Hedgehog and Segmentation

In many instances, remote signaling involves the transport of secreted molecules. The spread of Wingless within the embryonic epidermis of Drosophila was examined. Using two assays for Wingless activity (specification of naked cuticle and repression of rhomboid transcription), it was found that Wingless acts at a different range in the anterior and posterior directions. This asymmetry follows in part from differential distribution of the Wingless protein. Transport or stability is reduced within engrailed-expressing cells, and farther posteriorward Wingless movement is blocked at the presumptive segment boundary and perhaps beyond. The role of hedgehog in the formation of this barrier is demonstrated (Sanson, 1999).

It is proposed that asymmetric Wingless distribution ensures the establishment of well-differentiated cell fates on either side of the engrailed domain. Anteriorly, at the wingless source, rhomboid expression is repressed. In contrast, reduced Wingless movement and/or stability within the engrailed domain allows nascent posterior rhomboid expression. Around this time (stage 11), a barrier to Wingless that requires hedgehog signaling forms at the posterior of the engrailed domain and ensures that Wingless does not foray across and repress rhomboid. rhomboid then activates the Egfr pathway within its expression domain and in adjacent cells. It may be that rhomboid itself contributes to barrier formation and thus builds a line of defense against invasion by its repressor. In addition, activation of the EGF pathway by rhomboid would antagonize any Wingless leaking through. Denticle formation requires transcription of shavenbaby, which is under positive regulation by the Egfr pathway and negative regulation by the wingless pathway. Activated EGFR and the absence of Wingless posterior to the engrailed domain allow shavenbaby expression and hence denticle formation. At the anterior side, converse conditions exist, since Wingless is present at high levels and the Egfr pathway is inactive. Therefore, it is proposed that polarization of Wingless transport by engrailed and hedgehog guarantees the naked fate anterior to the engrailed domain and the denticle fate posteriorly, and thus establishes the anteroposterior polarity of each segment (Sanson, 1999 and references).

In the ventral abdominal region of the Drosophila embryo, wingless is expressed in single cell-wide stripes. To assess the range of Wingless in specifying the naked fate, these stripes were mapped onto the final cuticle pattern. Unexpectedly, the wingless stripes were found to be are eccentric within each expanse of naked cuticle. Naked cuticle is made over a distance of approximately 3-4 cell diameters anterior to the wingless source. In contrast, posterior to it, only the adjoining cells make naked cuticle; these cells are the most anterior of each engrailed stripe. The denticle fate of more posterior engrailed-expressing cells could be explained if they were unable to respond to wingless. Therefore the responsiveness to Wingless of all epidermal cells was assessed. To ask whether Wingless is required beyond one cell diameter posterior to its source, in individual cells the ability to respond to Wingless was removed and the phenotypic consequences was analyzed at single-cell resolution. Armadillo is a downstream effector of Wingless signal transduction and is also a component of Cadherin-based adhesion complexes. Armadillo is titrated out of its signaling pool when Shotgun (DE-Cadherin) is overexpressed, and in the embryonic epidermis, this leads to a phenocopy of a wingless mutation. Overexpressed Shotgun, therefore, blocks the response to Wingless in a cell-autonomous manner. Shotgun was overexpressed in single cells with the "Flp-on Gal4" system. Clones of cells expressing Gal4, which in turn activates expression of Shotgun and GFP, were induced during early stage 11 and scored at the end of embryogenesis. GFP-positive cells within naked regions make ectopic denticles, confirming that wingless signaling is required everywhere naked cuticle is made. All Shotgun-overexpressing cells located within belts make denticle of the type and size expected for their position. These results show that wingless signaling (via Armadillo) is not required in the presumptive denticle belts. Wingless signaling is only required within the naked domain, and this requirement is asymmetric relative to the wingless source (Sanson, 1999).

Two types of mechanisms could account for the asymmetry of wingless action: (1) the wingless protein itself could be unequally distributed in the anterior and posterior directions, or (2) Wingless could be distributed symmetrically, but downstream signaling would be repressed posterior to the source. Immunocytochemistry has revealed an asymmetric distribution of the wingless-containing vesicles within each segment at stage 11. By colabeling with anti-Engrailed, it has been shown that the posterior transition in Wingless distribution occurs at the interface with engrailed-expressing cells. Although Wingless protein can be detected in the wingless domain and anterior to it, only a small number of Wingless-containing vesicles can be seen in the most anterior row of engrailed cells, and none can be seen more posteriorly. Thus, Wingless distribution is asymmetric, and this could explain the asymmetry of wingless action (Sanson, 1999).

It is conceivable that undetectable yet active Wingless is present in cells posterior to engrailed stripes. To demonstrate the absence of active Wingless there, a functional assay was used, based on the finding that wingless signaling represses rhomboid expression. From stage 11 onward, rhomboid is expressed in stripes just posterior to each engrailed domain, and this expression is abolished by continuous and uniform expression of Wingless. Later ectopic expression, induced at late stage 11, inhibits rhomboid transcription only in the midventral region, and if sibling embryos are left to develop, they make ectopic naked cuticle in the same region. Therefore, Wingless can repress rhomboid transcription in the same time window as it specifies naked cuticle. Wingless is not only sufficient for rhomboid repression, it is also necessary since wingless null mutants have an additional rhomboid stripe in each abdominal segment. The position of these extra stripes relative to landmarks in the CNS suggests that they form at the anterior of the domain of extinct engrailed expression, where wingless would normally be expressed. Thus, in the wild type, the presence of Wingless at the anterior of each engrailed stripe maintains the silence of rhomboid expression there. Significantly, rhomboid is expressed posterior to the engrailed domain of wild-type embryos. Therefore, active Wingless is not present in these cells, at least at late stage 11; if it were, rhomboid would not be expressed. These cells are located only two cell diameters posterior to the Wingless source (Sanson, 1999).

