extradenticle
See the embryonic expression pattern of exd at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
With the exception of pole cells, where EXD is not found, EXD distribution during early embryogenesis is uniform. Protein levels decline prior to germ band extention. Levels increase again at stage 9 [Image]. Nonuniform expression patterns emerge and by stage 12, transcripts are detected in the head and thoracic regions, while levels decline posteriorly. exd is required for proper neurogenesis and is expressed in the CNS. Expression in the CNS and brain continues after expression is lost in the epidermis. exd is expressed in somatic and visceral mesoderm, where levels are high in anterior regions and low in posterior regions (Rauskolb 1993).
It is noteworthy that although Extradenticle protein's distribution is initially uniform but it is excluded from nuclei until gastrulation. During the extended germ band stage the protein remains predominantly cytoplasmic and does not accumulate in nuclei until germ band retraction. Nuclear accumulation occurs in a pattern that is highly regulated. For example, EXD is present at high levels within the nucleus of visceral mesoderm cells at the positions where the gastric caeca and all the midgut constrictions will form. EXD is also present throughout the underlying endoderm, but it is cytoplasmic at both ends of the midgut and only accumulates in nuclei in the central zone. The zone of nuclear accumulation of EXD in the endoderm, although broader than the region of labial expression, is similarly centered around parasegment 7. In the imaginal discs the nuclear accumulation of Extradenticle is also spatially regulated and, in the wing and leg discs, distal regions exhibit cytoplasmic Extradenticle, whereas proximally the protein is nuclear. In the wing imaginal disc, there are high levels of nuclear EXD in a ring around the area where the wing blade will form. This corresponds to the hinge region. In the notum EXD is nuclear in patches. EXD is cytoplasmic in the wing blade. It is suggested that this regulation of the sub-cellular localization of EXD is important for the interactions between EXD and the homeotic selector proteins and that EXD is not simply a ubiquitously available cofactor (Aspland, 1997).
Arthropod appendages are thought to have evolved as outgrowths from the body wall of a limbless ancestor. Snodgrass, in his Principles of Insect Morphology (1935), proposed that, during evolution, expansion of the body wall would originate the base of the appendages, or coxopodite, upon which the most distal elements that represent the true outer limb, or telopodite, would develop. The homeobox gene Distal-less (Dll), which is required in the Drosophila appendages for development of distal regions is now thought to promote formation of telopodite structures above the evolutionary ground-state of non-limb or body wall. Another homeobox gene, extradenticle, which is required for appropriate development of the trunk and the proximal parts of the appendages, represents a coxopodite gene. exd is transcribed in a pattern that surrounds and abuts Dll-expressing imaginal disc primordia in the ventrolateral epidermis of stage 14 Drosophila embryos. Early in embryogenesis, exd is broadly distributed throughout the embryo and colocalizes with Dll in the limb primordia. exd function is eliminated from the distal precursors in the developing limb and subsequently remains restricted to proximal precursors. This elimination is important because when ectopically expressed, exd prevents distal development and gives rise to truncated appendages lacking distal elements. This restriction of EXD protein to the peripheral parts of the disc is in contrast to its reported uniform mRNA distribution. EXD mRNA accumulates preferentially in the periphery of the leg disc, although lower levels are also detected in the central regions. This EXD mRNA in central regions may be responsible for the low levels of EXD protein detected in the cytoplasm, suggesting that the restriction of exd function to proximal leg parts may be controlled not only transcriptionally but also at the level of nuclear transport. The maintenance of exd expression during larval stages, contrary to Dll, does not require the hedgehog signaling pathway, suggesting that proximal regions of appendages develop independent of hh function. Targeting exd transcription to the distal limb, using a Dll promoter attached to the exd coding region, prevents distal leg development. Ectopic exd seems to interfere with cell proliferation in the central disc and results in cell death induction in the distal domain of the leg. Finally, in the crustacean Artemia, exd and Dll are expressed in comparable patterns as in Drosophila, suggesting a conserved genetic mechanism subdividing the arthropod limb (González-Crespo, 1996).
The varying results presented by mutant exd-clones has been studied to determine the role of EXD in adult patterns for different body parts. In some regions, exd- clones exhibit homeotic transformations similar to those produced by known homeotic mutations such as Ultrabithorax, labial, or Antennapedia. In other regions, the lack of exd causes novel homeotic transformations producing ectopic eyes and legs. Moreover, exd is also required for functions normally not associated with homeosis, such as the maintenance of the dorsoventral pattern, the specification of subpatterns in adult appendages or the arrangement of bristles in the mesonotum and genitalia (Gonzalez-Crespo, 1995).
