Interactive Fly, Drosophila

hedgehog


REGULATION (part 2/2)

Transduction of the hedgehog signal

Hedgehog is involved in the regulation of synthesis of two extracellular signaling molecules, DPP and WG. How are HH signals received and then transduced into the nucleus to regulate synthesis of HH targets?

Transduction of the hh-encoded signal is mediated by the activity of four other segment polarity genes: patched, fused, costal-2 and cubitus interruptus. Transcription of patched is regulated by the same pathway. There are cis-acting upstream elements of the ptc transcription unit that mediate this regulation (Forbes, 1993).

Patched has been implicated as the receptor for Hedgehog. In the most posterior row of cells in each parasegment, repression of wingless mediated by patched is neutralized by hedgehog, allowing wg expression. Thus excess Patched is capable of overcoming the neutralizing signal carried by hedgehog (Schuske, 1994). The true identity of the HH receptor is still unknown (Ingham, 1993).

Phosphorylation of Fused occurs in response to Hedgehog and cannot be blocked by activation of Protein kinase A, which is thought to be an antagonist of signaling from hedgehog. This suggests that Fused and Protein kinase A function downstream of Hedgehog but in parallel pathways that eventually converge downstream of Fused (Thérond, 1996).

Fused is an important component in the hedgehog pathway for induction of decapentaplegic, that is dpp is a target of the hh signal acting through Fused. Genetic evidence provides the support for involvement of Fused in hedgehog signaling. fu mutations rescue the phenotype due to ectopic expression of hh or to the lack of patched activity. fu is also required for the activation of engrailed caused when hh is ectopically activated in the wing disk. Although fu, cos-2 and ci probably form part of the same pathway that controls dpp expression, Protein kinase A probably controls dpp expression by a different pathway (Sánchez-Herrero, 1996).

Expression of wingless is normally maintained only in those cells receiving an extrinsic signal, encoded by hedgehog, that antagonizes the repressive activity of patched (Ingham, 1991). Unrestricted expression of ptc from a heat-shock promoter has no adverse effect on development of Drosophila embryos. The heat-shock construct can also rescue ptc mutants, restoring wg expression to its normal narrow stripe. The ectopic EN stripe fails to appear, but the normal one remains unaffected. These results imply that despite its localized requirement, the restricted expression of ptc does not itself allocate positional information (Sampedro, 1991).

The catalytic subunit of cyclic AMP-dependent protein kinase A (PKA) is involved in HH signaling. PKA is required for the correct spatial regulation of dpp expression during eye development. Loss of PKA function is sufficient to produce an ectopic morphogenetic wave marked by premature ectopic photoreceptor differentiation and non-autonomous propagation of dpp expression. PKA lies in a signaling pathway that controls the orderly temporal progression of differentiation across the eye imaginal disc (Strutt, 1995a). PKA is involved in wing and leg patterning downstream of HH as well (Lepage, 1995).

Hedgehog signaling is involved in the establishment of three different cell types in the segmentally repeated dorsal epidermis. A single row of cells, just posterior to the parasegmental division (type 1) produces large pigmented denticles. This is followed in the posterior by about two rows of cells (type 2) that secrete smooth cuticle. The next two or three rows of cells (type 3) produce thick pigmented hairs that are smaller and less pigmented that the Type 1 cells. Hedgehog acts in type 1 and 2 cells to antagonize the activity of Patched. Hedgehog acts in type 3 cells to antagonize the lines gene function (Bokor, 1996). Thus Hedgehog functions as a morphogen with developmental roles independent of its activity in regulating wingless and dpp expression through Patched.

Cubitus interruptus is an integral part of the Hedgehog pathway and is required to maintain expression of the wingless gene and to specify naked cuticle within each epidermal segment (Motzny, 1995).

The secreted Drosophila Hedgehog (Hh) protein induces transcription of specific genes by an unknown mechanism that requires the serpentine transmembrane protein Smoothened (Smo) and the transcription factor Cubitus interruptus (Ci). Protein kinase A (PKA) has been implicated in the mechanism of Hh signal transduction because it acts to repress Hh target genes in imaginal disc cells that express Ci. Changes in Ci protein levels, detected by an antibody that recognizes an epitope in the carboxy-terminal half of Ci, have been suggested to mediate the positive effects of Hh and the negative effects of PKA on Hh target gene expression in imaginal discs. The effects of PKA on Hh target genes were examined by expressing a mutant regulatory subunit, R*, to reduce PKA activity in embryos. The alterations of wingless, patched and ventral cuticle patterns due to PKA inhibition resemble those induced by low-level ubiquitous expression of Hh but are less pronounced than those elicited by high levels of Hh or strong patched mutations. A constitutively active mouse PKA catalytic subunit transgene (mC*) expressed in Drosophila embryos causes ectopic expression of wingless and patched . Responses to elevated PKA activity require smoothened and cubitus interruptus, but not hedgehog. The absolute requirement for Smo to observe transcriptional induction by PKA hyperactivity is consistent with two mechanisms: (1) either PKA acts on Smo, directly or indirectly, perhaps to uncouple it from the inhibitory influence of Ptc or, (2) alternatively, Smo has Hh-independent activity that acts in parallel with PKA to stimulate wingless and patched expression. There is considerable evidence that phosphorylation can alter activity of G protein-coupled receptors. (Ohlmeyer, 1997).