The asymmetric distribution of Wingless could be explained by decreased transport/stability either within the engrailed domain or at its posterior edge, where the segment boundary forms. To explore this, wingless was misexpressed directly in the engrailed domain (posterior to endogenous wingless) and it was determined whether the range of Wingless was shifted posteriorly. Wingless was expressed with the engrailed-Gal4 driver in otherwise wild-type embryos. The only effect on the cuticle pattern is the loss of row 1 denticles. Remarkably, no other denticles are lost. In particular, row 2 denticles are present even though they are adjacent to the Wingless-misexpressing cells. Thus wingless expressed at the anterior side of the presumptive segment boundary does not affect the fate of cells on the posterior side. To confirm this finding, rhomboid expression was used as an early molecular marker for the absence of Wingless. In the wild-type larva, rhomboid is expressed in the cells secreting rows 2-4. In en-Gal4/UAS-wg larvae, this expression is unchanged, indicating that the wingless pathway is not operative in the cells immediately posterior to the wingless-misexpressing cells. Thus, it appears that Wingless cannot cross the posterior edge of the engrailed domain. This was verified by looking directly at the distribution of Wingless protein in en-Gal4/UAS-wg embryos. In these embryos, Wingless is present within the domain of wingless misexpression, as expected. However, it is not detectable posterior to the engrailed-expressing cells. It is concluded that a barrier to Wingless protein movement exists at the presumptive segment boundary (Sanson, 1999).

Among various candidate genes, hedgehog was found to be required for the posterior barrier to Wingless. Assaying the range of Wingless in a hedgehog mutant is not straightforward, since wingless expression requires hedgehog signaling. Therefore, wingless expression was maintained artificially in hedgehog null mutants using en-Gal4. Normally the engrailed promoter also turns off in a hedgehog mutant for lack of wingless, but in en-Gal4/UAS-wg; hh- embryos, this is remedied by exogenous Wingless. Thus, a hedgehog-independent positive feedback loop is established between engrailed and wingless, and stripes coexpressing Wingless and Engrailed are obtained. The distribution of the Wingless protein in en-Gal4/UAS-wg; hh- embryos is different from that seen in en-Gal4/UAS-wg control embryos. Wingless spreads posterior to the engrailed domain as if a barrier had been lifted or Wingless movement enhanced. The resulting protein distribution is symmetrical, and this is reflected in the cuticle pattern: in contrast to en-Gal4/UAS-wg embryos, en-Gal4/UAS-wg; hh- embryos lack rows 2-4 and, instead, have an extra expanse of naked cuticle. At the positions where rows 5 and 6 normally form, lies a thin stripe of small denticles. Naked cuticle is specified equally in the anterior and posterior directions, as shown by marking the wingless-expressing cells with GFP. Thus, in the absence of hedgehog, wingless action is symmetric (Sanson, 1999).

Thus, loss of hedgehog signaling increases the range of Wingless. Now it was asked whether the converse is true. To assay the range of Wingless in the presence of excess hedgehog signaling, endogenous wingless must be removed because hedgehog signaling activates wingless expression and this would confuse the assay. Therefore, the en-Gal4/UAS-wg combination was used again, but this time in a wingless mutant background. The sole source of Wingless in these embryos is in the engrailed domain. The wingless mutant phenotype is significantly rescued: the normal alternation of denticle belts and naked cuticle is restored, and many belts are nearly wild type, except for the loss of row 1. In these embryos, the width of the band of naked cuticle is 4-5 cells, and this provides an assay for the anterior range of Wingless. This assay was validated with a version of Wingless expected to act only at short range. If a membrane-tethered form of Wingless is expressed instead of the wild-type protein, an expanse of naked cuticle only 1-2 cells wide is found. This demonstrates that Wingless has to be physically transported from cell to cell to specify a band of naked cuticle of the normal size, and that there is no relay mechanism. Next, the assay was used to find out the effect of increasing hedgehog signaling on the range of Wingless. Increased hedgehog signaling can be achieved either by overexpressing Hedgehog or by removing patched activity. wg- en-Gal4/UAS-wg embryos carrying in addition UAS-hedgehog have significantly narrower naked domains. Likewise, wg- ptc- en-Gal4/UAS-wg embryos have narrow naked bands as well. This suggests that excess hedgehog signaling reduces the range of Wingless, although excess Hedgehog signaling could also induce ectopic rhomboid, which would in turn antagonize Wingless signaling and bring about the loss of naked cuticle (Sanson, 1999).

It is known that Wingless sustains engrailed expression only in adjoining cells, suggesting that Wingless is not readily transported across the engrailed domain. This is supported by the asymmetric distribution of the protein. Immunostaining reveals the presence of Wingless anterior to its source, whereas very little is detected posteriorly; posterior to wingless-expressing cells, in the engrailed domain, some Wingless staining is found but only in the most anterior cells (nearest the Wingless source). Thus, engrailed-expressing cells appear to restrict Wingless movement. Restricted Wingless transport through the engrailed domain could be explained by the downregulation of a specialized transport receptor in the engrailed cells; the existence of such a receptor has been hypothesized. Alternatively, inefficient transport could follow from selective instability of Wingless or its sequestration within the engrailed domain. In wing imaginal discs, the stability and range of Wingless increase in response to overexpression of its receptor Frizzled2. By analogy, and conversely, Wingless might be particularly unstable within engrailed stripes for lack of a receptor there. Alternatively, the surface or extracellular matrix surrounding engrailed cells might trap Wingless and impede its movement. A receptor of the proteoglycan type could possibly mediate this activity. Indeed, in mutants for the gene encoding UDP-glucose dehydrogenase, that lack HSPGs, embryonic engrailed stripes are temporarily widened, implying an increased range of Wingless. Identification of the relevant receptors and their pattern of expression will be required to discriminate between the above alternatives (Sanson, 1999 and references).

Not only is Wingless movement restricted within the engrailed domain, but a barrier seems to exist at its posterior edge. This is especially evident in embryos that ectopically express Wingless in the engrailed domain. In these embryos, Wingless does not specify naked cuticle nor repress rhomboid posteriorly, even in adjacent cells. The lack of response is unlikely to be due to insufficient expression, since en-Gal4 is a robust driver. Also, uniform wingless expression (even at low levels and up to late stage 11) induces uniform naked cuticle (D) and represses rhomboid transcription. This suggests that all cells, including those posterior to each engrailed stripe, are responsive to Wingless (although it is formally possible that the latter cells are only responsive to autocrine signaling). Thus, the lack of posterior response in en-Gal4/UAS-wg embryos is probably because, in this experimental situation, Wingless does not reach posteriorly. Indeed, in the same embryos, immunostaining fails to detect Wingless protein posterior to the engrailed domain. The best interpretation of the results is that a barrier to Wingless movement exists at the segment boundary, although the possibility that movement is impeded throughout the rhomboid expression domain or that these cells are unable to respond to paracrine Wingless cannot completely excluded (Sanson, 1999).