Looking at genetic mosaics and a hypomorphic exd allele caused by a point mutation in the homeodomain furthered study of the role of exd in adult development. Loss of exd results in homeotic transformations: abdominal segments take on A5 or A6 segmental identity, antenna and arista transform to leg, and head capsule to dorsal thorax or notum. Proximal leg structures are particularly sensitive to the loss of exd, although exd does not influence the allocation of proximal positional values of the leg imaginal disc (Rauskolb, 1995).
Sex combs reduced (Scr) activity is proposed to be required cell nonautonomously for determination of tarsus identity, and Extradenticle (Exd) activity is required cell autonomously for determination of arista identity. Using the ability of Proboscipedia to inhibit the Scr activity required for determination of tarsus identity, it was found that loss-of-Exd activity is epistatic to loss-of-Scr activity in tarsus vs. arista determination. That is, loss-of-Exd activity produces tarsus when there is no Scr activity, suggesting that Exd functions downstream of Scr. This suggests that in the sequence leading to arista determination, Scr activity is OFF while Exd activity is ON, and in the sequence leading to tarsus determination Scr activity is ON, which turns Exd activity OFF. Immunolocalization of Exd in early third-instar larval imaginal discs reveals that Exd is localized in the nuclei of antennal imaginal disc cells and localized in the cytoplasm of distal imaginal leg disc cells. It is propose that Exd localized to the nucleus suppresses tarsus determination and activates arista determination. It is further proposed that in the mesodermal adepithelial cells of the leg imaginal discs, Scr is required for the synthesis of a tarsus-inducer, which, when secreted, acts on the ectoderm cells inhibiting nuclear accumulation of Exd, such that tarsus determination is no longer suppressed and arista determination is no longer activated (Percival-Smith, 1998).
Salivary gland formation in the Drosophila embryo is dependent on Scr. When Scr function is missing, salivary glands do not form, and when Scr is expressed everywhere in the embryo, salivary glands form in new places. Scr is normally expressed in all the cells that form the salivary gland. However, as the salivary gland invaginates, SCR mRNA and protein disappear. Homeotic genes, such as Scr, specify tissue identity by regulating the expression of downstream target genes. For many homeotic proteins, target gene specificity is achieved by cooperatively binding DNA with cofactors. Therefore, it is likely that Scr also requires a cofactor(s) to specifically bind to DNA and regulate salivary gland target gene expression. Two homeodomain-containing proteins encoded by the extradenticle and homothorax genes are also required for salivary gland formation. exd and hth function at two levels: (1) exd and hth are required to maintain the expression of Scr in the salivary gland primordia prior to invagination and (2) exd and hth are required in parallel with Scr to regulate the expression of downstream salivary gland genes. Scr regulates the nuclear localization of Exd in the salivary gland primordia through repression of homothorax expression, linking the regulation of Scr activity to the disappearance of Scr expression in invaginating salivary glands (Henderson, 2000).
To determine if Exd cooperates with Scr to control salivary gland gene expression, the accumulation of two early salivary gland proteins, CrebA and Trh, was examined in embryos lacking exd function. Zygotic loss of exd function results in a reduction in the number of salivary gland cells expressing CrebA and Trh, as well as a decrease in the level of protein made in these cells. This reduced level of salivary gland protein expression is not as severe as the one seen in Scr mutant embryos. Unlike SCR, EXD mRNA is supplied maternally and, thus, the maternal contribution may partially compensate for the loss of zygotic function. To test this possibility, the maternal contribution of exd was removed using the FLP-FRT system. In embryos lacking maternal exd function, salivary gland expression of CrebA and Trh is at wild-type levels. However, salivary gland expression of CrebA and Trh is completely absent in embryos lacking both the maternal and the zygotic contributions of exd. Thus, exd is required for embryonic salivary gland gene expression. Moreover, zygotically provided exd can rescue the loss of maternally provided exd and maternally provided exd can partially compensate for zygotic loss of exd (Henderson, 2000).
Since Scr, exd, and hth are required for salivary gland formation, the mRNA and/or protein expression patterns of these genes during normal salivary gland formation were examined. During stages 9 and 10, when salivary gland gene expression is established, Scr and hth are expressed in the salivary gland primordia, as well as other tissues, and Hth and Exd are nuclear. During stage 11, after the establishment of early salivary gland gene expression, the salivary glands begin to invaginate. At this stage, there are several changes in the expression and/or localization of these genes and/or proteins in the salivary gland cells: Scr and hth transcripts disappear, Hth protein disappears, and Exd protein becomes cytoplasmic (Henderson, 2000).
