Mutations of the hedgehog gene are generally embryonic lethal, resulting in a lawn of denticles on
the ventral surface. In strong alleles, no segmentation is obvious and the anterior-posterior polarity of
ventral denticles is lost. Temperature shift analysis of a temperature-sensitive allele indicates an
embryonic activity period for hedgehog between 2.5 and 6 hr of embryonic development (at 25
degrees) and a larval/pupal period from 4 to 7 days of development (at 25 degrees). Mosaic
analysis of hedgehog mutations in the adult cuticle indicates a series of defined defects associated
with the failure of appropriate hedgehog expression. In particular, defects in the distal portions of
the legs and antenna occur in association with homozygous mutant hedgehog clones in the posterior
compartment of those structures (Mohler, 1988).
In the trunk of the Drosophila embryo, the segment polarity genes are initially activated by the pair-rule
genes; later, the segment polarity genes maintain one another's expression through a complex network of cross-regulatory
interactions. These interactions, which are critical to cell fate specification, are similar in each of the
trunk segments. To determine whether segment polarity gene expression is established differently
outside the trunk, the regulation of the genes hedgehog (hh), wingless (wg), and engrailed
(en) was studied in each of the segments of the developing head. The cross-regulatory relationships
among these genes, as well as their initial mode of activation in the anterior head are significantly
different from those in the trunk. In addition, each head segment exhibits a unique network of segment
polarity gene interactions. It is proposed that these segment-specific interactions evolved to specify the
high degree of structural diversity required for head morphogenesis (Gallitano-Mendel, 1997).
The proposed interactions betweeh hh, wg and en are described below.
1. The intercalary segment. In this cephalic segment, hh expression is en-independent. In addition, ptc mutations cause the loss of wg rather than ectopic wg expression The dependence of wg, en, and hh expression on ptc indicates a unique role for segment polarity genes in the intercalary segment. Unlike wg action in the trunk and gnathal segments, wg restricts rather than maintains en and hh expression in this segment. Finally, en expression, as it occurs in the trunk, depends on hh function. However, this dependence cannot be mediated through wg, since wg does not maintain en expression in the intercalary segment.
2. The antennal segment. As in the trunk, hh antennal expression depends on en, while wg expression requires hh. The requirement for hh is presumably mediated through ptc, which represses wg in this segment. Unlike in the trunk, wg restricts the expression domains of both en and hh. As in the intercalary segment, regulation of en by hh is wg- independent.
3. The ocular segment. In this segment, hh is en-independent and wg expression does not require hh. Although the wg domain (the head blob) does not expand in ptc mutant embryos, noncontiguous ectopic wg expression appears in its vicinity. Unlike its action in the trunk and the other head segments, wg is required to initiate en expression in the ocular segment. However, hh expression still expands in wg mutant embryos (as in the intercalary and antennal segment). As in the intercalary and antennal segments, regulation of en by hh does not depend on wg.
It is concluded that cross-regulatory interactions among the segment polarity genes in the anterior head are very different from those in the posterior head and trunk segments. The mode of patterning of the anterior head (the acron and cephalic segments) is thought to be more ancient than that of the posterior head (the gnathal segments). This distinction appears to be reflected in the segmentation mechanism used by certain present day short germ insects and primitive arthropods. In these organisms, the early germ band includes only the acron, cephalic segments, and tail. Gnathal and trunk segments are generated later in embryogenesis by a progressive budding process (Gallitano-Mendel, 1997).
Drosophila Toll-like receptor (now termed 18 wheeler), encodes a protein containing multiple LRRs (leucine-rich motifs) in its presumed
extracellular domain, and a single transmembrane segment with homology to the
cytoplasmic domain of the interleukin 1 receptor in its presumed intracellular domain.
The pattern of tlr expression at the extended germ band stage is characterized by 15
transverse stripes in the gnathal and trunk segments, with four patches of expression
corresponding to head segments and an additional patch of expression in the
presumptive hindgut. The segmentally repeated TLR stripes in the trunk overlap both the
Wingless and Engrailed stripes and thus span the parasegment boundary. The TLR stripes
require pair rule gene function for their establishment and later become dependent
upon segment-polarity gene function for their maintenance. Segmental modulation of
tlr expression later in the tracheal system is dependent upon the function of the
homeotic genes of the bithorax complex. The tlr gene also is prominently expressed in
the imaginal discs. In the eye disc, this expression occurs in two stripes at the anterior
and posterior margins of the morphogenetic furrow; this expression is consistent with
a genetic interaction between a tlr mutation and an eye-specific allele of hedgehog. These data suggest a role for tlr in interactions between cells at critical
boundaries during development (Chiang, 1994).
Localized or ubiquitous expression of the N-terminal domain of HH, a biologically active form of the protein that lacks the normal lipophilic modification, causes an expansion of wingless expression, ventral cuticle defects including a rectangular rather than trapezoidal shape for the denticle belts and loss of denticle diversity, dorsal cutical defects and embryo lethality. This suggests a role for HH autoprocessing in spatial regulation of hedgehog signaling (Porter, 1996).
The hedgehog (HH) family of ligands plays an important instructional role in metazoan development. HH proteins are initially produced as approximately 45-kDa full-length proteins, which undergo an intramolecular cleavage to generate an amino-terminal product that subsequently becomes cholesterol-modified (HH-Np). It is well accepted that this cholesterol-modified amino-terminal cleavage product is responsible for all HH-dependent signaling events. Contrary to this model this study shows that full-length forms of HH proteins are able to traffic to the plasma membrane and participate directly in cell-cell signaling, both in vitro and in vivo. It was also possible to rescue a Drosophila eye-specific hh loss of function phenotype by expressing a full-length form of HH that cannot be processed into HH-Np. These results suggest that in some physiological contexts full-length HH proteins may participate directly in HH signaling and that this novel activity of full-length HH may be evolutionarily conserved (Tokhunts, 2010).