The notion that Wingless movement is blocked at the forming segment boundary contrasts with an earlier proposal that Wingless spreads symmetrically. According to this view, posterior to its source, Wingless signaling is antagonized by active EGFR. The Egfr pathway is activated within and near the rhomboid stripe, which lies just posterior to the segment boundary. However, it is proposed that this segmental activation, which requires rhomboid, occurs after formation of the restrictions to Wingless movement. If wingless protein were present in the rhomboid cells at late stage 11, rhomboid expression would not be allowed there since wingless has been shown to repress rhomboid transcription. Subsequent establishment of rhomboid expression would further counteract activation of the Wingless pathway in prospective denticle belts (Sanson, 1999).

It is suggested that two mechanisms restrict posterior Wingless movement. The first restriction occurs within the engrailed domain and is unlikely to be under hedgehog control, since engrailed cells are not thought to respond to Hedgehog. Rather, engrailed could implement this restriction by controlling a gene involved in Wingless transport, sequestration, or stability. By contrast, the barrier at the posterior of the engrailed domain requires hedgehog signaling. Wingless produced ectopically in the engrailed domain of hedgehog mutants is allowed to invade posteriorly located cells and induce naked cuticle there. The finding that the same effects are seen in cubitus interruptus mutants indicates that the hedgehog signaling pathway is involved. The role of the hedgehog pathway is confirmed by "gain-of-function" experiments. Loss of patched results in overactivation of the hedgehog pathway and so does excessive hedgehog expression. Both situations reduce the range of Wingless in the anterior direction as if the spread of the protein were reduced. It is presumed that, in the wild type, a downstream Hedgehog target is upregulated at the posterior of each engrailed/hedgehog stripe and this would lead to Wingless destabilization or a block to transport there (Sanson, 1999).

Each abdominal segment produces a large dorsal cuticular plate (the tergite) and a smaller ventral plate (the sternite). Each tergite can be divided into three regions: an acrotergite that contains undecorated sclerotized cuticle, a central region containing an array of microchaetes, and a posterior region that contains a dark pigment band as well as a row of large macrochaetes at the posterior edge of this posterior region. All of the tergite, except the acrotergite, is covered with trichomes. For convenience, the posterior boundary of the tergite is defined to be the posterior edge of the pigment band. The intertergal cuticle is unpigmented and composed of an anterior trichome-bearing region (the posterior hairy zone or PHZ) and a posterior region of naked cuticle (the intersegmental membrane or ISM). All trichomes and bristles in the abdomen are oriented from the anterior to the posterior. The tergite and anterior portion of the PHZ develop from the anterior dorsal histoblast nest; the rest of the PHZ and the ISM develop from the posterior dorsal nest (Kopp , 1997a).

Hedgehog protein secreted by posterior compartment cells plays a key role in patterning the posterior portion of the anterior compartment in adult abdominal segments. Loss of function of hh in the hh(ts2) mutant causes the loss of posterior tergite characteristics in the anterior compartment, whereas ectopic expression driven by hs-hh or the gain-of-function allele hh(Mir) causes transformation of anterior structures toward the posterior. hh-expressing clones in the anterior compartment induce surrounding wild-type cells to produce posterior tergite structures, establishing that HH functions nonautonomously. The effects of pulses of ectopic expression driven by hs-hh indicate that bristle type and pigmentation are patterned by HH at widely different times in pupal development. The primary polarization of abdominal segments is symmetric. This symmetry is strikingly revealed by ectopic expression of engrailed. As expected, this transforms anterior compartment cells to a posterior compartment identity. However, ectopic en expression causes an autonomous reversal of polarity in the anterior portion of the anterior compartment, but not the posterior portion. By determining the position of polarity reversal within en-expressing clones, a cryptic line of symmetry can be defined that lies within the pigment band of the normal tergite. This line appears to be retained in hh(ts2) mutants raised at the restrictive temperature, suggesting it is not established by hh signaling. It is argued that the primary role of hh in controlling polarity is to cause anterior compartment cells to reverse their interpretation of an underlying symmetric polarization. Consistent with this, it is found that strong ectopic expression of hh causes mirror-symmetric double posterior patterning, whereas hh loss of function can cause mirror-symmetric double anterior patterning (Kopp, 1997a).

This patterning function of Hh is mediated by optomotor-blind. omb- mutants mimic the effects of loss-of-function alleles of hh: structures from the posterior of the anterior compartment are lost; often this region develops as a mirror image of the anterior portion. Structures from the anterior part of the posterior compartment are also lost. In the pupa, omb expression in abdominal histoblasts is highest at or near the compartment boundary, and decreases in a shallow gradient toward the anterior. This gradient is due to activation of omb by Hh, secreted by posterior compartment cells. In contrast to imaginal discs, this Hh signaling is not mediated by dpp or wg. Several hh gain-of-function alleles have been described that cause ectopic expression of omb in the anterior of the segment. Most of these cause the anterior region to develop with posterior characteristics without affecting polarity. However, an allele that drives high level ubiquitous expression of omb (QadroondFab) causes the anterior tergite to develop as a mirror-image duplication of the posterior tergite, a pattern just the opposite of that seen in omb- mutants. The Qd Fab allele has a dramatic effect on both polarity and bristle patterning. In Qd Fab hemizygotes and heterozygotes, the anterior tergite and intersegmental membrane (ISM) are deleted and replaced with a mirror-image duplication of the posterior tergite and PHZ. Ectopic macrochaetes are often, but not always, present at the anterior edge of the duplicated tergite structures, and sometimes also in the central tergite. The lines of polarity reversal are not fixed precisely with respect to cuticular pattern. In the most extreme phenotype, polarity is reversed exactly in the middle of the tergite and in the middle of the PHZ. More frequently, the line of polarity reversal is shifted anteriorly in the tergite and posteriorly in the PHZ. The phenotype is stronger in hemizygous males than heterozygous females, and is stronger in more posterior segments. The intertergal region is often compressed, and the dorsal longitudinal muscles underlying the tergites show irregular spacing and attachment sites (Koop, 1997b).

omb alleles cause defects that are reciprocal to those of the Qd alleles. Hemizygotes for omb loss-of-function alleles mostly die as late larvae or early pupae; only a small percentage survive to the late pharate adult stage. Among the latter, the loss of structures that lie within the posterior region of the anterior compartment and the anterior region of the posterior compartment have been observed. In many hemisegments, especially those more anterior in the animal, posterior tergite and PHZ are deleted and replaced with a mirror-image duplication of the anterior tergite. This phenotype is exactly reciprocal to the phenotype of Qd Fab (Koop, 1997b).

