopic Hth does repress the function of Dll and perhaps other genes, and in doing so abolishes distal leg formation. Another possibility is that the main role of Hth in interfering with distal leg development is the nuclear localization of Exd. It has been shown that when the ectopic expression of Exd was driven by Dll-Gal4, it was able to induce the same phenotype of leg truncation as ectopic Hth. When Exd was expressed along the AP border it was able to disrupt distal leg formation to a lesser degree. Another observation in support of this view is that a defective Hth, in which the homeodomain was inactivated by a mutation, was able to interfere with distal leg development when ectopically expressed, driven by Dll-Gal4. This effect was probably caused by the ability of the defective Hth to induce ectopic nuclear localization of Exd. Furthermore, in contrast to the effects caused by ectopic Hth, the ectopic expression of Exd or the homeodomain-defective Hth in the developing eye and antenna did not generate abnormal phenotypes. This suggests that in the eye and antenna the transcriptional activity of Hth is required, whereas, in the specific context of distal leg, the transcriptional activity of Hth may be less relevant (Inbal, 2001 and references therein).

Drosophila proprioceptors (chordotonal organs) are structured as a linear array of four lineage-related cells: a neuron, a glial cell, and two accessory cells, called cap and ligament, between which the neuron is stretched. To function properly as stretch receptors, chordotonal organs must be stably anchored at both edges. The cap cells are anchored to the cuticle through specialized lineage-related attachment cells. However, the mechanism by which the ligament cells at the other edge of the organ attach is not known. The identification of specialized attachment cells is reported that anchor the ligament cells of pentascolopidial chordotonal organs (lch5) to the cuticle. The ligament attachment cells are recruited by the approaching ligament cells upon reaching their attachment site, through an EGFR-dependent mechanism. Molecular characterization of lch5 attachment cells demonstrates that they share significant properties with Drosophila tendon cells and with mammalian proprioceptive organs (Inbal, 2004).

In an attempt to characterize the origin and fate of ch attachment cells, the distribution was examined of alpha85E-tubulin (alpha85E-tub) in ch organs. This minor alpha-tub variant is known to be expressed in the cap cells and the adjacent attachment cells, as well as in the ligament cells of lch5 organs. Close inspection of the distribution of this protein in mature embryos and first instar larvae revealed another alpha85E-tub-expressing cell in close proximity to the ventral edge of the ligament cells. Rarely, two such cells were observed. These large cells appeared to be good candidates to function in the attachment of ligament cells. Indeed, further analysis demonstrated that these cells are localized within the epidermal layer and are connected to the ventral edges of the ligament cells via Integrin-mediated adhesion, as suggested by the high concentration of the Integrin ßPS subunit in the contact site between these two cell types. In addition, these cells possess many features that are typical of other types of attachment cells. To avoid confusion, the attachment cells that anchor the cap cells are referred to as CA (cap attachment) cells and to the attachment cells that anchor the ligament cells as LA (ligament attachment) cells (Inbal, 2004).

To find whether the presence of ligament cells is sufficient to induce the formation of LA cells regardless of their position, embryos were examined in which the ligament cells were abnormally localized. Mutations in abdominal-A (abd-A), homothorax (hth), and ventral veinless (vvl) result in frequent dorsal localization of lch5 organs. lch5 organs that fail to localize to their correct position in these mutants do not have LA cells. However, since the protein products of abd-A, hth, and vvl are normally expressed in the ectoderm, it is possible that, in their absence from the ectoderm of mutant embryos, LA cells cannot develop, regardless of the positioning of ligament cells. To assess specifically the influence of ligament cell positioning, an inducible Hth antimorph (En-Hth1-430) was used that can phenocopy hth loss of function. Expression of this antimorph in ch organs under the regulation of ato-Gal4 results in a high percentage of abnormally oriented lch5 organs. Except for their abnormal positioning, lch5 organs in these embryos appear to be fully differentiated, as judged by their ability to express typical markers, such as Repo, alpha85E-tub, and Sr. In ato-Gal4/UAS-En-Hth1-430 embryos, no LA cells could be observed in abdominal segments that exhibited abnormally oriented lch5 organs. Altogether, these data suggest that lch5 ligament cells recruit their attachment cells and that this process is restricted spatially, perhaps due to competence of cells in the attachment site region (Inbal, 2004).

Effects of Mutation: Homothorax and brain development

During early brain development in Drosophila a highly stereotyped pattern of axonal scaffolds evolves by precise pioneering and selective fasciculation of neural fibers in the newly formed brain neuromeres. Using an axonal marker, Fasciclin II, the activities of the extradenticle (exd) and homothorax (hth) genes are shown to be essential to this axonal patterning in the embryonic brain. Both genes are expressed in the developing brain neurons, including many of the tract founder cluster cells. Consistent with their expression profiles, mutations of exd and hth strongly perturb the primary axonal scaffolds. Furthermore, mutations of exd and hth result in profound patterning defects of the developing brain at the molecular level, including stimulation of the orthodenticle gene and suppression of the empty spiracles and cervical homeotic genes. In addition, expression of eyeless is significantly suppressed in the mutants except for the most anterior region. These results reveal that, in addition to their homeotic regulatory functions in trunk development, exd and hth have important roles in patterning the developing brain through coordinately regulating various nuclear regulatory genes, and imply molecular commonalities between the developmental mechanisms of the brain and trunk segments, which were conventionally considered to be largely independent of one another (Nagao, 2000).