PKA inhibition, like Hh, leads to increased "carboxy-terminal" Ci staining and Hh target gene expression in embryos. Hh and Smo can stimulate target gene expression at constant Ci levels; increased PKA activity can induce ectopic Hh target gene expression in a manner that requires Smo and Ci activity but does not involve changes in Ci protein concentration. Nevertheless, elevated PKA suppresses the elevation of Ci-C-terminal antibody staining normally elicited by Hh at the borders of each Ci expression stripe. This suggests a branching pathway of Hh signal transduction downstream of Smo and that PKA exerts opposite effects on the two branches. Two PKA targets (direct targeting of Smo and targeting of Ci) with opposing actions on Hh target gene expression can account for the initially surprising observation that both PKA inhibtion and PKA hyperactivity induce wingless and patched expression in embryos. The negative target, relevant to regulating Ci protein levels, is sensitive almost exclusively to reduction of PKA activity. Hh signaling in embryos does not depend on cAMP-dependent regulation of PKA activity (Ohlmeyer, 1997).

The Cubitus interruptus controls the transcription of Hedgehog (Hh) target genes. A repressor form of Ci arises in the absence of Hh signalling by proteolytic cleavage of intact Ci, whereas an activator form of Ci is generated in response to the Hh signal. These different activities of Ci regulate overlapping but distinct subsets of Hh target genes. To investigate the mechanisms by which the two activities of Ci exert their opposite transcriptional effect, the imaginal disc enhancer of the dpp gene, which responds to both activities of Ci, has been dissected. Within a minimal disc enhancer, the DNA sequences have been identified that are necessary and sufficient for the control by Ci. The same sequences respond to the activator and repressor forms of Ci; their activities can be replaced by a single synthetic Gli-binding site. The enhancer sequences of patched, a gene responding only to the activator form of Ci, effectively integrate also the repressor activity of Ci if placed into a dpp context. These results provide in vivo evidence against the employment of distinct binding sites for the different forms of Ci and suggest that target genes responding to only one form must have acquired distant cis-regulatory elements for their selective behavior (Muller, 2000).

For the minimal dpp and brinker enhancers, a Hh-independent activator input has been postulated that causes a basal expression level. This transcriptional activity can either be synergistically enhanced by Ci[act] or suppressed by Ci[rep], depending on which form of Ci is prevailing and thus predominantly binding to the Gli/Ci-binding site. This scenario represents the simplest case of a Hh target gene, one that responds to both forms of Ci. To achieve selective responsiveness to only one form of Ci, as in the cases of hh and ptc, additional cis-acting elements must have evolved. At present, the cis elements that make some Ci target genes different from others are unknown. However, a firm case can be made for the relevance of this yet unknown mechanism. LetÂ’s consider first the hh gene, which is particularly interesting because it responds effectively and selectively to Ci[rep]. hh expression levels can neither be increased in the posterior compartment nor ectopically induced in the anterior compartment by overexpression of constitutively active forms of Ci. But even very low levels of Ci[rep] suffice to repress hh expression in anterior cells near the AP boundary, despite the presence of high levels of Ci[act]. This was revealed by the observation that anterior ci mutant clones located close to the AP boundary ectopically express the hh gene. If Ci controls hh directly, by binding to Gli sites of the hh gene, the hh promoter must be configured to assemble a transcriptional complex that is unable to effectively interact with Ci[act] (Muller, 2000 and references therein).

Drosophila Hedgehog (Hh) is secreted by Posterior (P) compartment cells and induces Anterior (A) cells to create a developmental organizer at the AP compartment border. Hh signaling converts Fused (Fu) to a hyperphosphorylated form, Fu*. Anterior border cells of wing imaginal discs contain Fu*. Unexpectedly, P cells also produce Fu*, in a Hh-dependent and Ptc-independent manner. Increasing Ptc, the putative Hh receptor expressed specifically by A cells, reduces Fu*. These results are consistent with proposals that Ptc downregulates Hh signaling and suggest that a receptor other than Ptc mediates Hh signaling in P cells of imaginal discs. It is concluded that Hh signals in these P cells and that the outputs of the pathway are blocked by transcriptional repression (Ramírez-Weber, 2000).