Ubiquitous expression of hh causes double-posterior patterning similar to that of Qd gain of function alleles. omb- alleles suppress this effect of ectopic hh expression and posterior patterning becomes independent of hh in the QdFab mutant. These observations indicate that omb is the primary target of hh signaling in the adult abdomen. However, it is clear that other targets exist. One of these is likely to be Scruffy, a novel gene, which acts in parallel to omb. To explain the effects of omb alleles, it is proposed that both anterior and posterior compartments in the abdomen are polarized by underlying symmetric gradients of unknown origin. It is suggested that omb has two functions: (1) it specifies the development of appropriate structures both anterior and posterior to the compartment boundary and (2) it causes cells to reverse their interpretation of polarity specified by the underlying symmetric gradients (Koop, 1997b).

Hedgehog, but not Odd skipped, induces segmental grooves in the Drosophila epidermis

The formation of segmental grooves during mid embryogenesis in the Drosophila epidermis depends on the specification of a single row of groove cells posteriorly adjacent to cells that express the Hedgehog signal. However, the mechanism of groove formation and the role of the parasegmental organizer, which consists of adjacent rows of hedgehog- and wingless-expressing cells, are not well understood. This study reports that although groove cells originate from a population of Odd skipped-expressing cells, this pair-rule transcription factor is not required for their specification. It was further found that Hedgehog is sufficient to specify groove fate in cells of different origin as late as stage 10, suggesting that Hedgehog induces groove cell fate rather than maintaining a pre-established state. Wingless activity is continuously required in the posterior part of parasegments to antagonize segmental groove formation. These data support an instructive role for the Wingless/Hedgehog organizer in cellular patterning (Mulinari, 2009).

It has been reported that segmental groove formation requires the activity of engrailed (en) and hh and that en has a function that is independent of its role in hh activation. More recently, it was been found that en is not expressed in groove cells, thus creating a non-cell-autonomous requirement for en. To address this issue, the role of hh and en in segmental groove formation was reinvestigated (Mulinari, 2009).

It was found that segmental grooves do not form in hh mutants. When hh was overexpressed, the four to five cell rows posterior to the Hh source constricted apically, elongated their apical-basal axis and took on a shape characteristic of segmental groove cells. Very similar cell behavior was observed in patched (ptc) mutants or when activated Ci, which mediates hh activity, was expressed. These observations suggest that Hh can organize segmental groove formation. No cell constrictions were observed in the ventral epidermis, indicating that a different mechanism might regulate cell shape there (Mulinari, 2009).

To address the proposed hh-independent function of en, en, invected (inv) double mutants were investigated in which hh expression was maintained using prd-Gal4. Segmental grooves were rescued in these mutants, suggesting that en is not required for segmental groove formation independent of its role in hh activation. By contrast, it was found that en represses groove cell behavior when ectopically expressed together with hh. A previous study that reported a requirement of en in groove formation was based on the analysis of en, inv, wg triple mutants, in which hh expression was maintained but did not rescue groove formation. This result was confirmed, but it is proposed that wg may be required in en mutants to allow the morphological differentiation of grooves (Mulinari, 2009).

Analysis of ptc mutants, or embryos overexpressing hh, reveals that a broad region of cells posterior to the en expression domain are specified as groove cells. However, groove-like invaginations form only at the edges of these regions. This is even more obvious in double mutants of ptc and the segment polarity gene sloppy paired 1 (slp1), which is required for maintained wg expression. In slp1, ptc mutants, wg expression fades prematurely and Hh signaling is constitutively active. This results in a substantial expansion of the number of groove cells. However, furrows differentiate only at the edges of groove cell populations. It is proposed that the morphological differentiation of segmental grooves can occur only at the interface between groove and non-groove cells (Mulinari, 2009).

To test this, wg, ptc double mutants were used in which Hh signaling is active throughout the epidermis and all cells take on a groove fate. Interestingly, these embryos did not differentiate grooves. A similar observation has been reported in en, inv, wg mutants, in which hh expression is sustained, leading to the suggestion that en might be required for groove specification (Mulinari, 2009).

Analysis of cell behavior in wg, ptc mutants showed, however, that cells throughout the tissue constrict their apices but fail to form invaginating furrows. The failure of wg, ptc mutants and en, inv, wg; UAS-hh embryos to differentiate grooves might be due to the absence of non-groove cells in the epidermis and the concomitant absence of an interface with groove cells (Mulinari, 2009).

The pair-rule gene odd is initially expressed in 4- to 5-cell wide stripes in even-numbered parasegments. At early gastrulation, odd expression expands to segmental periodicity and is subsequently refined to a single row of prospective groove cells located posterior to en. Continued expression of odd in these cells requires hh. In odd5 mutant embryos, grooves are unaffected in odd-numbered parasegments, but partially missing in even-numbered parasegments, and residual grooves coincide with regions in which odd expression is detectable (Vincent, 2008). These observations have been interpreted as indicating that groove fate might be specified prior to the requirement of Hh and differentiation of the groove. Thus, the later activity of Hh might not induce, but merely maintain, groove cell identity that has been pre-established in the odd-expressing cell population (Vincent, 2008). However, this hypothesis is based on the presumption that odd has a function in groove cell specification and this has not been demonstrated (Mulinari, 2009).

Residual grooves in odd5 mutants have been attributed to the hypomorphic nature of the odd5 allele; however, the molecular lesion in odd5 is unknown. Therefore the nucleotide sequence of odd5 was determined and a substitution was found that mutates codon 84 from CAG to a TAG stop codon. The resulting truncated peptide, which lacks all four putative zinc fingers encoded by wild-type odd, is no longer restricted to the nucleus but uniformly distributed in the cell. Thus, odd5 is likely to be a null allele (Mulinari, 2009).

To exclude the possibility that groove formation may be rescued by read-through of the stop codon in odd5 mutants, or that odd may be required redundantly, segmental grooves were investigated in Df(2L)drmP2 mutants, in which odd and its sister genes drumstick (drm) and sister of odd and bowl (sob) are entirely deleted. In these embryos, normal grooves formed in odd-numbered parasegments in the complete absence of odd function (Mulinari, 2009).