In the course of embryonic brain development both EXD and HTH proteins became clearly detectable by early stage 12 in many of the delaminating cephalic neuroblasts. Strong nuclear expression is particularly evident in the deuto- and trito-cerebrum neuroblasts, but less prominent expression is also detectable in most of the protocerebrum neuroblasts. These patterns are maintained in almost identical manners since the brain neuromeres were formed by division of the cephalic neuroblasts. As development proceeds further, EXD and HTH localize in several domains in the brain: high level expression is maintained for both proteins in most of the neural cells in the deuto- and trito-cerebrum anlagen (neuromeres b2 and b3); in the mediolateral regions of the b1 neuromere and most of the cells of the subesophageal ganglia. Both EXD and HTH localize in the nucleus in the developing brain neurons, as confirmed by colocalization with nuclear transcription factors. The apparent identical expression of EXD and HTH in the developing brain has been confirmed by double staining with anti-EXD and anti-HTH antibodies or a HTH-lacZ reporter. Moreover, EXD immunoreactivity in the brain is lost in the hth mutant whereas cytoplasmic EXD is still detectable in the epidermis. Likewise, HTH expression is dependent on the activity of exd, since virtually all the HTH immunoreactivity is lost in both the epidermis and the brain in exd mutant (Nagao, 2000).

The expression patterns of EXD and HTH in developing brain neuromeres are partly reminiscent of the patterns of fiber tract founder clusters. Examinations of embryos double stained with anti-HTH antibody and anti-FAS II antibody demonstrate that many of the cells in the fiber tract founder clusters indeed express the HTH protein. This coexpression is already seen by the middle of stage 12 when the first set of the Fas II clusters in the brain becomes evident. Significant coexpression is seen in the fiber tract founder cluster D/T, which is located in neuromere b3, the tritocerebrum anlage: this stage is marked by the lab gene. Despite the fact that the HTH pattern becomes more restricted in later stages, the HTH expression in the fiber tract founder clusters is largely maintained. In particular, HTH is expressed at significant level in the D/T and P1 clusters. Similar overlapping expression in the fiber tract founder clusters is detected for the EXD protein. Coexpression of FAS II, HTH, and EXD is also seen in the developing optic lobe primordia (Nagao, 2000).

In order to gain insights into their functions, the expression patterns of EXD and HTH in the developing brain were further examined in conjunction with known neuraxial patterning genes. In the proto- and deuto-cerebrum anlagen, the immunoreactivity of the EXD protein only partially overlaps with otd transcripts except for the dorsally located cells in neuromere b1, which express both genes at high levels. In contrast to otd, the EXD immunoreactivity largely overlaps with the EMS immunoreactivity in neuromeres b2 and b3). EMS is predominantly expressed in the anterior parts of neuromeres b2 and b3. EMS and EXD colocalize in many of the b2 and b3 cells with the exception of some of the most anterior cells of each neuromere, which clearly express EMS but EXD only faintly. Coexpression of the two genes is also detected in neuroblasts. In the tritocerebrum anlagen, EXD immunoreactivity overlaps with the lab-lacZ expression, which localizes in the posterior part of the b3 neuromere. EXD immunoreactivity also overlaps with the DFD immunoreactivity in the mandibular and the anterior half of the maxillary neuromeres. Similarly, the hth-lacZ expression, which is identical to the endogenous hth and exd expression patterns in double staining, overlaps with the SCR immunoreactivity in the posterior half of the maxillary neuromere and the anterior half of the labial neuromere (Nagao, 2000).

Thus exd and hth genes are coexpressed in many of the neurons of the fiber tract founder clusters, suggesting that the activities of these genes are intrinsically required for axonal programming of the tract founder cluster neurons. This is particularly evident for the D/T cluster, in which Fas II expression is largely dependent on exd and hth. Most of the Eyeless patterns, including those that partially overlap with the fiber tract founder clusters, are suppressed in the mutants. Given these results, it is likely that the intrinsic axonal programs of the fiber tract founder clusters are altered in the exd and hth mutants. Intriguingly, in addition to the apparent defects in the primary axonal scaffolds, mutations in the exd and hth genes result in gross anatomical defects in the developing brain. Notably, both mutations cause abnormal positioning of the brain commissure at more posterior positions (in neuroaxis), suggesting widespread regional patterning defects in the mutant brains. In support of this notion, molecular neuroanatomical analyses have revealed alterations to the expression patterns of many of the regional patterning genes, including stimulation of otd and suppression of ems, in the developing brain neuromeres. Similarly, in accordance with the anatomical defects at the cervical junction, expression of the anterior HOM-C genes lab, Dfd and Scr are significantly suppressed in the mutant brains. Furthermore, consistent with the anatomical abnormalities, both engrailed expressing cells en-b1 and Brain segment homeobox (Bsh) are up-regulated in the mutant brains with ectopic cell clusters in more posterior positions. Thus the strong defects in the embryonic axonal scaffolds in the exd and hth mutant brains are likely to be caused by combined defects in intrinsic neural programming of the fiber tract founder neurons and in extrinsic patterning of the brain neuromeres that provide the substrate for the axonal extension of the fiber tract founder clusters (Nagao, 2000).

Effects of Mutation: Homothorax and the eye disc

In Drosophila the eye-antennal disc gives rise to most adult structures of the fly's head. Yet the molecular basis for its regionalization during development is poorly understood. homothorax is shown to be required early during development for normal eye development and is necessary for the formation of the ventral head capsule. In the ventral region of the disc are homothorax and wingless involved in a positive feedback loop necessary to restrict eye formation. homothorax is able to prevent the initiation and progression of the morphogenetic furrow without inducing wingless, which points to homothorax as a key negative regulator of eye development. In addition, the iroquois-complex genes are shown to be required for dorsal head development, antagonizing the function of homothorax in this region of the disc (Pichaud, 2000).

The eye-antennal disc is a compound imaginal disc that gives rise to several different parts of the fly head: the head capsule (ventral and dorsal, including the ocellar region), that surrounds the eye, plus the antenna and maxillary palp. Because the gene homothorax (hth) has been implicated in limiting the eye field, its pattern of expression throughout the development of the eye-antennal disc was examined and its requirements in the head were examined in detail. Early in development, during the second instar larval stage, hth is weakly expressed in all the cells of the eye-antennal disc. The same widespread expression pattern is seen for wg. During the third instar larval stage, as the eye field is patterned in a posterior-to-anterior direction, the expression of hth regresses anteriorly and laterally. By late third instar, hth remains strongly expressed in the prospective head capsule, antennal regions and more weakly in the maxillary palp primordium. In the developing eye field, hth is expressed 10-15 cell diameters ahead of the morphogenetic furrow (MF), but it is switched off thereafter. In the differentiated eye, hth is found in the pigment cells in the posterior region of the eye field (Pichaud, 2000).