It is speculated that the two most likely reasons for the current results are that the full-length unprocessed HH proteins retain some substantial level of activity or that the activity of some small undetectable amount of inefficiently processed HH-Np is being observed. The former model is favored because of analyses of HH-U proteins, which are unable to process into their cholesterol-modified forms. However, it remains possible that these full-length HH-U proteins are subject to some small amount of nonspecific proteolytic cleavage that liberates an active amino-terminal product, which would then be responsible for the bulk of activity observed in the study. The results are not consistent with the latter hypothesis for three main reasons: (1) the quantitative nature of the assays used, both in vitro and in vivo, to determine the activity of the full-length HH proteins are inconsistent with the majority of the activity observed being contributed by a small undetectable fraction of the protein, (2) it was possible to specifically immunoprecipitate active SHH-U using two distinct antibodies to the SHH carboxyl-terminal domain, and (3) the holoprosencephaly mutant SHH-T267I, which should be subject to the same putative nonspecific proteolytic clipping as SHH-U and SHH-D222N, exhibited negligible activity (Tokhunts, 2010).
Contrary to the current results, it has been previously suggested that full-length HH proteins are not active. The reasons for the apparent discrepancy between the results of the various groups involved are not known, but it is speculated that the level of expression and type of assay used may be important to visualize the activity of full-length HH proteins (Tokhunts, 2010).
The ability to assay the activity of HH-U proteins was limited to assays that involved cell-cell contact, with the activity contributed by any SHH-U in the conditioned media being negligible. Consistent with this observation, SHH-U was only internalized by PTC when cells expressing each construct were in direct contact. The ability to rescue the ommatidia defect of hh1 flies might also be due to more localized signaling, because HH is othought to act over small distances in the fly eye. This suggests that the biological functions of HH-U proteins might be limited to those that do not require HH to act far from its site of synthesis. Although it is generally accepted that long and short range HH signaling is differentially regulated, this research has focused on how other distinct proteins direct the processed, cholesterol-modified form of HH proteins through different mechanisms. The current results broaden this discussion, because it is suggested that unprocessed full-length HH proteins might be directly responsible for a subset of localized HH signaling. In this model, long and short range HH signaling would be controlled by distinct forms of HH proteins, with long range signaling regulated solely by the cholesterol-modified processed protein and short range signaling controlled, at least in part, by full-length unprocessed HH proteins. Consistent with this model, no significant accumulations of full-length HH proteins were observed in tissues thought to depend primarily on ability of HH to signal over extended distances, such as chicken limb buds. In contrast large amounts of full-length HH protein were detected in Drosophila embryos, in which HH is thought to act in a more localized manner. In conclusion, the results suggest that the relationship between HH processing and activity is more complex than previously thought and that in some biological contexts the full-length forms of HH family members may play a significant physiological role (Tokhunts, 2010).
In Drosophila embryos cubitus interruptus activity is both necessary and sufficient to drive expression of HH-responsive genes, including wingless, gooseberry and patched. To demonstrate that ci is required for transduction of the HH signal, expression of wg was examined in ci null embryos when HH is ubiquitously expressed under control of a heat-shock promoter (Hs-hh). In Hs-hh embryos, wg is expressed ectopically in anteriorly expanded stripes. In ci mutants Hs-hh does not induce ectopic expression of wg. Similar results were obtained for gsb. CI is a sequence-specific DNA binding protein that drives transcription from a wingless promoter in transiently transfected cells. CI binds to the same 9 bp consensus sequence -TGGGTGGTC- as mammalian Gli and Gli3. Alteration of a single nucleotide in the core sequence prevents binding. CI activates transcription from a 5-kb fragment of the wg promoter. CI binding sites in the wg promoter are necessary for this transcriptional activation of. CI element maps to a distal 1-kb region of the 5-kb fragment. The wg promoter sequence has 10 possible Gli consensus binding sites, with three pairs of sites in the distal 1.2 kb. When putatitive CI binding sites are mutagenized, mutant fragments show a greater than 90% reduction in CI-dependent transcriptional activation. Mutagenesis of these sites completely eliminates an electrophoretic mobility shift caused by binding of CI to unmutagenized sites (Van Ohlen, 1997).
Hedgehog (Hh) is an important morphogen involved in
pattern formation during Drosophila embryogenesis and
disc development. cubitus interruptus encodes a
transcription factor responsible for transducing the hh
signal in the nucleus and activating hh target gene
expression. Previous studies have shown that Ci exists in
two forms: a 75 kDa proteolytic repressor form and a 155
kDa activator form. The ratio of these forms, which is
regulated positively by hh signaling and negatively by PKA
activity, determines the on/off status of hh target gene
expression. Exogenous expression of Ci that is mutant for four
consensus PKA sites, CI(m1-4), causes ectopic expression
of wingless in vivo and a phenotype consistent with wg
overexpression. Expression of CI(m1-4), but not Ci(wt),
can rescue the hh mutant phenotype and restore wg
expression in hh mutant embryos. When PKA activity is
suppressed by expressing a dominant negative PKA
mutant, the exogenous expression of Ci(wt) results in
overexpression of wg and lethality in embryogenesis,
defects that are similar to those caused by the exogenous
expression of CI(m1-4). In addition,
in cell culture, the mutation of any one of the three serine-containing
PKA sites abolishes the proteolytic processing
of Ci. PKA is shown to directly phosphorylate
the four consensus phosphorylation sites in vitro. Taken
together, these results suggest that positive hh and negative
PKA regulation of wg gene expression converge on the
regulation of Ci phosphorylation (Chen, 1999).