Next even-numbered parasegments were investigated in which grooves are partially missing. odd encodes a transcriptional repressor that regulates the expression of other segmentation genes in the early embryo. In odd mutants, derepression of the en activator fushi-tarazu in even-numbered parasegments results in the formation of an ectopic en stripe posterior to the normal stripe. Simultaneously, wg expands anteriorly and becomes expressed adjacent to the ectopic en-expressing cells. This results in the formation of an ectopic parasegment boundary with reversed polarity. Thus, the outward-facing edges of both en stripes are genetically anterior and lined by wg-expressing cells that do not form grooves. The inward-facing edges of the normal and ectopic en stripes fuse in some areas, and these corresponded to areas in which grooves were missing, as cells that were genetically posterior to en and could respond to the Hh signal had been replaced by en-expressing cells. The fusion of normal and ectopic en stripes was more severe in Df(2L)drmP2 mutants; however, islands of invaginating groove cells could still be observed, demonstrating that groove fate is specified in the absence of odd, drm and sob function in all parasegments. It is concluded that all cells that are genetically posterior to en are specified as groove cells in the absence of odd function and the partial absence of grooves in even-numbered parasegments in odd mutants is a secondary consequence of the pair-rule phenotype of these embryos. The slightly more severe pair-rule phenotype seen in Df(2L)drmP2 mutants might be due to a contribution from one of the odd sister genes, most likely sob, to pair-rule function, or could be caused by low-level read-through of the stop codon in the odd5 allele (Mulinari, 2009).

Finally, to investigate whether odd is sufficient to trigger cell shape changes, a UAS-odd transgene was expressed either alone or together with hh in the epidermis. No induction of groove cell behavior other than that triggered by hh was observed. Together, the data show that odd plays no essential role in groove cell specification and that odd paralogs are unlikely to act redundantly in this process (Mulinari, 2009).

The identification of odd as a groove cell marker led Vincent to suggest that groove fate might be specified prior to Hh requirement and that Hh may merely maintain groove fate instead of having an inducing role (Vincent, 2008). This study demonstrate that grooves are specified in the absence of odd function; however, this could be due to an odd-independent, early-acting mechanism present in the cells from which grooves arise (Mulinari, 2009).

In order to address whether groove fate is pre-established in the odd-expressing cell population, it was asked if groove fate could be induced in cells of a different origin at a later point in time. lines (lin) mutants were used in which late wg expression is altered resulting in the formation of an ectopic segment boundary at the anterior edge of the en domain in the dorsal epidermis. Importantly, the early expression of pair-rule or segment polarity genes is not affected (Mulinari, 2009).

In lin mutants, ectopic expression of the groove marker odd was initiated at stage 12 in a single row of groove-forming cells anterior to en that are derived from a previously non-odd-expressing cell population that does not contribute to grooves in the wild type. Ectopic grooves require hh as they were not induced in hh, lin double mutants, and ectopic odd expression was not induced in this background. An increase in hh levels in lin mutants resulted in the specification of groove fate in all cells except those expressing en. These results suggest that hh is sufficient, late in development, to specify groove cell fate in cell populations of different origins and that earlier-acting factors present in the population of odd-expressing cells posterior to en are not required. Very similar results have been reported by Piepenburg (2000), who showed that segment border cells form solely in response to the Hh signal that emanates from the en domain (Mulinari, 2009).

The findings are consistent with the role of Hh in the regulation of cell shape in other systems. Thus, during Drosophila eye development, Hh has been shown to control cell shape in the morphogenetic furrow, and Hh activation in other tissues is sufficient to induce apical constriction and groove formation. It is likely that Hh plays a similar role in tissue morphogenesis in other organisms. During neural tube closure in vertebrates, cells undergo similar shape changes involving apical-basal elongation and apical constriction, which is likely to be in response to Hh sources in the notochord and floor plate. Accordingly, knockout of sonic hedgehog is associated with defects in neural tube closure in mice. These observations suggest that Hh might be a principal inducer of cell shape across species (Mulinari, 2009).

It has previously been established that wg antagonizes the activity of hh in the specification of segment border cells (Piepenburg, 2000). However, it is not clear whether wg has a similar role in segmental groove formation, and a late requirement of wg to antagonize Hh-mediated groove specification has been questioned (Vincent, 2008). To investigate a direct role of wg in groove specification, a dominant-negative form of the transcription factor pan (panDN), which suppresses Wg signaling, was expressed. For this, pnr-Gal4, which initiates expression in the dorsal epidermis at stage 10-11 and thus does not affect early wg function, was used. Embryos that express panDN formed a single row of ectopic groove cells anterior to the en domain, confirming the results in lin mutants. Strikingly, inactivating Wg signaling and increasing Hh levels at the same time by co-expression of panDN and hh resulted in the expansion of groove fate to all cells except those expressing en. These results show that Wg signaling is required after stage 10 to repress groove specification anterior to en, thus making the activity of Hh asymmetric. These results also confirm observations that Hh is sufficient to induce groove fate in cells from different positions along the anterior-posterior axis and suggest that groove fate is not determined before stage 10 (Mulinari, 2009).

To confirm the ability of wg to repress groove fate, wg was expressed posterior to en in cells that normally take on groove fate. This resulted in the loss of Odd from many cells, suggesting that wg indeed antagonizes hh activity. Interestingly, these cells still formed grooves. However, these grooves appeared much earlier than segmental grooves, suggesting that they are ectopic parasegmental grooves caused by ectopic wg expression, as recently suggested (Larsen, 2008). Together, these data therefore support the contention that Wg signaling is required to repress Hh-mediated induction of groove fate after stage 10, thus permitting the formation of segmental grooves posterior, but not anterior, to en in the wild type (Mulinari, 2009).