The role of hth in the head structures was examined using mosaic analysis. Consistent with hth expression pattern in late third instar stage, hth mutant clones induced at any time during larval life autonomously produce ectopic eyes only in the ventral head capsule. The frequency and size of ectopic eyes is generally greater with hthB2, a weak allele, than with strong alleles such as hthC1 or hthP2. The ventral head region (gena and rostral membrane) is reduced as a consequence of the production of ectopic eyes, and the maxillary palps are frequently absent or abnormal in hth- clones. Conversely, ectopic expression of hth in clones in the eye can cause the eye to be split by tissue resembling ventral head capsule. When hth-M- clones are induced during first instar larval stage, large mutant clones in the head can be recovered. These clones result in ventral overgrowths of eye tissue and in the loss of ventral and dorsal head structures. Nevertheless, the rim of cuticle surrounding the eye (the orbital and postorbital area) is preserved, indicating that other factors are responsible for the specification of these structures. hth- clones induced later in development cause the autonomous transformation of the dorsal part of the head capsule into mesonotum (dorsal second thoracic segment) (Pichaud, 2000).

hth- clones affecting dorsal head regions never induce ectopic eyes. These phenotypes are equivalent to those described for the loss of extradenticle (exd), consistent with a role for hth in the nuclear localization of the Exd protein. In addition, mutant clones in the antenna cause its autonomous transformation to leg tissue (Pichaud, 2000).

An analysis was carried out to see if a particular cell types are absent in mutant hth- clones in eyes of living flies and in tangential sections of eyes containing clones. Occasionally incomplete ommatidia were observed with missing photoreceptors and fewer bristles, most of the time at the interface between clonal and wild-type tissue in the mosaic eyes. In addition, hth mutant clones in the eye are normally pigmented, indicating that pigment cells are present, even though hth is expressed in this cell type. In hth mosaic eyes the ommatidia frequently show an orthogonal shape, instead of the normal hexagonal one. This shape is likely to be the result of incorrect cell stacking, probably due to disorganized ommatidia. Interestingly, in hthB2 mosaic eyes the induction of ectopic equators was observed occurring parallel to the endogenous equatorial axis, accompanied by frequent inversion of ommatia polarity along the anterior-posterior (A/P) axis. These defects are both cell- and non-cell-autonomous (Pichaud, 2000).

The progression of the MF orchestrates a wave of cell differentiation that gives rise to the different cell subtypes found in the adult eye. The product of the gene wingless (wg) limits the expansion of the differentiating eye, allowing head capsule development. Since hth also limits eye development in the ventral head, a test was performed to see if hth and wg regulation is linked. In third instar eye-head region, hth is strongly expressed in the prospective regions of the dorsal and ventral head, where it overlaps with wg expression. Large ventral hth-M1 mutant clones induced in the prospective head capsule region cause the formation of ectopic eyes, visualized by the de novo expression of Elav, and the loss of wg expression. Ectopic eye differentiation starts at the margins and progresses inward, in agreement with the ectopic eye tissue seen in the adult. By contrast, large dorsal hth clones do not produce ectopic eyes and wg expression is not affected. Nonetheless, removal of hth from the dorsal head results in the loss of the ocellar region. It has been shown that this region requires the expression of wg and its downstream target orthodenticle (otd). Thus, it is possible that, in the absence of hth, wg expression is subtly reduced. Alternatively, hth could act in parallel to, or downstream of wg to specify this dorsal head region. These results suggest that, in the ventral part of the eye- head region, hth helps defining the territory of the disc that will become head, probably in part by maintaining wg, but does not do so in the dorsal region. In the absence of hth, wg is lost ventrally and ectopic eyes are generated (Pichaud, 2000).

As the eye field is patterned, hth expression is repressed several rows of cells ahead of the MF, and it is upregulated at the margins of the disc. This dynamic pattern of expression resembles wg expression, which is detected in all the cells of the eye-antennal disc during second instar larval, but is later restricted to the margins. During late third instar, hth expression straddles that of wg. This observation raises the possibility that hth could be also controlled by wg. To test this hypothesis, clones were generated ectopically expressing a membrane-tethered Wg form (teth-Wg), that cannot diffuse. In teth-Wg expressing clones located anterior of the furrow, Hth levels are increased. This is not the case when the clones are induced in regions immediately in front of or posterior to the furrow. A test was performed to see if blocking the wg pathway could lead to modifications of hth expression. This issue was addressed by producing ectopic clones of a dominant negative form of dTCF (Pangolin), a nuclear factor required for the transduction of the wg signal. In these clones hth expression is strongly reduced in the presumptive head cuticle region both ventrally and dorsally. This result shows that wg is necessary to maintain hth expression in the presumptive head regions of the eye-head disc. Conversely, when hth is expressed ectopically in clones, it is unable to initiate wg expression. However, ectopic hth upregulates wg in regions where it (and hth) are already expressed. Also, ectopic expression of hth can block furrow initiation without inducing wg. These observations raise the possibility that hth mediates, at least partially, the eye-repressing function of wg (Pichaud, 2000).