It can be determined whether PKA phosphorylates
consensus PKA target sites in vitro. Ci fragments of wild type Ci and of
CI(m1-4) that contain the four PKA sites (aa441-1065) were fused to
GST. Two-dimensional tryptic phosphopeptide
maps of the expressed fusion proteins were generated. There are at least 13 phosphopeptides that
are labeled by PKA in the wild-type Ci peptide. In vitro, PKA
can recognize RxS/T, the subset RRxS/T, RxxS/T and
RKxxS/T. The phosphorylation of S is preferred 40:6 over T
and in vivo, the RRxS
site is preferred 2:1 over the others. The four
consensus RRxS/T sites in Ci were chosen for mutation because they would probably
be the preferred phosphorylation sites in vivo. Scanning the
Ci fragment for all possible consensus PKA sites, it was found that all of the phosphopeptides can be accounted for by the
number of PKA consensus sites in the fusion protein. Three
of the strong spots and two weaker spots that are present in
the wild-type fragment are missing in the mutant fragment,
demonstrating that PKA can specifically and directly
phosphorylate the four RRxS/T consensus PKA sites in vitro.
The two weak spots are difficult to distinguish and may
represent only one spot or incomplete digestion of a single
peptide. GST alone was not phosphorylated (Chen, 1999).
What of the positive regulation of Ci activity by hh?
Because the genetic data suggests that hh
does not regulate PKA directly, it may be that hh
affects the phosphorylation state of Ci by activating a
phosphatase, or through changing the accessibility of Ci to a
phosphatase. In support of this idea is the observation that the
phosphatase inhibitor, okadaic acid, stimulates Ci proteolysis,
even in the presence of a Hh signal.
Hh signaling stimulates fu kinase activity to transform full-length
Ci to a transcriptional activator. It may also be that fu activity renders full-length
Ci inaccessible to PKA phosphorylation (Chen, 1999).
The Hedgehog (Hh) signal has an inductive role during Drosophila development. Patched is part of the Hedgehog-receptor
complex and shows a repressive function on the signaling cascade, which is alleviated in the presence of Hh. The first dominant gain-of-function allele of patched has been identified: Confused (patchedCon). Analysis of the patchedCon allele has uncovered novel features of the reception and function of the Hh signal. At least three different regions of gene expression
were identified and a gradient of cell affinities was established in response to Hh. A new state of Cubitus interruptus activity,
responsible for the activation of araucan and caupolican genes of the iroquois complex, is described. This state has been shown to be independent of Fused kinase
function. In the disc, patchedCon behaves like fused mutants and can be rescued by Suppressor of fused mutations.
However, fused mutants are embryonic lethal while patchedCon is not, suggesting that Patched could interpret Hedgehog
signaling differently in the embryo and in the adult (Muller, 2000).
Thus ptcCon has partially impaired Hh-signaling
transduction, interpreting the surrounding Hh concentration
that reaches the cell as lower than it really is. Changes
in Hh concentration alter Hh target gene expression in
ptcCon cells and, subsequently, the ptcCon phenotype, indicating that ptcCon affects the interpretation of Hh levels. The lesion of the ptcCon protein is located in the first
extracellular loop of the Ptc protein, which, in vertebrates,
is involved in binding Shh. A putative explanation for this
would be that ptcCon binds Hh less efficiently, impeding the
proper transduction of the signal. The
transduction of the Hh signal can be interpreted as a balance between Ptc
protein interacting with Hh to open the pathway and Ptc
protein interacting somehow with Smo to block the pathway.
The interaction between Ptc and Hh and between Ptc
and Smo could take place inside the cell in distinct subcellular
compartments. Hh could sequester Ptc to avoid the
negative, direct or indirect, interaction with Smo. If this
were the situation, given that ptcCon binds Hh less efficiently, the result would be more Ptc protein interacting with Smo. The
increase in Ptc-Smo interaction could impede the release or
modification of Smo to transduce the signal. This explanation
also accounts for the dominant effect of ptcCon. In a
heterozygotic fly, both forms of Ptc would be present. One
of them, ptcCon, would have less affinity for Hh, which
would reduce the reception of Hh at the A-P border. Thus,
A cells would receive less Hh because ptcCon competes with
the wild-type protein for the reception of Hh. The high Hh
levels that induce some responses such as anterior En
expression would not be read, provoking the dominant
phenotype of ptcCon (Muller, 2000).
Depending on the domain where a ptcCon clone is
located, the results of blocking the Hh signal are different. The specification of vein 3 has been a subject of debate
due to its morphogenetic implications. Some lines of evidence
suggest that vein 3 differentiation depends upon the
presence of high levels of Dpp. Nevertheless, ectopic expression of Dpp does
not affect vein 3 or promote differentiation in a genetic
background in which Hh signaling is impaired. In ptcCon and fu clones, dpp
is not expressed and yet both types of clones differentiate
vein 3 when the Hh concentration is sufficient to
induce a response. When a dose of hh is removed, ptcCon mutant cells do not differentiate vein 3. It follows that Hh,
and not Dpp, specifies the location of vein 3, and Dpp has a
permissive role in establishing a broad, competent domain
for vein 3 differentiation. The results presented here confirm
that Hh forms a concentration gradient in the A
compartment and strongly suggest that Hh acts as a morphogen in the wing disc to pattern the central region of the wing (Muller, 2000).