Hh signaling in the Drosophila embryonic eye field

The function of the Dpp and Hh signaling pathways in partitioning the dorsal head neurectoderm of the Drosophila embryo has been analyzed. This region, referred to as the anterior brain/eye anlage, gives rise to both the visual system and the protocerebrum. The anlage splits up into three main domains: the head midline ectoderm, protocerebral neurectoderm and visual primordium. Similar to their vertebrate counterparts, Hh and Dpp play an important role in the partitioning of the anterior brain/eye anlage. Dpp is secreted in the dorsal midline of the head. Lowering Dpp levels (in dpp heterozygotes or hypomorphic alleles) results in a 'cyclops' phenotype, where mid-dorsal head epidermis is transformed into dorsolateral structures, i.e. eye/optic lobe tissue, which causes a continuous visual primordium across the dorsal midline. Absence of Dpp results in the transformation of both dorsomedial and dorsolateral structures into brain neuroblasts. Regulatory genes that are required for eye/optic lobe fate, including sine oculis (so) and eyes absent (eya), are turned on in their respective domains by Dpp. The gene zerknuellt (zen), which is expressed in response to peak levels of Dpp in the dorsal midline, secondarily represses so and eya in the dorsomedial domain. Hh and its receptor/inhibitor, Patched (Ptc), are expressed in a transverse stripe along the posterior boundary of the eye field. Hh triggers the expression of determinants for larval eye (atonal) and adult eye (eyeless) in those cells of the eye field that are close to the Hh source. Eya and So, which are induced by Dpp, are epistatic to the Hh signal. Loss of Ptc, as well as overexpression of Hh, results in the ectopic induction of larval eye tissue in the dorsal midline (cyclopia). The similarities between vertebrate systems and Drosophila are discussed with regard to the fate map of the anterior brain/eye anlage, and its partitioning by Dpp and Hh signaling (Chang, 2001).

At the onset of gastrulation, the anlage that gives rise to the anterior brain (protocerebrum) and the eye, roughly defined by the expression of otd, extends from the cephalic furrow to the anlage of the foregut. In the dorsoventral axis, the anlage crosses the dorsal midline; laterally it reaches to ~50% of egg diameter where it is bounded by the ventral neurectoderm. During gastrulation and germband elongation, the anlage splits up into different components that can be recognized morphologically and with the help of molecular markers. Three main domains, the head midline ectoderm, protocerebral neurectoderm and the visual primordium, can be distinguished (Chang, 2001).

A narrow strip straddling the dorsal midline gives rise to the medial portion of the head epidermis. In the acephalic larva, these cells (and most other cells of the head epidermis) are folded inside the animal to form the dorsal pouch (Chang, 2001).

The lateral part of the head neurectoderm produces the neuroblasts that form the central protocerebrum, the major compartment of the insect brain that includes associative centers such as the mushroom bodies and central complex. A narrow domain within the dorsomedial protocerebrum is the anlage of the so-called pars intercerebralis, which contains clusters of neuroendocrine cells producing various neuropeptides. The neuroendocrine neurons project their axons in a peripheral nerve that leaves the brain and reaches the corpora cardiaca, a neurohemal organ located close to the heart. The pars intercerebralis-corpora cardiaca system is highly reminiscent of the vertebrate hypothalamus-pituitary axis, and this similarity extends to the embryonic origin of the corpora cardiaca. Thus, the corpora cardiaca arise as invaginations from the foregut. Their embryonic origin has been well documented in Manduca sexta; in Drosophila, the corpora cardiaca, along with precursors of the stomatogastric (i.e. autonomic) nervous system, also invaginate from the foregut (Chang, 2001).

The visual primordium, defined molecularly by the expression of so, is wedged in between the midline ectoderm and the protocerebral neurectoderm in the posterior head. During gastrulation and germband extension, cells of the visual primordium move laterally and are subdivided into the larval and adult eye primordia and the inner and outer optic lobe. The optic lobe and larval eye form a triangular placode that invaginates. The posterior lip of this invagination, marked by the expression of FasII, represents the primordium of the lamina and medulla, and gives rise to the lobula complex. The larval eye, or Bolwig's organ, labeled by FasII and mAb22C10, develops at the lateralmost tip of the optic lobe placode. The cells that will become the eye imaginal disc (adult eye) are anterior and dorsal to the optic lobe placode and can be recognized by the expression of eyeless (Chang, 2001).

Hh is expressed in metameric stripes that coincide with the posterior compartment of each segment. In the head, hh expression in the stage 5-7 embryo forms a wide stripe in front of the cephalic furrow. This stripe, which crosses the dorsal midline, includes the future antennal segment and posterior part of the visual anlage. As germ band extension proceeds, hh expression disappears from the dorsal midline and two separate bands are parceled out (antennal stripe, pre-antennal or occular stripe). The pre-antennal stripe overlaps with the lateral boundary of the visual primordium. Towards the late extended germband stage, the Hh head domain decreases in size and expression level. During stage 11 and early 12, only a small cluster of cells corresponding to the precursors of the larval eye located laterally in the visual primordium remain hh positive (Chang, 2001).

Hh signaling is negatively regulated by Ptc, a membrane linked protein that, by binding to Hh ligand, becomes inactivated in cells receiving high levels of Hh. Ptc expression in the head resembles hh expression at an early stage. A wide antennal/pre-antennal stripe traverses the head in front of the cephalic furrow. During germband extension, this domain splits into two stripes. At the late extended germ band stage, ptc remains expressed in a large domain that corresponds to the anterior optic lobe (Chang, 2001).

Loss of hh results in a strong reduction of the head midline epidermis, a reduction in the size of the brain and optic lobe, and the total absence of the larval and adult eye primordium. Temperature-sensitive shift experiments of hhts2 embryos indicate that the phenocritical period for Hh function in Bolwig's organ development is between 4 and 7 hours. Aside from the larval eye, the primordium of the compound eye, which is marked from stage 12 onward by the expression of eyeless (ey), is also affected by the loss of hh. Heatshock induced overexpression of hh, as well as loss of ptc, causes an increase in larval eye neurons and optic lobe precursors. Interestingly, ectopic Hh activity is able to induce optic lobe and Bolwig's organ tissue in the head midline and thereby generate a cyclops phenotype similar to the condition described above for partial reduction of dpp. Applying heatshocks at different times of development indicates that the phenocritical period for the Hh induced cyclops is early, between 2.5 and 5 hours. Thus, heat pulses administered during this time cause fusion of the optic lobe and, at a lower frequency, of the larval eye without significantly increasing the number of optic lobe and larval eye cells. By contrast, later heat pulses (after 5 hours) lead to larval eye/optic lobe hyperplasia but no concomitant cyclops phenotype (Chang, 2001).

The finding that both loss of Hh and Dpp cause the absence of visual structures, and ectopic expression of Hh and partial loss of Dpp cause transformation of head midline epidermis into visual primordium, begs the question of how the two signaling pathways interact. In Drosophila compound eye development, hh expression is required to turn on dpp expression. To establish whether a regulatory relationship exists between Hh and Dpp signaling, the expression of dpp and pMAD was examined in the background of hh loss of function, as well as hh, ptc and Cubitus interruptus (Ci) expression in the background of dpp loss of function. Cells in which Dpp signaling is activated can be visualized by an antibody against phosphorylated MAD (pMAD) protein. Dpp RNA expression and pMAD are normal in a stage 5-9 hh-null background, indicating that Hh is not required to activate Dpp signaling in the embryonic head (Chang, 2001).