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 Fasciclin II, an axonal marker, the activities of the extradenticle (exd) and homothorax (hth) genes have been 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).
The Hox/homeotic genes encode transcription factors that generate segmental diversity during Drosophila
development. At the level of the whole animal, they are believed to carry out this role by regulating a large number of
downstream genes. This study addresses the unresolved issue of how many Hox target genes are sufficient to define the
identity of a single cell. Focus was placed on the larval oenocyte, which is restricted to the abdomen and induced in response
to a non-cell autonomous, transient and highly selective input from abdominal A (abdA). Hox mutant rescue
assays were used to demonstrate that this function of abdA can be reconstituted by providing Rhomboid (Rho), a processing factor for the EGF receptor ligand, secreted Spitz. Thus, in order to make an oenocyte, abdA regulates just one principal target, rho, that acts at the top of a complex hierarchy of cell-differentiation genes. These studies strongly suggest that, in at least some contexts, Hox genes directly control only a few functional targets
within each nucleus. This raises the possibility that much of the overall Hox downstream complexity results from cascades of indirect regulation and
cell-to-cell heterogeneity (Brodu, 2002).
Oenocytes are present in clusters of approximately six cells in each of the abdominal segments A1-A7. In the thorax, there is no Egfr induction around the chordotonal organ precursor called C1 and no specific serial homolog of the oenocyte. In order to score unambiguously the presence of oenocytes in a range of different genetic backgrounds, a panel of seven immediate-early, early and late markers were identified. To determine why oenocyte formation is restricted to the abdomen, embryos lacking various Hox genes or extradenticle (exd), which encodes a Hox co-factor, were examined. These experiments indicate that oenocyte formation requires exd and abdA but not two other Hox genes that are also expressed in the abdomen: Antp and Ubx. To assess whether oenocytes form in the absence of all Hox functions, the T1 segment was examined in embryos lacking Sex combs reduced (Scr) and Antp activities. No oenocytes are produced in this context, and therefore these cells are not part of the ground state. However, the ground state does contain both the signaling and responding cell types involved in oenocyte induction: C1 and the Sal-positive dorsal ectoderm (Brodu, 2002).
Genetic analysis shows that Engrailed has both negative and positive targets. Negative regulation is expected from a factor that has a well-defined repressor domain but activation is harder to comprehend. VP16En, a form of En that has its repressor domain replaced by the activation domain of VP16, has been used to show that En activates targets using two parallel routes, by repressing a repressor and by being a bona fide activator. The intermediate repressor activity has been identified as being encoded by sloppy paired 1 and 2 and bona fide activation is dramatically enhanced by Wingless signaling. Thus, En is a bifunctional transcription factor and the recruitment of additional cofactors presumably specifies which function prevails on an individual promoter. Extradenticle (Exd) is a cofactor thought to be required for activation by Hox proteins. However, in thoracic segments, Exd is required for repression (as well as activation) by En. This is consistent with in vitro results showing that Exd is involved in recognition of positive and negative targets. Moreover, genetic evidence is provided that, in abdominal segments, Ubx and Abd-A, two homeotic proteins not previously thought to participate in the segmentation cascade, are also involved in the repression of target genes by En. It is suggested that, like Exd, Ubx and Abd-A could help En recognize target genes or activate the expression of factors that do so (Alexandre, 2003).
Wg
signaling contributes to the activation of En's positive targets. The temporal aspect of this requirement has not been investigated, but earlier results
suggest that it is probably transient. Note
that Wg signaling is irrelevant to repression by En and that, even in cells
that are within the range of Wg, repression and activation (of distinct
targets) coexist. For example, in the normal domain of en expression,
ci is repressed and hh is activated. Therefore, Wg signaling
does not convert En from an activator to a repressor. Perhaps Wg signaling
helps the recruitment, on specific targets, of a cofactor needed to mask the
repressor domain of En, while at the same time providing an activation domain.
One candidate cofactor that could be regulated by Wg is the homeodomain
protein encoded by exd, a known cofactor of Hox gene activity in
vivo. However, Exd is not an
activation-specific cofactor and more work is therefore needed to understand
how Wg signaling contributes to the activating function of En (Alexandre, 2003).