In the abdomen, most morphogenetic functions are mediated
by Hh, and although other morphogenetic molecules
might exist, Dpp does not seem to have a role in patterning
the abdomen. In ptcCon discs, dpp is not expressed and this may account for the lack of growth in these discs. Nevertheless, the larvae
reach the third larval instar stage and the discs are similar in size
to those from the second larval instar. Thus, Dpp activation
in response to Hh seemed to function only after the second
larval instar to promote growth and patterning of the discs. Hh may have evolved as the primary morphogen of adult structures and it was not until the advent of appendages during evolution that Dpp was recruited for long-range
patterning of structures. This may be due to a need for a
higher diffusion capacity to pattern the new structures
(wings, antennae, and legs) (Muller, 2000).
Hh is also responsible for inducing a change in cell
affinity. Lack of Smo completely abolishes Hh signaling
and, consequently, impedes the change in A-cell affinity. Although the involvement of Hh and Smo in this process has been clear, that of the Hh-receptor Ptc has
not. There is the possibility of a second signaling pathway,
dependent on Smo but not on Ptc, which would mediate the
responses for changing cell affinity. In this study it is concluded that
the establishment of the lineage restriction border (LRB) depends upon the correct Ptc perception of the Hh signal. The mechanism by which the LRB arises raises a further question: why do A cells responding to Hh not form a
restriction border with A cells not responding to Hh? ptcCon clones close to the P compartment present straight
boundaries with both A and P cells, indicating that the cell
affinity of ptcCon cells is different from that of both
populations of cells. In ptcCon cells, there is a weak
response to Hh, which may be responsible for a discrete
change in cell affinities in ptcCon cells, making them different
from both the P cells, which do not respond to Hh, and
the adjacent A cells, which do respond to Hh. When a copy of hh is removed, ptcCon clones take up more posterior
positions and adopted more wiggly boundaries with P cells,
indicating that their cell affinity is more similar to that of
P cells. Changes in cell affinities seem to form in a gradient
fashion, with different changes in response to different
concentrations of Hh. Adjacent A cells receiving the Hh
signal may have such similar cell affinities that no restriction
border forms between A cells. A similar mechanism
has been suggested to occur in the abdomen of Drosophila (Muller, 2000).
In ptcCon clones, a unique experimental situation is presented
in that reception of Hh signaling is severely impaired,
allowing the accumulation of Ci in the cytoplasm without
the activation of dpp. ptcCon
clones in the wing differentiate vein 3 when close to the P compartment and substitute vein 4 for vein 3. This is in accordance with the
activation of Caup in ptcCon
clones, which is involved in determining vein 3 in the wing
imaginal disc. When lowering the concentration of Hh by
removing a copy of hh, vein 3 is not induced in ptcCon
clones and the levels of cytoplasmic Ci are low,
similar to smo clones that do not differentiate vein 3. In the same line, ptcCon clones close to but not touching the A-P border do not
develop vein 3 nor express Caup. Since ptcCon
cells interpret high Hh levels as low, these
results ascribe the role of determining the position and
differentiation of vein 3 to low levels of Hh. Furthermore,
Ci accumulation in the cytoplasm indicates the activation
of Ci to induce expression of Caup and differentiation of vein 3 (Muller, 2000).
The fact that ptcCon imaginal discs reach second larval
instar suggests that it is not until this stage that the
responses to Hh affected by ptcCon are needed. However,
there is still a paradox: if fu clones behave like ptcCon clones, why are smo and fu mutants embryonic lethal while ptcCon is not? It is proposed that ptcCon affects a function of Ptc that is needed only in larval stages, perhaps to interact with another protein, providing further refinements to Hh-signaling
interpretation. Alternatively, in the embryo, another
protein may participate in the Hh-receptor complex
(so far formed by Ptc and Smo) by binding to Ptc through a
domain not affected by the ptcCon mutation. Evidence for
the existence of other proteins involved in receiving the Hh
signal is provided by the embryonic ptc;hh double-mutant
phenotype, which is not identical to that of ptc, indicating
that Ptc alone does not receive the Hh signal in the embryo. A putative candidate, Hip, has been recently found in vertebrates. Hip is a membrane protein that binds Hh with the same affinity as that of Ptc and, similar to Ptc, is
expressed and modified by Hh. Since ptcCon would affect a
domain of Ptc needed only in larval stages, Ptc function in
embryos would be unaltered (Muller, 2000).
Ci is involved in controlling the transcription of Hh
target genes. It has been recently proposed that Hh controls
both the repressing and the activating functions of Ci. Apart
from negatively regulating the generation of a repressor
form of Ci (Ci-75), Hh controls the activation of Ci. Only two forms of
Ci are detected in a Western blot: a 75-kDa form which bears repressor activity and a 155-kDa form which seems to act as an activator. Two activation states for Ci have been described, both of which are probably modifications of the Ci-155 form.
One is responsible for inducing en and the other for inducing ptc and dpp. Neither of these responses is produced in the absence of fu or in ptcCon cells (Muller, 2000).
The unmasking of a third level of apparent
Ci activity is reported that is independent of the other two levels. This new state of Ci activity is responsible for the
activation of iro and the differentiation of vein 3 in the
wing. The other two levels of Ci activity arise from high
levels of Hh and depend on Fu activity. The new state of Ci
is activated by low levels of Hh and is Fu independent.