These experiments suggest that hth is necessary to maintain wg expression, but not sufficient for its de novo induction. Starting during late second or early third instar larval stage wg and hth seem to be engaged in a positive regulatory feedback loop that might be important for the development of the ventral head capsule. This feedback loop could be responsible for the upregulation of hth in the ventral and dorsal head capsule while hth is required to maintain wg only in its ventral part (Pichaud, 2000). hth is able to block eye differentiation in a wg-independent manner. One possibility is that hth acts by repressing dpp, which is expressed in the furrow. Alternatively, hth could perform its function through the repression of genes downstream of dpp. To test these possibilities, ectopic clones of cells over-expressing hth were generated and their effect on both dpp expression and furrow progression was analyzed. Small hth expressing clones in the furrow do not repress dpp, and do not allow photoreceptor differentiation (reported by the neuronal specific marker Elav). hth expressing clones induced just anterior to the furrow delay furrow propagation: the hth- cells receive signals from the furrow, since they are able to turn on dpp-lacZ expression, and the furrow is able to advance over them, leaving some hth+ cells behind it. Nevertheless, the furrow is retarded and the hth+ cells posterior to it do not differentiate as photoreceptors. These results show that hth can block MF movement downstream of dpp (Pichaud, 2000).

hth seems to be mainly involved in the maintenance of wg and the repression of eye development only at the ventral margin of the eye-head region. However, hth is expressed both ventrally and dorsally. Therefore, there must be other genes involved in setting up this asymmetry in the head. Candidates for such factors are the iroquois-Complex (iro-C) homeobox genes -- araucan, caupolican, and mirror -- because their expression is restricted to the dorsal half of the eye primordium. The iro-C genes seem to position the equator, a narrow domain where the Notch (N) signaling pathway is activated and where N signaling triggers initiation of eye differentiation. Also, they have been been shown to be under the control of pannier, a GAGA-family transcription factor-encoding gene. The expression of an ara/caup reporter was mapped in the dorsal eye-head primordium relative to hth, and to pannier, which is expressed in the dorsal-most region of the head capsule. In late third instar eye-antennal discs, hth is expressed in the dorsal fold that gives rise to the dorsal head capsule and in the peripodial membrane. The domain of hth expression overlaps with pnr (monitored with a Gal4 insertion line in this gene in the dorsalmost part of the head capsule region, and with iro-C more ventrally in a thin strip of cells. At this larval stage, iro-C and pnr-Gal4 do not overlap, and wg is expressed in the iro-C domain. In order to map the hth/iro-C co-expression domain in discs to the adult head structures, the ara/caup reporter adult heads were stained in X- gal solution. Staining was observed in the orbital region of the dorsal head, in agreement with the fate map of the eye-antennal disc. Since in hth- clones the orbital region is unaffected, the iro-C genes could be determining this dorsal head structure (Pichaud, 2000).

Thus, in the dorsal region of the eye disc, wg is under the control of pnr. In an analogous manner, hth must be kept on in the ventral margin of the disc to stop eye differentiation (as evidenced by the ectopic eyes in the presence of hth mutation) and to maintain wg expression in this region. wg is expressed at a higher level in the dorsal region of the presumptive head cuticle than in the ventral region. Removal of wg from the dorsal edge of the disc results in ectopic furrow generation, whereas this effect is less penetrant in the ventral part of the disc. In that respect, the removal of hth in the ventral part of the disc 'mimics' the effect of removing wg dorsally, by producing large eye overgrowths. One possibility is that both wg and hth have to be removed from the ventral edge of the disc in order to efficiently generate ectopic furrow, suggesting that wg might not be the only activator of hth expression in the prospective ventral head region. hth, downstream of or in parallel to wg, would then repress eye formation in this region of the disc. In this model hth maintains wg in the ventral region of the disc, while pnr maintains wg dorsally. In turn, wg upregulates hth expression in both the dorsal and the ventral head prospective regions of the disc. hth expression in the eye field may be independent of any wg input. Analysis of hth minus clones has also revealed the formation of ectopic equators and inversion of ommatidia polarity along the A/P axis. These observations are consistent with the role of hth in limiting eye formation by repressing MF triggering (Pichaud, 2000).

Repression of hth is a prerequisite to allow neuronal differentiation. In clones expressing ectopic hth, dpp is still expressed. Therefore, it is likely that hth prevents eye formation downstream of dpp. This could happen through a disruption of the hh/dpp feedback loop by a direct repression of hh, or through an inhibition of proneural genes such as atonal (Pichaud, 2000).

To analyze the role of iro-C in dorsal head development, iro-C function was removed in clones of cells carrying a deficiency for ara, caup and mirr (iroDFM3), or only ara and caup (iroDFM1), and the phenotypic consequences were examined in adult heads. Only results for iroDFM3 clones will be described, since iroDFM1 clones give similar results. iro-C- clones cause a series of phenotypes, adding progressively more 'ventral-type' tissue in the following order: dorsal eye overgrowth or ectopic dorsal eyes; overgrowth of ventral type of cuticle (ptilinum and rostral membrane); ectopic antennal pouches; antennae and maxillary palps. The extra head structures are produced autonomously, but the eyes can be composed of both mutant and wild-type ommatidia. The ectopic structures, which can duplicate the full complement of ventral structures, all grow from the orbital region of the head. The rest of the dorsal head is displaced by the overgrown tissue. The orbital region fate maps to the domain where hth and iro-C expressions overlap. These results show that the iro-C genes are required to repress the proliferation of a group of dorsal cells, that otherwise would grow with ventral head identity, and may contribute to assign them a dorsal head ('orbital-region') identity (Pichaud, 2000).

iro-C- clones in discs frequently produce overgrowths, in agreement with the structures produced dorsally. However, hth and wg expressions are not substantially altered in iro-C- clones. Since removal of ara, caup and mirr produces the same phenotypes as removal of only ara and caup, it is concluded that mirr is dispensable for suppressing ventral identity in the dorsal head. Alternatively, mirr expression could be under the control of ara and caup (Pichaud, 2000).

Homothorax switches function of Drosophila photoreceptors from color to polarized light sensors

Different classes of photoreceptors (PRs) allow animals to perceive various types of visual information. In the Drosophila eye, the outer PRs of each ommatidium are involved in motion detection while the inner PRs mediate color vision. In addition, flies use a specialized class of inner PRs in the 'dorsal rim area' of the eye (DRA) to detect the e-vector of polarized light, allowing them to exploit skylight polarization for orientation. Homothorax plays a critical role for DRA development: hth is expressed specifically in maturating inner PRs of the DRA and maintained through adulthood. homothorax is both necessary and sufficient for inner PRs to adopt the polarization-sensitive DRA fate instead of the color-sensitive default state. Loss of hth results in the transformation of the DRA into color-sensitive ommatidia, and misexpression of hth forces color-sensitive inner PRs to acquire the typical features of polarization-sensitive DRA cells. Homothorax increases rhabdomere size and uncouples R7-R8 communication to allow both cells to express the same opsin rather than different ones as required for color vision. Homothorax expression is induced by the Iroquois complex and the Wingless (Wg) pathway. However, crucial Wg pathway components are not required, suggesting that additional signals are involved (Wernet, 2003).