The expression of hh and ptc is normal in early embryos mutant for dpp. Since ptc is a downstream target of Hh signaling, this result strongly suggests that Dpp signaling is not required to activate the Hh cascade. To show more directly whether this cascade is interrupted, the antibody AbN, which recognizes both the full-length Ci protein and the cleaved repressor form (Ci75) was used in the background of a dpp-null mutation. According to the present model, Hh function consists of preventing the cleavage of the Ci protein to generate the repressor form, which is able to enter the nucleus and inhibit transcription of target genes such as ato and/or hh. In a mutation of Ci that produces only the repressor form or in eye clones that lack hh, a higher level of Ci can be detected in the cells. In dpp-null embryos, cytoplasmic Ci signal in the visual primordium of stage 7 embryos is at the same level as in wild type, indicating that Dpp is not required for Hh signal to go through. However, it should be conceded that it is very difficult to quantify, in embryonic tissues as opposed to cultured cells, expression levels using the Ci antibodies available, which leaves open the possibility that Dpp might have a quantitative effect of on the strength of the Hh signal reaching the nucleus (Chang, 2001).

Taken together, these findings suggest that no direct interaction exists between Hh and Dpp signaling, and that the antagonistic effect of Hh and Dpp on the formation of visual structures is most probably based upon an indirect interaction between the two signaling pathways that involves the expression of the eye genes so and eya (Chang, 2001).

Hh is positively required for the visual system. Loss of this gene causes the absence of the larval eye, as well as the adult eye primordium. This phenotype is reminiscent of the later requirement of Hh for the initiation of cell differentiation in the larval eye imaginal disc. Increased expression of Hh, as well as absence of the inhibitor of Hh function, Ptc, results in a cyclops phenotype (Chang, 2001).

In view of these results, it is speculated that the interaction between Dpp and Hh is indirect and requires the function of so, eya and possibly other 'early eye genes' -- according to this model, Dpp activates so and eya in the eye field. Slightly later, expression of so and eya is lost dorsomedially, due to repression by Zen at this level. In a second step, the expression of Hh (which comes on later than Dpp) triggers larval eye fate in cells close to the Hh source. The response of a cell to Hh, that is, its expression of ato, depends on its previously expressing so and eya. Finally, Ptc inhibits the range of Hh action, similar to its alleged function in the trunk and imaginal discs (Chang, 2001).

A model is proposed to explain the phenotypes resulting from manipulating Dpp, Hh and Ptc expression:

  1. In wild type, Hh can activate larval eye only in cells expressing so and eya. No larval eye develops in the dorsal midline because so is down regulated in this region rapidly, and Hh 'has no opportunity' to overcome the ptc mediated inhibition and induce visual system at an early stage when so is still present in the dorsal midline (Chang, 2001).
  2. In ptc-, Hh is able to induce larval eye fate in the dorsal midline because it is not inhibited at the early stage when so is still expressed dorsomedially (Chang, 2001).
  3. Heatshock-induced Hh expression at an early stage (stage 5; around 3 hours) has the same effect, overcoming the ptc-mediated inhibition and inducing larval eye dorsomedially (Chang, 2001).

The topology in which different derivatives of the anterior brain anlage are laid out in the early embryo exhibits considerable similarity to that of vertebrates. To appreciate this similarity, one needs to remember that the neurectoderm of insects does not invaginate. As a result, early embryonic tissues located in the dorsal midline (i.e. the head midline ectoderm) of the fly embryo remain where they are, i.e. mid-dorsally, whereas in vertebrates, they form the ventral midline of the neural tube. This inverse topology may explain in part why dorsomedial structures in Drosophila share several functional and molecular similarities with the ventral forebrain in vertebrates. For example, both give rise to neuroendocrine centers (the pars intercerebralis of the insect brain, hypothalamus of vertebrates). In both vertebrates and insects, cells that start out as epithelial placodes in the foregut anlage anteriorly adjacent to the eye field, form neurohemal structures (anterior pituitary in vertebrates, corpora cardiaca in insects) that become innervated by the neuroendocrine neurons derived from the midventral/mid-dorsal brain. The topological similarity between the eye field in Drosophila and vertebrates extends to the location of the eye. In both systems, the eye maps close to the midline and genetic manipulations affecting the midline result in the fusion of the eyes (cyclopia) (Chang, 2001).

The dorsal location of the eye field and protocerebral neurectoderm in Drosophila, as well as all extant arthropods, is not easy to reconcile with the hypothesis that the chordate body plan is derived from an arthropod/annelid-like ancestor whose dorsoventral axis is reversed, although it does not directly contradict this idea. Thus, eye field and protocerebral ectoderm of ancestral arthropods might have actually occupied a ventral position in front of the stomodeum, and subsequently shifted dorsally. However, given that no comparative-structural or fossil evidence exists for such a shift, an alternative hypothesis can be offered: the CNS of the ancestor of chordates (deuterostomes) and arthropods/annelids (protostomes) may have been restricted to the head of the animal where also sensory receptors (eyes, statocysts, chemoreceptors) are concentrated. In support of this view, nerve cells in many groups of platyhelminths, in particular Acoels (considered as the sister group of bilaterians according to recent molecular-phylogeneitc data), are exclusively derived from the anterior pole of the embryo. From this primitive anterior ganglion of the bilaterian ancestor, the protocerebrum/eye field of present day bilaterians is directly derived, with no change in dorsoventral axis. In the trunk region, which originally lacked central neurons, a central nervous system was 'added' that followed different patterns during evolution. In the line leading to higher protostomes, ganglia located ventrally were added, whereas a dorsal trunk neurectoderm formed in chordates (Chang, 2001).