Thus, Hh signaling activates two different pathways
through inhibition of Ptc function. Fu would be involved in
mediating transduction of the signal in one of these pathways.
The second pathway would modify Ci to activate it in
a Fu-independent manner. It has been suggested that low levels of Hh activate a
new form of Ci, named 'Ci default', which does not depend
on Fu activity (Muller, 2000).
In the mesoderm of Drosophila embryos, a defined number of cells segregate as progenitors of individual
body wall muscles. Progenitors and their progeny founder cells display lineage-specific expression of
transcription factors but the mechanisms that regulate their unique identities are poorly understood. The homeobox genes ladybird early and ladybird late are shown to be expressed in only one muscle progenitor and its progeny: the segmental border muscle (SBM) founder cell and two precursors of adult muscles. lb activity is associated with all stages
of SBM formation, namely the promuscular cluster, progenitor cell, founder cell, fusing myoblasts and syncytial fiber. The segregation of the ladybird-positive progenitor requires coordinate action of neurogenic genes and an
interplay of inductive Hedgehog and Wingless signals from the overlying ectoderm. The SBM progenitor corresponds to the most
superficial cell from the promuscular cluster,
thus suggesting a role for the overlying
ectoderm during its segregation. . Since epidermal Wg and Hedgehog (Hh) signaling has been shown to
influence muscle formation, the SBM-associated lb expression was examined in embryos
carrying hh and wg thermosensitive mutations. Wg and Hh signalings, mutually dependent at this time, are shown to be required for the promuscular lb activity and/or the segregation of SBM
progenitors. The initial influence of these signals is no longer observed later in
development. In addition to signals from the epidermis, the
activity of the mesodermal gene tinman, initially expressed in the whole trunk mesoderm, is involved in the early events of myogenesis. In tin - embryos, the formation of SBM promuscular clusters and segregation of lb-positive progenitor cells are strongly affected, leading to the absence of
the majority of SBM fibers. During promuscular cluster
formation, since tin expression becomes restricted to the dorsal mesoderm, its influence on ventrolaterally located SBMs is likely to be indirect and mediated via an
unknown factor. The lack of neurogenic gene function, known to be
involved in cell-cell interactions during lateral inhibition, generates the opposite phenotype. Mastermind - and Enhancer of split - embryos fail to restrict promuscular lb expression to only one cell; in consequence, they display a hyperplastic lb pattern in later stages (Jagla, 1998).
During Drosophila embryogenesis, mesodermal cells are
recruited to form a stereotyped pattern of about 30
different larval muscles per hemisegment. The formation
of this pattern is initiated by the specification of a special
class of myoblasts, called founder cells, that are uniquely
able to fuse with neighbouring myoblasts. The COE transcription factor Collier plays a role in the
formation of a single muscle (muscle DA3[A] in the abdominal segments; DA4[T] in the thoracic segments T2 and T3). Col
expression is first observed in two promuscular clusters (in
segments A1-A7), corresponding to two progenitors and
then their progeny founder cells, but its transcription is
maintained in only one of these four founder cells, the
founder of muscle DA3[A]. This lineage-specific restriction
depends on the asymmetric segregation of Numb during
the progenitor cell division and involves the repression of
col transcription by Notch signaling. In col mutant
embryos, the DA3[A] founder cells form but do not
maintain col transcription and are unable to fuse with
neighbouring myoblasts, leading to a loss-of-muscle
DA3[A] phenotype. In wild-type embryos, each of the
DA3[A]-recruited myoblasts turns on col transcription,
indicating that this conversion, accomplished by the DA3[A] founder cell, induces the naive myoblasts to express founder cell distinctive patterns of
gene expression, activating col itself. Muscles DA3[A] and DO5[A] (DA4[T] and DO5[T] respectively)
derive from a common progenitor cell, the DA3[A]/DO5[A] progenitor. However, ectopic expression
of Col is not sufficient to switch the DO5[A] to a
DA3[A] fate. Together these results lead to a proposal that
specification of the DA3[A] muscle lineage requires both
Col and at least one other transcription factor, supporting
the hypothesis of a combinatorial code of muscle-specific
gene regulation controlling the formation and
diversification of individual somatic muscles (Crozatier, 1999a).
The col-expressing promuscular clusters and progenitor cells
have a distinctive position, as defined relative to morphological
landmarks and ectodermal Engrailed (En) expression. The DA3[A]/DO5[A] progenitor cell lies underneath the anterior epidermal compartment, whereas the DT1[A]/DO4[A] progenitor cell lies on the anterior edge of the posterior
compartment, consistent with mapping of the primordium for
the somatic mesoderm. Since Wingless (Wg) and Hedgehog (Hh) signaling have been
shown to be required for mesoderm segmentation and
formation of a subset of muscle founder cells, col expression was analyzed in wg
and hh mutant embryos. At stage 10, both mutant embryos
show changes in mesodermal col expression: rather than being
restricted to specific clusters in the anterior compartment, it
appears almost continuous along the anteroposterior axis. Therefore, both wg and hh signalings appear to restrict col transcription to specific clusters. Lack of Wg or Hh
activity does not seem, however, to impede specification of the
DA3[A]/DO5[A] progenitor, which is singled out in the mutant as well as the wild-type embryos. It was noticed, however, that, while the
DA3[A]/DO5[A] progenitor appears to be specified normally,
more than one cell is singled out from the DT1[A] /DO4[A]
cluster in hh mutant embryos (Crozatier, 1999a).