Each ommatidium contains 8 photoreceptor cells (PRs: R1 to R8). Rhabdomeres of outer PRs (R1 to R6) span the whole retina and their axons project to the lamina (L) part of the optic lobe. Inner PRs (R7 and R8) are located on top of each other and both project axons to the medulla. Ommatidia fall into three categories based on rhabdomere morphology and opsin expression. Inner PRs of pale and yellow ommatidia produce rhabdomeres of small diameter and can be distinguished by their characteristic opsin expression (p: rh3/rh5 versus y: rh4/rh6). Specialized ommatidia are found exclusively in the DRA of the adult eye, manifesting large inner PR rhabdomere diameters and rh3 expression in both R7 and R8 (Wernet, 2003).

To identify genes controlling late PR maturation events, a GAL4 enhancer trap screen was performed in adult PRs using GFP as a reporter gene. One of the insertions was expressed in a single row of ommatidia along the dorsal head cuticle. In some locations, two (but never more) positive rows of ommatidia were observed. The projections of the GAL4-positive cells to the optic lobe were visualized with UAS-lacZ; all marked axons terminate in the dorsalmost part of the medulla with projections to both R7 and R8 layers, indicating that GAL4 was expressed exclusively by inner PRs in the DRA. The insertion was determined to be in the second intron of homothorax. To verify that the observed GAL4 expression pattern in developing DRA inner PRs was indeed that of endogenous Hth protein, pupal retinas (48 hr after puparium formation, APF) were stained with an antibody against Hth. Hth expression was always detected in one, at most two rows of ommatidia and only at the dorsal rim of the pupal retina. The majority of positive ommatidia expressed Hth in two cells per cluster, which were identified as R7 and R8 because of their stereotypical positioning as compared to the landmark svp-lacZ. Ommatidia with only one Hth expressing cell could also rarely be observed without showing any obvious preference toward R7 or R8. Hth expression is maintained throughout adulthood and is coexpressed with the R7 UV-opsin Rh3, which is the only opsin expressed by both inner PRs of the DRA. Rh3-expressing R7 cells outside of the DRA are always negative for Hth. Therefore, Hth is a highly specific marker for the polarization-sensitive inner PRs of the DRA (Wernet, 2003).

The role of Hth in DRA development represents a specific example illustrating how late PR differentiation events specify the three ommatidial subtypes. It provides further evidence that establishment of terminal PR fates in p, y, or DRA ommatidia is achieved by consecutive determination steps. In this model, early PR cell fate decisions (i.e., determination of the 8 types of PRs) and projection to the optic lobes occur in the third instar imaginal disc. The distinction between inner and outer PRs is controlled by spalt: Salm represents the earliest marker expressed in both R7 and R8 starting at third instar larval stages and maintained to adulthood. Loss of both salm and salr results in transformation of adult inner into outer PRs; the inner PR rhodopsin genes (rh3-rh6) are replaced by the outer PR rh1, although most axons still maintain their projections to the medulla. R7 and R8 are further distinguished from each other by expression of Prospero and Senseless, respectively. The distinction between the three classes of ommatidia appears to be achieved later; hth expression in the DRA is only initiated during early pupation. It is proposed that only those cells that express Sal are competent to face another cell fate decision at the beginning of pupation. The inner PRs of ommatidia close to the dorsal rim come under the influence of a DRA inducing signal that includes Wg and express Hth, whereas in all other ommatidia two different pairs of color-sensitive PRs develop in a stochastic manner. Consistent with this model, the outer PRs, which do not express Sal, are not transformed by forced expression of Hth, and Hth expression is lost in salm:salr double mutants. The crucial decision made by inner PRs between color sensors or polarization detectors therefore depends uniquely on their position within the retina (Wernet, 2003).

Inactivation of Hth function results in the transformation of the DRA into atypical color-sensitive ommatidia expressing Rh3 in R7 and Rh6 in R8. Overexpression of both activated Armadillo and dominant-negative Hth (GMR>ArmS10+hthHM) also results in all dorsal R7 cells expressing rh3 and all underlying R8 cells expressing rh6. This further suggests that Wg activity directs the inner PRs toward a DRA program but that without Hth function, the DRA program cannot be executed: inner PR rhabdomeres do not become larger and Rh3 is not expressed in R8. But why are atypical color-sensitive Rh3/Rh6 ommatidia always formed? Loss of Hth might not allow the full program of color PR specification to be activated at the dorsal rim, since p and y subtypes are not distinguished stochastically. R7 ommatidia always choose expression of rh3; R8, which are not properly instructed by R7, choose rh6 and not coupled expression of rh5. These results are consistent with the model that Rh6 is the ground state for R8, since in the absence of R7 (sev) the vast majority of R8 express Rh6. By extrapolation, rh4 was therefore suggested to be the ground state in R7. However, the results suggest that Rh3 might in fact represent the ground state in R7. A gene has recently been identified that is both necessary and sufficient for the expression of Rh4 in R7, presumably by distinguishing yR7 (rh4) from the ground state pR7 (rh3). It is therefore proposed that the Rh3/Rh6 pair represents the combination of independent R7 and R8 'ground states' upon which PR subtype decisions are imposed; the stochastic choice made by R7 outside the DRA is usually linked to communication from R7 to R8, resulting in coupling of rh3/rh5 in p and rh4/rh6 in y subtypes. It appears that this process is suppressed in DRA inner PRs, even when Hth function is lacking, suggesting that the high Wg levels activating Hth at the dorsal rim might also repress the subtype decisions of color-sensitive ommatidia as well as communication between R7 and R8 (Wernet, 2003 and references therein).

