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 full-length unprocessed hedgehog protein is an active signaling molecule

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).

Transduction of the HH signal

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).

Hedgehog and muscle development

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).

Hedgehog and terminalia

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

Continued: hedgehog Effect of mutation part 2/3 | part 3/3

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

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