This study reports the expression pattern of Dll in the genital disc, the requirement of Dll activity for the development of the terminalia and the activation of Dll by the combined action of the morphogenetic signals Wingless (Wg) and Decapentaplegic (Dpp). In Drosophila, the terminalia comprise the entire set of
internal and external genitalia (with the exception of the
gonads), and includes the hindgut and the anal structures. They arise from a single imaginal disc of ventral
origin which is of complex organization and shows bilateral symmetry. The genital disc
shows extreme sexual dimorphism. Early in development,
the anlage of the genital disc of both sexes consists of three
primordia: the female genital primordium (FGP); the male
genital primordium (MGP), and the anal primordium (AP).
In both sexes, only two of the three primordia develop: the
corresponding genital primordium and the anal primordium.
These in turn develop, according to the genetic sex, into
female or male analia. The undeveloped genital primordium
is the repressed primordium (either RFP or RMP,
for the respective female and male genital primordia) (Gorfinkiel, 1999).
During the development of the two components of the anal primordium -- the hindgut and the analia -- only the latter is dependent on Dll and hedgehog (hh) function. The hindgut is defined by the expression of the homeobox gene even-skipped. The lack of Dll function in the anal primordia transforms the anal tissue into hindgut by the extension of the eve domain. Meanwhile targeted ectopic Dll
represses eve expression and hindgut formation. The Dll requirement for the development of both anal plates in males and only for the dorsal anal plate in females, provides further evidence for the previously held idea that the analia arise from two primordia. In addition, evaluation was made of the requirement for the optomotor-blind (omb) gene which, as in the leg and antenna, is located downstream of Dpp. These results suggest that the terminalia show similar behavior as the leg disc or the antennal part of the eye-antennal disc, consistent with both the proposed ventral origin of the genital disc and the evolutive consideration of the terminalia as an ancestral appendage (Gorfinkiel, 1999).
Hh signal is required to form the genital and
anal structures but not the hindgut.
In the leg and antennal discs, the expression of Dll
depends on the Hh signaling pathway. Using the hh
ts2
allele, it was observed that in the genital disc, Hh is also
required for Dll activation: after 4 days at the restrictive
temperature, the genital discs are very small and show
no Dll expression. In the same hh ts2 larvae, residual Dll expression can be detected in the trochanter region
of the leg disc.
However, eve expression in the anal primordia is maintained and occupies most of the reduced genital
disc. This result indicates that Dll, but not eve expression,
depends on Hh and that all the terminalia with the exception
of the hindgut require Hh function.
To further analyze this hh requirement for Dll activation,
the effect of smoothened (smo) lack of function was examined.
In smo2 cells, Hh reception is impeded because smo is a
component of the Hh receptor complex.
In the genital disc, Dll expression only disappears in smo2
clones when the clone is large enough to cover most of the
Dll expression domain. Accordingly,
eve expression is also ectopically activated in smo2 mutant cells; although in Dll2 cells
eve cannot be activated in certain regions of the clones. These results indicate once again that Dll is
dependent on Hh function while eve is not (Gorfinkiel, 1999).
Large smo2 clones close to the A/P compartment transform some structures of the external genitalia and analia. In the female genitalia, smo2
clones duplicate the
long bristle of the vaginal plates and clones
in T8 to produce tissue overgrowth with y2
bristles. Large smo2
clones reduce the female dorsal anal
plate, whereas the female ventral anal plate is rarely
affected. Some clones produce segregated tissue
in the female analia labelled with y bristles in the perianal
region. However, small clones or clones
located outside the A/P compartment border have no effect. In the male genitalia, smo2
clones duplicate the genital arc, part of the claspers and the hypandrium bristle. All these structures are located close to the A/P
compartment border. As in Dll2 clones, large smo2
clones
delete the anal plate in males. In both
males and females, only when the clone is large enough
can Dll expression not be activated in the disc primordia,
giving rise to the Dll2
phenotype. This result suggests that
only in large smo2
clones both wg and dpp are not activated
and therefore are unable to induce Dll expression (Gorfinkiel, 1999).
The hh requirement for the analia but not for the hindgut
has also been confirmed by the ectopic expression of Cubitus
interruptus (Ci). ci encodes a transcription factor that acts
as an activator of the target genes of the Hh pathway. The overexpression of Ci in the
anal primordia of cad-GAL4/UAS-ci flies, leads to the
enlargement and fusion of the anal plates. Accordingly, the Dll expression domain in the genital disc is expanded to cover most of the primordia and the
eve domain is reduced. This again demonstrates
the complementary and exclusive nature of the eve and Dll
domains in the anal primordia (Gorfinkiel, 1999).
The requirement for the Hh signal in Dll activation might
be mediated by Wg and Dpp signals. This occurs in other
ventral discs. Dll expression arises at the juxtaposition of
Wg and Dpp expressing cells as revealed by double staining
for Dll and Dpp, and Dll and Wg. In both genital and anal
primordia, Dll expressing cells overlap those that
express wg and dpp. It has been previously
reported that the ectopic expression of both Wg and Dpp
produces several phenotypic alterations in both female and
male terminalia. Similar types of
transformations are also induced by the lack of function of
either patched (ptc) or Protein kinase A (Pka). In these
mutants, the Hh pathway is constitutively active giving
rise to the derepression of Wg and Dpp. The lack
of Pka function in the genital disc induces ectopic Dll. This Dll induction requires both Wg and Dpp
signals in the same cells since Dll is not activated in Pka2;dpp2 and in Pka2;wg2
double mutant clones, as occurs in other discs of ventral origin (Gorfinkiel, 1999).