Expression of Hth in inner PRs is sufficient to induce the DRA fate both morphologically (increase in rhabdomere diameter) as well as molecularly (monochromacy by expression of Rh3 in both R7 and R8 and repression of Sens in R8), although the genetic programs activated by Hth remain unknown. One of the major roles of Hth is to translocate Exd into the nucleus where Hth and Exd form transcriptional complexes with HOX proteins. Consistent with this, Exd is localized to the nuclei in inner PRs of the DRA, but not in color-sensitive ommatidia, suggesting that Hth and Exd function together. Whether Hth and Exd directly repress Sens in R8 cells of the DRA is currently being investigated, since loss of Sens expression seems to be essential for DRA R8 cells to escape the typical color-sensitive R8 fate and for switching to the DRA R8 fate with its R7-type rh3 expression. A better understanding of Hth function in vivo is of great importance because mammalian homologs of Hth (Meis1a) cooperate with HOX factors to induce acute myeloid leukemia although direct association with HOX factors might not always be necessary. Since no HOX proteins have been implicated in Drosophila eye development, DRA development represents an attractive model system for identifying new factors interacting with Hth and Exd in vivo (Wernet, 2003).

Although the IRO-C genes have been suggested to act only before the MF, the current experiments reveal that IRO-C genes are able to induce dorsal-specific morphological changes at later time points. Evidence has been found that members of the IRO-C complex indeed act as selector genes to specify the dorsal compartment of the developing eye. They fulfill at least two additional typical features proposed for such selector genes: persistence of expression and induction of transformations when misexpressed in the ventral compartment (Wernet, 2003).

caup persists at very low levels during pupal stages before returning to high levels in adults. One possible explanation for such transient downregulation could be that high levels of IRO-C genes are toxic for the developing PRs. Indeed, massive cell death is observed when ara, caup, or mirr are overexpressed under the control of a strong GMR-GAL4 driver. Weaker drivers expressed posterior to the MF, however, give rise to healthy PRs and a ventral rim area. Therefore, during early pupal stages, low levels of dorsally expressed IRO-C genes might restrict induction of Hth expression to the dorsal half of the rim. The results suggest that the IRO-C complex acts together with a factor induced by high levels of Wg signaling. Indeed, overexpression of both ArmS10 and ara posterior to the morphogenetic furrow induces Hth expression in inner PRs throughout the eye. Since loss of all three IRO-C genes does not result in a loss of the DRA, a fourth unknown factor might be partially redundant with the IRO-C genes, or alternatively the deficiency used to eliminate the three genes might bear residual activity (Wernet, 2003).

Although activation of the Wg pathway strongly induces DRA throughout the IRO-C compartment, the DRA develops normally when Fz and DFz2, dsh, or TCF are inactivated. It is possible that low levels of wild-type protein persist long enough in the clones for DRA development to proceed, although this is unlikely considering the late onset of Hth expression. Therefore, redundant factors might exist, such as the Derailed receptor which has recently been shown to mediate Wnt5 function. Alternatively, another diffusible factor could act in parallel with the Wg/Fz pathway to induce the DRA, possibly acting downstream of Wg as a 'relay signal'. Indeed, cell nonautonomous inductive effects downstream of both wg and Arm have been reported to influence cell fate determination at the periphery of the fly retina, including the DRA (Wernet, 2003).

In summary, hth is both necessary and sufficient for changing the function of PRs from color vision toward polarized light detection, thus switching the perception associated with a given PR subtype. Hth therefore represents an important tool to further understand how terminal PR differentiation processes depend on spatial cues as opposed to the stochastic choice between color-sensitive ommatidial subtypes in the main part of the fly retina. In the future, it will be interesting to understand how the molecular targets of hth affect DRA cell properties and to investigate how the eyes of different species adapted their PRs to respond best to different environments (Wernet, 2003).

A new role for hth in the early pre-blastodermic divisions in Drosophila

In Drosophila, the preblastodermic syncytial nuclear divisions occur very fast. In this short period of time chromosomes must condense, segregate and decondense, in conditions governed by maternally provided RNAs and proteins. This report shows that the Homothorax (Hth) transcription factor is maternally provided and that its function is necessary for the proper assembly of the centric/centromeric heterochromatin during preblastodermic divisions. Embryos lacking the hth maternally-derived transcript, show abnormal localisation of the centromeric CID protein, and aberrant chromosomal segregation. In this syncytial context, Hth presumably acts together with its partner Extradenticle (Exd) and the RNA PolII, to facilitate transcription of satellite repeats. The transcripts derived from these sequences are needed for t

TALE-class homeodomain transcription factors, homothorax and extradenticle, control dendritic and axonal targeting of olfactory projection neurons in the Drosophila brain

Precise neuronal connectivity in the nervous system depends on specific axonal and dendritic targeting of individual neurons. In the Drosophila brain, olfactory projection neurons convey odor information from the antennal lobe to higher order brain centers such as the mushroom body and the lateral horn. This study shows that Homothorax (Hth), a TALE-class homeodomain transcription factor, is expressed in many of the antennal lobe neurons including projection neurons and local interneurons. In addition, HTH is expressed in the progenitors of the olfactory projection neurons, and the activity of hth is required for the generation of the lateral but not for the anterodorsal and ventral lineages. MARCM analyses show that the hth is essential for correct dendritic targeting of projection neurons in the antennal lobe. Moreover, the activity of hth is required for axonal fasciculation, correct routing and terminal branching of the projection neurons. Another TALE-class homeodomain protein, Extradenticle (Exd), is required for the dendritic and axonal development of projection neurons. Mutation of exd causes projection neuron defects that are reminiscent of the phenotypes caused by the loss of the hth activity. Double immunostaining experiments show that Hth and Exd are coexpressed in olfactory projection neurons and their progenitors, and that the expressions of Hth and Exd require the activity of each other gene. These results thus demonstrate the functional importance of the TALE-class homeodomain proteins in cell-type specification and precise wiring of the Drosophila olfactory network (Ando, 2011).