Irrespective of which of the two aforementioned hypotheses regarding topology of the neural fate map will turn out to be correct, the high degree of conservation of signaling pathways and regulatory genes controlling the patterning of the fate map in Drosophila and vertebrates emphasizes how 'close' the body plans manifested during early embryogenesis still are. Dpp/BMP and Hh/Shh signaling are centrally involved in head patterning in both systems, and could have exerted this role already in the bilaterian ancestor. However, it is also true that the impact of Dpp and Hh signaling on midline and eye structures seems very different in chordates and arthropods, which makes the independent recruitment of the two signaling pathways into head patterning in these phyla a distinct possibility. In chordates, loss of Hh results in a cyclops phenotype and holoprosencephaly, since high levels of Hh are required for hypothalamus and optic stalk. Hh positively regulates Pax2, a regulatory gene expressed in and required for the optic stalk. In the Drosophila embryo, excess function of Hh causes cyclopia. Moreover, Hh has a positive effect on the Pax6 homolog, eyeless; ey expression requires the presence of the Hh signal (Chang, 2001).

When comparing the expression pattern of conserved regulatory genes, such as otd, tll, so and many others in anterior brain and eye development of fruit flies and vertebrates, one is also struck by the high number of similarities. These similarities indicate that the bilaterian ancestor might have possessed a head in which photoreceptors, various brain structures and neuroendocrine cells were laid out in a way reminiscent of the pattern found in present day taxa. This obviously does not imply the existence of complex organs, such as the eye, pituitary or brain structures. What it does imply is that the bilaterian ancestor had an anterior ‘neurectoderm’ in which clusters of cells with the basic properties of photoreceptors, pigment cells, neuroendocrine cells or central neurons were positioned in a pattern reminiscent of the modern pattern formed by the progenitors of these structures in different animals. During evolution, these cell types diversified further and became shaped by morphogenetic movements into more complex organs. For example, in the chordates (including urochordates and cephalochordates), the anterior neurectoderm invaginated to form a tube that included all cells with the fate of photoreceptors, pigment cells and target neurons. In vertebrates these cells then evaginated as the optic cup, induced lens and other structures from the outer ectoderm and formed an eye. In the evolutionary line leading to arthropods, cells with the fate of photoreceptors and pigment cells were separated at an early developmental stage from cells destined to become optic target neurons. The former remained in the outer ectoderm and became organized into a compound eye, while the latter delaminated along with other neural stem cells to form the brain. The stage is set for comparative studies of eye morphogenesis and gene expression that will elucidate in more detail how a simple visual system changed into the various types of eyes that can be observed in extant animal groups (Chang, 2001).

Hedgehog and tracheal development

The elaborate branching pattern of the Drosophila tracheal system originates from ten tracheal placodes on both sides of the embryo, each consisting of about 80 cells. Simultaneous cell migration from each tracheal pit in six different directions gives rise to the stereotyped branching pattern. Each branch contains a fixed number of cells. Previous work has shown that in the dorsoventral axis, localized activation of the Dpp, Wnt and EGF receptor pathways subdivides the tracheal pit into distinct domains. The role of the Hedgehog (Hh) signaling system in patterning the tracheal branches is presented in this study. Hh is expressed in segmental stripes abutting the anterior border of the tracheal placodes. Induction of patched expression, which results from activation by Hh, demonstrates that cells in the anterior half of the tracheal pit are activated. In hh-mutant embryos migration of all tracheal branches is absent or stalled. These defects arise from a direct effect of Hh on tracheal cells, rather than by indirect effects on patterning of the ectoderm. Tracheal cell migration can be rescued by expressing Hh only in the tracheal cells, without rescuing the ectodermal defects. Signaling by several pathways, including the Hh pathway, thus serves to subdivide the uniform population of tracheal cells into distinct cell types that will subsequently be recruited into the different branches (Glazer, 2001).

This work has identified four distinct, consecutive roles for the Hh pathway in tracheal morphogenesis. (1)The earliest function of Hh is to participate in allocating the correct cell number to several tracheal pits, most notably pit number 3, where only half the number of tracheal cells is observed in hh mutants. The determination of the position and number of cells in the tracheal placodes is highly regulated. Segmental differences in cell number are observed. Most placodes contain 80 cells, while the first placode contains 150 cells, and the third contains only 50 cells. In the dorsoventral axis, it appears that the activity of the Egfr pathway in the ventral ectoderm, and the Dpp pathway in the dorsal ectoderm, restrict the position of the placodes to the central region. The cues for determination of the placodes in the anteroposterior axis are only partially known. Indications for repression of tracheal fates by Wingless have been reported. Since most of the placodes form normally in hh mutants, Hh signaling is not involved in providing global cues for the position of the placodes. The defects observed in tracheal cell number could imply that Hh collaborates with homeotic genes in the specification of segment-specific placode cell number, particularly in placode number 3. (2) The second defect is observed at stage 11 in all metamers, during the invagination of the tracheal pits. In hh mutants, six to eight cells remain on the ectoderm. Similar defects were observed in mutants for the EGF receptor pathway. (3) From stage 12 onwards, the most severe tracheal defect is the lack or significant impairment of migration. The reduction in cell number in the tracheal pits caused by the two earlier Hh functions cannot account for this severe defect. Tracheal pits containing half of the normal number were shown to migrate normally. Thus, the migration defect represents a distinct function of Hh. In addition to defects in the migration of the branches anteriorly, it was noted that in hh mutant embryos, the lateral trunk posterior branch is also stalled. Although the induction of the Hh target genes ptc and Complex1 (an enhancer trap line expressed in all anterior tracheal cells at stage 13) in the LTp cells was not observed, it is possible that these cells require a lower level of Hh signaling (Glazer, 2001).

(4) The Hh pathway is required for patterning the terminal cells in the dorsal branches. Compromising the activity of the pathway reduced the extent of terminal branching, while activation of the Hh pathway in the tracheal cells gave rise to an excess of terminal cells in the dorsal branch. It has previously been shown that high levels of Btl activation induce terminal cell fates, through the expression of Pointed P1. Thus, the leading cells that are closest to the Bnl source become terminal cells. During the migration of the dorsal branch cells, Bnl activation is high in the leading cell and low in the trailing ones. Hh expression is poised directly over the migrating cells, and may provide a uniform level of activation in these cells. Hh signaling could provide a dorsal branch 'context' for these cells, and function in conjunction with the signals elicited by Bnl. It is interesting to note that similar effects on terminal cell fates were observed in other branches such as the ganglionic branch, following expression of these constructs. Late Hh expression, however, is not positioned in close proximity to these branches, raising the possibility that these late abnormalities may reflect Hh influence at an earlier stage (Glazer, 2001).

Hedgehog and neural development

Continued: hedgehog Effect of mutation part 3/3 | back to part 1/3

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

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