In the male repressed primordium (RMP) of the female
genital disc, wg is expressed but not dpp. Consequently, Dll
is not expressed because Dll is only activated in cells that
express both dpp and wg. Ectopic Dpp expression in the wg
expression domain driven by the MS248-GAL4 line
induces Dll 'de novo' in the RMP, which
shows an increase in size. However, these changes do not
allow the development of adult structures from this primordium since there is no activation of the male specifc cyto-differentiation genes because the genetic sex has not changed. Dll
is not activated in the repressed female primordium (RFP)
of the male genital disc despite the fact that, in this primordium, both wg and dpp are normally expressed. This activation does not occur even if the levels of Dpp are increased.
These results suggest that specific genes expressed in the
RFP can exert a negative control of Dll expression (Gorfinkiel, 1999).
In order to find other genes involved in the development
of the terminal structures, the expression
pattern and the functional requirement for optomotor-blind
(omb) were examined. This gene encodes a protein with a DNA-binding
domain (T domain) and behaves
as a downstream gene of the Hh pathway in other imaginal
discs. In the genital disc,
Omb is detected in the dpp expression domains, abutting
the wg expressing cells. This behaviour
of omb expression is similar to that found in the leg and
antennal discs. In the genital
disc, omb is also regulated by the Hh signaling pathway
since Pka2
clones also ectopically express omb.
The phenotypes produced due to omb lack of
function using the allele omb282 were examined; homozygous females for this allele could not be obtained
but some male pharates were analyzed. In males, the dorsal
bristles of the claspers and the hypandrium bristles are
absent. Also, the hypandrium is
devoid of hairs and the hypandrium fragma is reduced. Surprisingly, the anal plates are mostly
somewhat enlarged in the ventral region and reduced in the
dorsal areas. The structures affected in omb2
are
duplicated when omb is overexpressed in the dpp domain
using the dpp-GAL4/UAS-omb combination. In males, the
dorsal bristles of the clasper and the hypandrium
bristles are duplicated. These phenotypes are
similar to the ones obtained as a result of ectopic Dpp (Gorfinkiel, 1999).
The hindgut of the Drosophila embryo is subdivided into three major domains, the small intestine, large intestine, and rectum, each of
which is characterized by specific gene expression. The expression of wingless, hedgehog, decapentaplegic, and engrailed corresponds to the generation or growth of particular domains of the hindgut. wg, expressed in the prospective anal pads, is necessary for activation of hh in the adjacent prospective rectum. hh is expressed in the prospective rectum, which forms anterior to the anal pads, and is necessary for the expression of dpp at the posterior end of the adjacent large intestine. wg and hh are also necessary for the development of their own expression domains, anal pads, and rectum, respectively. dpp, in turn, causes the growth of the large intestine, promoting DNA replication. en defines the dorsal domain of the large intestine, repressing dpp in this domain. A one-cell-wide domain, which delineates the anterior and posterior borders of the large intestine and its internal border between the dorsal and ventral domains, is
induced by the activity of en. A model is proposed for the gene regulatory pathways leading to the subdivision of the hindgut into domains (Takashima, 2001).
The term 'tissue compartments' can be used to indicate the domains of the gut. In this report, the term 'domain' is used in order to avoid confusion with
the term 'developmental compartment', which has been defined
by clonal analysis of the wing disc. To clarify the use of
anatomical descriptions, the organization of the hindgut
domains, as revealed by specific gene expression patterns is described. The most anterior domain of the hindgut, which is just posterior to the midgut, is the small
intestine. The small intestine is followed by the large intestine, then the rectum. The large intestine is further subdivided into a ventral and a dorsal domain. A one-cell-wide domain, which was designated as h4, forms at the anterior and posterior borders of the large intestine, as well as at the border between the dorsal
and ventral domains of the large intestine. The
cells in these regions are designated collectively 'border cells'. Until the
end of stage 12, the hindgut tube is situated on the midline of
the body, and is left-right symmetric. During early stage 13,
the hindgut rotates to the left, resulting in the original dorsal
and ventral domains coming to face the left and right side of
the body, respectively. The orifice of the rectum (the anal
slit) is surrounded by the anal pads, the development of
which is tightly linked to that of the hindgut (Takashima, 2001).
wg, hh, and dpp are expressed in the hindgut of the Drosophila embryo. The expression patterns of these genes have been re-examined in detail to define their exact spatial relationship. wg is expressed throughout the proctodeum at stage 9, then soon becomes restricted to two separate
regions: (1) the primordium of the anal pads, which
surrounds the posterior opening of the hindgut, and (2) a
narrow ring anterior to the small intestine. The expression in these two
domains persists throughout embryogenesis (Takashima, 2001).
hh is expressed throughout the hindgut primordium at
stage 10. Subsequently, as in the case of wg, the expression
is divided into two separate regions at stage 11: the region just posterior to the anterior wg domain, which corresponds to the small intestine, and the posterior-most region of the hindgut, which corresponds to the prospective rectum and is situated just anterior to the anal pads (Takashima, 2001).