Two types of activities have been ascribed to Exd. According to the selective binding model, Exd could help En recognize positive
targets and assemble a transcription complex. Alternatively, or in addition,
Exd could mask the repressor domain of En and, at the same time, recruit an
activator (the so-called activity regulation model). Adding a functional activation domain to En (as in VP16En) does not
override the need for Exd. This gives in vivo support to the selective binding
model and is consistent with in vitro studies, which have shown that Exd and
En can dimerize and bind DNA cooperatively. Cooperativity requires the eh2 domain of En,
a domain that is left intact in VP16En. Because VP16En
requires Exd for in vivo activity, it is concluded that the N-terminal half of En,
which is absent in VP16En, is not required for the interaction with Exd (Alexandre, 2003).
In thoracic segments, VP16En requires exd to act
on all En targets, positive and negative. This is the first indication that
Exd could be involved in negative (as well as positive) target recognition by
En. Indeed in thoracic segments wild-type En requires Exd for repression of
its natural targets. This had presumably not been noticed previously because
endogenous expression of En is lost in the absence of Exd. That Exd could be
involved in repression is consistent with in vitro studies with PBX proteins
and earlier suggestions from in vivo work with Drosophila. Because Exd is required for both repression and activation, the
issue of what distinguishes activated targets from repressed ones remains
unresolved. Throughout the present study, it has been found that the two En-positive
targets, en and hh, are expressed identically in a variety
of experimental conditions. It may therefore be that the regulatory regions of
these two genes might contain unique features that make them positive
targets (Alexandre, 2003).
En must be capable of activating transcription in the
appropriate context. Because En harbors a robust repressor domain, it is
likely that one or several cofactor(s) mask this domain and recruit an
activation function and, it is unlikely that Exd alone
provides such an activity. Nevertheless, the possible role of Hth is worth
discussing. In vitro, Hth binds DNA as a part of a ternary complex with Exd
and a Hox protein. Intriguingly, overexpression of an activator form of Hth
(VP16Hth) phenocopies the overexpression of wild-type Hth (VP16Hth mimics
overactive Hth). This suggests that the normal role of Hth is to bring an
activation domain to a complex -- a conclusion that contradicts the
observation that Hth is required for both repression and activation by En. One
way to resolve this paradox would be to suggest that Hth has two distinct
roles: to help target recognition on negative and positive targets and, in
addition, to bring an activation domain onto positive targets. Of course
activation by En could also involve as yet unidentified activating cofactors.
Further progress will require the identification, within natural targets, of
enhancers that confer either activation or repression. Comparing these sites
and subsequent mutational and biochemical analysis could lead to a molecular
understanding of what distinguishes negative from positive targets (Alexandre, 2003).
The most unexpected aspect of these results is that, in abdominal segments,
the Hox proteins Ubx and Abd-A are involved in repression by En. In formal
genetic assays, Ubx and Abd-A can substitute for Exd in helping En act on
negative targets. In the absence of Ubx, Abd-A and Exd, En can no longer
repress target genes. By contrast, two other Hox proteins (Antp and Abd-B)
appear not to be involved in En function. Antp does
not help En repress targets in vivo even though its homeodomain differs from
that of Abd-A at only five positions. Likewise, Abd-B, a more distantly
related Hox protein, is also unlikely to participate in En function. It is concluded that the role of Ubx and Abd-A in repression by En is
specific (Alexandre, 2003).
How could ectopic Ubx or Abd-A allow En to repress targets in the absence
of Exd? It could be that this is mediated by wholesale transformation of
segmental identity [although such transformation would have to be
exd/hth-independent. Alternatively, Ubx and Abd-A could have a more
immediate involvement in En function. One can envisage that they could
regulate an as yet unidentified corepressor of En (although such regulation
would not require Exd). Alternatively, and more speculatively, Ubx and Abd-A
could serve as cofactors themselves in regions of the embryo where Exd levels
are low. Again, molecular analysis of negative targets will be needed to
discriminate these possibilities (Alexandre, 2003).
Homeotic genes have not been previously implicated in En function despite
many years of genetic analysis of the Bithorax complex. It is suggested that the
role of Ubx and Abd-A in En function has been overlooked previously because,
in the absence of these two genes, Exd is upregulated in the presumptive
abdomen and thus takes over as a repression cofactor. However, the present
results establish that homeotic genes do participate in the segmentation
cascade and link two regulatory networks previously thought to be
independent (Alexandre, 2003).
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extradenticle: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation
date revised: 20 September 2007
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