In addition to the regulatory functions as homeotic cofactors, hth and exd have important functions in the determination of the developmental identity of the antennal segment; removing the function of exd or hth transforms the antenna into leg-like structures, and ectopic expression of hth can trigger antennal development elsewhere in the fly. In addition, the activities of hth and exd are required for the development of the embryonic axonal tracts that pioneer the projections between the deutocerebrum and the dorsal parts of the protocerebrum. Consequently, loss of hth or exd results in marked perturbations of the axonal scaffolds in the developing brain (Ando, 2011).

This study has shown that Hth is expressed in many of the AL neurons including local interneurons and PNs. Hth and Exd are coexpressed not only in postmitotic neurons but also in the progenitors of the anterodorsal and lateral AL lineages. Loss of either hth or exd causes loss of the lateral lineage, in which hth plays an essential role in suppression of apoptosis. Moreover, it has been shown that hth and exd are required for precise dendritic and axonal targeting of olfactory PNs. Mutations of hth result in profound targeting defects in many of the on-target glomeruli such as VA1lm, VA3 and D. They also cause ectopic innervation in many of the off-target glomeruli. The dendritic defect of exd1 mutation is reminiscent of the phenotype of hth mutations, with reduced number of GH146-expressing cells yet exhibiting selective ectopic innervation in the off-target DA1 glomeruli. The functional importance of hth and exd was further confirmed in post-mitotic neurons by the analyses of single-cell clones of the anterodorsal DL1 and the lateral DA1 neurons. Apart from dendritic defects, hth and exd mutations cause severe axonal defasciculation, misrouting and aberrant ventrolateral extensions. As with hth mutant clones, exd mutant clones exhibit an increase in the numbers of the LH terminal branches. Moreover, it was shown that coexpression of Hth and Exd is essential for efficient expression of either protein in the olfactory PNs, recapitulating the interdependence of the expression of the two proteins in the embryonic brain. These results suggest that exd and hth could positively regulate each other expression in the olfactory PNs. Alternatively, stability of Hth and Exd in the olfactory PNs could depend on the interaction of the two proteins as suggested in the antennal and leg discs (Ando, 2011).

Although Hth is co-expressed with Acj6 and Lola in both developing and adult ad-PNs, loss of hth function fails to alter Acj6 and Lola expression. Conversely, mutation of neither acj6 nor lola abolishes Hth expression. In agreement with independent regulatory mechanisms, the dendritic phenotype of hth PNs diverges from the phenotypes of either acj6 or lola mutant PN clones. Thus, in contrast to the severe innervation defects in VA1lm, VA3, and D of the hth mutant clones, acj66 mutant clones show only mild defects for VA1lm and D, and only VA3 is severely affected. Similar differences can be noted for single-cell clones, in which hth but not acj6 mutant clones exhibit complete switching of DL1 specificity (Ando, 2011).

With partial commonality with hth mutant PNs, lola mutant PNs exhibit severe defects in multiple glomeruli. Nonetheless, lola and hth mutant clones exhibit different degree and spectra of glomerular mistargeting. While both hthP2 and hthP1-K6-1 ad-NB clones fail to innervate the VA1lm glomerulus, most of the lola ad-NB clones completely innervate VA1lm. In addition, only 23% of the lola single-cell clones lack dendritic innervation in DL1 while the majority of the hth single-cell clones fails to innervate the correct target. It is also noteworthy that, unlike the mutations of hth or exd, loss of the lola activity dose not eliminate the lateral PN lineage. In addition to these LOF phenotypes, GOF mistargeting phenotypes are also different between hth, acj6, and lola mutations. Similar differences in dendritic phenotypes can be noted with mutations of other transcription factors that have been shown to be involved in PN specification. These results are thus consistent with the notion that different transcription factors control the targeting specificity of olfactory PNs via distinct intrinsic programs that regulate diverse repertoires of downstream genes, even though they are coexpressed during development (Ando, 2011).

In the development of vertebrate spinal motor neurons, homeodomain transcription factors play important roles in the generation and determination of diverse neuronal subtypes. In particular, Hox proteins act as central mediators of the intrinsic programs that shape motor neuron subtype identity and target muscle specificity. Hox proteins not only influence the identity of motor neuron columns but also control the initial specificity of motor axon projections via specific transcriptional cascades that determine the expression profiles of the cell surface guidance receptors such as Eph family proteins. Combinatorial expression of Hox proteins and a cofactor, Meis 1, the vertebrate homolog of Hth, determine the specificity of the motor neuron pool subtypes (Ando, 2011).

The result that Hth and Exd are expressed in many of the Drosophila AL neurons suggests that Hth and Exd are unlikely candidates as lineage specific or cell-type specific regulators by themselves. Rather, it is more likely that these TALE-class homeodomain transcription factors control the identity of the AL neuromere in collaboration with other transcription factors that regulate the wiring specificity of individual neurons. In the development of antenna, hth and exd genetically interact with Distalless, which encodes another homeodomain transcription factor. Although Distalless is expressed in the antennal olfactory neurons that innervate the AL glomeruli, it is expressed neither in the olfactory PNs nor in the local interneurons. Unlike the more posterior parts of the central nervous system, none of the homeotic proteins are expressed in the Drosophila deuto- and protocerebrum neuromeres except for Probosipedia that is expressed in a small number of cells at the posterior deutocerebrum. On the other hand, studies on the embryonic brain have shown that Ems plays an essential role in the development of the deutocerebrum primordia that give rise to the ALs. In addition, the activity of ems is required for the generation of the lateral PNs and for precise dendritic targeting of the ad-PNs. Intriguingly, the amino acid sequence Tyr-Pro-Trp, located in the immediate upstream of the Ems homeodomain, partially matches the YPWM motif of the homeotic proteins that are bound by Exd/Pbx proteins. Although the current data demonstrate that Ems expression is independent of the hth activity, the phenotypic commonality that both hth and ems are required for the generation of the lateral but not the ad-PNs suggests a cooperative interaction of the Hth, Exd and Ems proteins in the regulation of down stream programs that regulate the proliferation of the lateral progenitors. In addition, Hth and Exd could cooperatively function with non-homeodomain transcription factors in the AL neurons as demonstrated by the ternary interaction between the Pbx1, Meis1 and MyoD proteins on the downstream target genes that regulate myogenic differentiation (Ando, 2011).

