homothorax
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).
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 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 Gonzˇlez-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).
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).
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).
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