Strong defects occur in the hindgut of wg mutant embryos, and it has been
argued that such defects are likely due to an early effect on
cell proliferation. The effects of the hypomorphic mutation wg17en40 on development of hindgut
patterning was examined in detail. In this mutant, proctodeal invagination
is almost normal until stages 9-10, but much of the proctodeum, except the anterior-most region including the small intestine, begins to degenerate after the onset of germband shortening, resulting in a very tiny epithelial sac. The anal pads, which express wg, also degenerate in this mutant. The expression of hh in the
prospective rectum is abolished in this wg mutant, while
the expression of hh in the small intestine is not affected. The hindgut of a null allele of wg (wgPY40) shows
essentially the same defects though overall morphology of
the embryo is affected more severely. This result suggests
that hh expression in the prospective rectum is activated by
wg signaling. To prove this, the effect of ectopic expression
of wg was examined by using the GAL4-UAS system. The UAS-wgts strain, in which functional wg can be induced at the permissive temperature of 17°C, was
mated with the byn-GAL4 strain, in which GAL4 is
expressed throughout the whole hindgut and anal pads. It
was found that hh expression is expanded throughout
hindgut upon misexpression of wg. Except for a
short anterior portion corresponding to the small intestine,
the lumen of the hindgut is characteristically enlarged,
with no morphological boundary between the large intestine
and rectum. Similar results were obtained when an active
form of Armadillo is misexpressed throughout the hindgut. These results strongly
suggest that the ectopic wg activity induces hh expression at
the prospective large intestine, and the latter develops as a
part of the rectum. Conversely, in the hh mutant, there are no
drastic changes in anal pad development or wg expression (Takashima, 2001).
en expression in the dorsal domain of the large intestine, in contrast to that of dpp and hh, is not affected in wg embryos. These results suggest that the defects of the hindgut in wg mutants are partly mediated by failure of hh expression in the future rectum. It should be noted that the defects of the large intestine in the wg mutant are more drastic than those in either hh or dpp mutants. There may exist some pathway of wg action that is not mediated by hh and dpp (Takashima, 2001).
hh is expressed in the prospective rectum and small intestine after stage 11. The hindgut of the hh mutant embryo is shorter than that of wild-type (i.e. about 70% that of wild-type at stage 16. After stage 12 in wild-type embryos, the prospective rectum is recognized by a slightly enlarged lumen at the posterior end of the proctodeum. In hh embryos, the rectum is initially almost normal in size at early stage 12, but after early stage 13 it begins to degenerate, and becomes scarcely recognizable at stage 16. Consequently, the posterior border of en expression in the dorsal domain is more proximate to the orifice. The small
intestine is also reduced in hh mutant embryos, but this defect is
not so drastic when compared with that of the rectum. Growth of the large intestine, which occurs in wild-type embryos after stage 12, is suppressed in hh
mutants, resulting in a short hindgut. In hh embryos, dpp
expression in the region just anterior to the prospective
rectum becomes very weak, but dpp expression in the ventral
domain of the large intestine is not affected or, if anything,
appears to be enhanced. A dpp mutation, in
contrast, does not affect hh expression in the future rectum. These results indicate that hh expression in the prospective rectum is necessary for the development of the rectum itself, and also for sustaining the normal dpp level
in the posterior end of the large intestine. Inductive effects of
hh on dpp expression in the large intestine has been demonstrated
by the ectopic expression of hh. Ectopic expression of hh in
the posterior half of the large intestine by mating the UAS-hh
strain with the hairy-GAL4 strain, which expresses GAL4 in
the posterior half of the hindgut including most of the large intestine, results in markedly expanded dpp expression in the posterior portion of the large intestine, including both the ventral and dorsal regions (Takashima, 2001).
dpp is expressed in two overlapping
regions of the large intestine; these regions appear to be
regulated independently. dpp expression at the posterior
end of the large intestine depends on hh activity in the
adjacent rectum, whereas the weak expression of dpp in
the ventral domain of the large intestine is not affected
in the hh mutant. In the dorsal domain of the large intestine,
where dpp is not expressed except in the posterior-most
portion, en is expressed throughout development. Double
staining for En protein and dpp mRNA reveal that the
en-domain and the dpp-domain do not overlap.
To analyze the regulatory relationship between dpp and en,
dpp expression was examined in an en mutant,
in which en and its paralog invected (inv) are deficient.
Expression of dpp expands to the dorsal domain of the
large intestine in the en mutant, but overall
morphology of the hindgut is almost normal except for
a slight overgrowth. Repression of dpp by en is also
demonstrated by ectopic expression of en. When en is
expressed throughout the hindgut with the GAL4-UAS
system, dpp expression in the hindgut becomes very weak
except in the posterior-most portion of the large intestine, where the hh signal from the adjacent rectum activates dpp expression (Takashima, 2001).
It should be noted that wg and hh mutations result in a
short hindgut, and these mutations are associated with the
reduction of dpp expression in the large intestine. It is very
likely that suppression of the growth of the large intestine
correlates with the decrease in dpp expression. The effect of dpp mutation on the development of the hindgut was therefore examined (Takashima, 2001).
The hindgut of the dpp mutant embryo is of almost normal length based on observation of its overall morphology. However, by in situ hybridization with a byn probe, which detects the whole hindgut and anal pads,
strong homozygous dpp mutant embryos show a significantly shorter hindgut. In these embryos, the anal pads and posterior abdomen are abnormally internalized, forming a tube-like structure continuous to the hindgut orifice. dpp mutation does not affect hh expression in the small intestine or rectum, and these parts develop almost normally. The short hindgut observed in dpp mutants could be a consequence of the failure of normal growth of the large intestine. Consistent
with this idea, when dpp is ectopically expressed throughout the hindgut by the GAL4-UAS system, excessive growth of the hindgut is induced. Excessive
growth is observed only when the patched-GAL4, in which GAL4 strongly expressed throughout hindgut in stages 9-11, is used as a driver (Takashima, 2001).
Hedgehog and segmentation
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Biological Overview
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