Four Exd-related proteins (Pbx1, Pbx2, Pbx3, and Pbx4) and five Hth-related proteins (Meis1, Meis2, Meis3, Prep1 and Prep2) are found in vertebrates. Mutations of the Pbx/Meis genes cause homeotic transformations in the hindbrain, mimicking the LOF phenotypes of the Hox genes expressed in the anterior neuromeres. In addition, as with the Drosophila homologs, these vertebrate TALE-class homeodomain proteins are expressed in more anterior brain structures during development. In the mouse brain, Meis1/2 and Pbx1/2/3 are expressed in the developing telencephalon, and Meis2 and Pbx1/2 are expressed in the entire dorsal mesencephalon, regulating the expression of the cell-surface EphA8 receptor to a specific subset of cells. In zebrafish development, Pbx4 functionally interacts with Engrailed to pattern the midbrain-hindbrain and diencephalic-mesencephalic boundaries. Moreover, Pbx3 and Meis1 are coexpressed with Rnx, an orphan Hox protein, in the ventral medullary respiratory center to regulate the development and/or functions of inspiratory neurons. Given the cross-phylum commonality in the glomerular organization and neuronal connectivity between the Drosophila and vertebrate olfactory systems, it would be important to determine the functional significance of the TALE-class homeodomain transcription factors in the control of the cell type specificity in the vertebrate brain (Ando, 2011).

Extradenticle and homothorax control adult muscle fiber identity in Drosophila

This study had identified a key role for the homeodomain proteins Extradenticle (Exd) and Homothorax (Hth) in the specification of muscle fiber fate in Drosophila. exd and hth are expressed in the fibrillar indirect flight muscles but not in tubular jump muscles, and manipulating exd or hth expression converts one muscle type into the other. In the flight muscles, exd and hth are genetically upstream of another muscle identity gene, salm, and are direct transcriptional regulators of the signature flight muscle structural gene, Actin88F. Exd and Hth also impact muscle identity in other somatic muscles of the body by cooperating with Hox factors. Because mammalian orthologs of exd and hth also contribute to muscle gene regulation, these studies suggest that an evolutionarily conserved genetic pathway determines muscle fiber differentiation (Bryantsev, 2012).

These results demonstrate a dramatic effect upon muscle fiber identity of the two factors Exd and Hth. The fact that muscle fiber type can be profoundly influenced by the activity of the two genes defines a central mechanism for the control of fiber identity and begins to expose the entire fiber specification pathway (Bryantsev, 2012).

A recent study identified salm as a controller of transition from tubular leg muscle to the fibrillar fiber type. Tubular leg muscles could be transformed into the fibrillar type by ectopic expression of salm. This study has expand these observations to show that the mechanism of Salm action is less straightforward: salm is expressed in the tubular jump muscle, suggesting that its pro-fibrillar action may require cooperation with additional factors. The current data suggest that Salm cofactors could be Exd and Hth: their absence in the jump muscle prevents this muscle from acquiring a fibrillar fiber phenotype despite its expression of salm; also, ectopic expression of salm in leg muscles promotes fibrillar fate, perhaps because the leg muscles also express exd and hth (Bryantsev, 2012).

It is also noted that, in the flight muscles, Exd and Hth maintain their localization in the absence of Salm. Moreover, despite the sustained accumulation of Exd and Hth, loss of Salm nevertheless results in transformation of the flight muscles toward a tubular fate. This indicates that Exd and Hth have at least some requirement for Salm to promote fibrillar muscle fate, and it will be interesting in the future to identify the respective roles of these factors directly interacting with other fiber-specific enhancers (Bryantsev, 2012).

This study also provides a direct mechanistic link between the determinants of fibrillar fate, exd/hth, and the actin gene characteristic of the flight muscles, Act88F. Whether fibrillar muscle genes are direct targets of Exd/Hth or Salm, or both, is yet to be determined; nevertheless, the identification of fiber-specific enhancers will provide new mechanistic insight into this process (Bryantsev, 2012).

Since diverse fiber types are characteristic of many vertebrate muscles, these findings may relate directly to vertebrate myogenesis. In zebrafish, slow muscle fate is promoted by the activities of PBX and MEIS, which are the vertebrate orthologs of Exd and Hth, respectively (Maves, 2007). In mice, PBX and MEIS are cofactors for myogenic determination genes, where they facilitate transcription factor binding to nonconsensus target sites, and this effect might function to fine tune muscle fiber fate (Heidt, 2007). Thus, diverse lines of evidence suggest a robust and conserved mechanism for fiber type specification, acting through PBX1/Exd and MEIS/Hth (Bryantsev, 2012).

Gene duplication, lineage specific expansion and sub-functionalization in the MADF-BESS family patterns the Drosophila wing-hinge

Gene duplication, expansion and subsequent diversification are features of the evolutionary process. Duplicated genes can be lost, modified or altered to generate novel functions over evolutionary time scales. These features make gene duplication a powerful engine of evolutionary change. This study explores these features in the MADF-BESS family of transcriptional regulators. In Drosophila melanogaster, the family contains sixteen similar members, each containing an N-terminal, DNA binding MADF domain and a C-terminal, protein interacting, BESS domain. Phylogenetic analysis shows that members of the MADF-BESS family are expanded in the Drosophila lineage. Three members, that were named hinge1 (CG9437), hinge2 (CG8359) and hinge3 (CG13897) are required for wing development, with a critical role in the wing-hinge. hinge1 is a negative regulator of Wingless expression and interacts with core wing-hinge patterning genes such as teashirt, homothorax and jing. Double knockdowns along with heterologous rescue experiments are used to demonstrate that members of the MADF-BESS family retain function in the wing-hinge, in spite of expansion and diversification over 40 Million years. The wing-hinge connects the blade to the thorax and has critical roles in fluttering during flight. MADF-BESS family genes appear to retain redundant functions to shape and form elements of the wing-hinge in a robust and failsafe manner (Shukla, 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).

homothorax: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity | Protein Interactions | Developmental Biology | References

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

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