Consistent with expectations, Fu* is absent from hh and smo mutant embryos in which Hh signal transduction is blocked, and it accumulates in mutant embryos lacking Ptc, a negative regulator of Hh signaling. These studies confirm Fu* as an indicator of Hh signaling. In addition, ectopic expression of ptc in discs results in a fu phenocopy and abolishes Fu* from the disc. This indicates that Fu* embodies the active form of Fu. However, identification of the cells in normal wing discs that make Fu* did not conform to expectations (Ramírez-Weber, 2000).

Both Fu and Fu* are present in the A cells that express high levels of ptc at the A/P compartment border. In contrast, only Fu is detected in A cells located away from the compartment border near the disc flank, and only Fu* is detected in P cells. The quantitative conversion of Fu to Fu* in P cells shows that all of the Fu protein is responsive to Hh and indicates that P cells transduce the Hh signal. This latter conclusion contradicts a fundamental tenet of Hh signaling -- that the cells that produce Hh do not transduce the Hh signal. P cells do not express Hh target genes such as ptc and dpp, so it had been assumed that they are refractory to Hh. If the Hh signal transduction pathway is indeed active in P cells, as the presence of Fu* suggests, then the output of the pathway must be blocked at some downstream step. This is an unorthodox means of regulating a signal transduction pathway (Ramirez-Weber, 2000).

Although the Hh pathway is active in P cells, Fu function is not required for normal development of the P compartment, and Hh signaling has no apparent role. It is proposed that the Hh signaling pathway does not reach transcriptional fruition in P cells due to the activity of Engrailed (En). En is expressed in P cells and induces these cells to express hh. P cells, as well as their neighbors in the A compartment, respond to Hh, initiate the Hh signal transduction cascade, and generate Fu*. In A border cells, Hh signal transduction modulates Ci to upregulate dpp and ptc expression. In contrast, En represses ci expression in P cells, thereby preventing a transcriptional response (Ramirez-Weber, 2000).

Several related observations support this model of Hh signaling in P cells. (1) When En is absent from P cells, ci, dpp, and ptc are activated. Presumably, En directly represses ci in normal P cells, and the expression of ci in the mutant cells mediates the induction of dpp and ptc as an indirect consequence of Hh signaling. It is also possible that En plays a direct role in repressing dpp and ptc, but the patterns in which dpp and ptc are induced at the periphery of en mutant clones suggests that their expression is dependent upon Hh. (2) Hh seems to influence the activity of Ci when ci is expressed ectopically in P cells. Hh regulates Ci activity in part by converting Ci to an activator form (CiAct) and by inhibiting its conversion to a repressor form (CiRep). When the full-length Ci protein is made ectopically in P cells, dpp and ptc are activated in a smo-dependent manner, and hh, a target of CiRep, is not repressed. These observations indicate that CiAct is functional in these cells and that CiRep is not. Both are hallmarks of Hh signaling. Using a temperature-sensitive allele of hh, the data with Fu* show that the state of the Hh signaling pathway is not constitutively activated in P cells, but that it reflects the activity of Hh (Ramirez-Weber, 2000).

Ptc protein and ptc RNA have been detected only in A cells, so a role for Ptc in suppressing activation of the Hh pathway in the P cells of imaginal discs seems unlikely. For technical reasons, this could not be tested directly by examining Fu* in ptc- P disc cells, so the possibility cannot be ruled out that P cells express ptc RNA and protein at levels that can not be detected. However, since the level of Ptc in P cells is much less than Smo, any model in which Ptc suppresses Smo signaling in the absence of Hh would require that Ptc act catalytically to silence Smo. If Ptc does act catalytically, it is not obvious why the much higher levels of Ptc in the A cells at the border fail to prevent Hh signaling. Moreover, the fact that overexpression of ptc depresses Hh signaling suggests that the relative levels of Hh and Ptc are important and directly influence Hh signaling. It therefore seems more likely that Hh signaling in discs is mediated by a Hh binding protein other than Ptc (Ramirez-Weber, 2000).

Previous work has shown that in embryos the Hh signal transduction pathway becomes Hh independent in the absence of Ptc. Several different ptc;hh allele combinations were examined, RNAi phenocopies of hh and ci were made in ptc mutants, and Fu* was independently monitored. In each assay, the results are consistent with the proposal that Hh signal transduction pathway is activated independently of Hh in ptc mutant embryos. This behavior contrasts with P disc cells, which are Hh dependent and Ptc independent (Ramirez-Weber, 2000).

Two issues that may be relevant to this apparent contradiction are the role of Ptc and the mechanisms involved in transporting Hh from producing to receiving cells. Hh is presumed to bind Ptc, although no binding studies with the Drosophila proteins have been described. In the work reported here, indirect evidence for a Hh-Ptc interaction is provided. Hh adopts a diffuse distribution in P cells and a particulate appearance in A cells. Ptc and Hh colocalize to these particles and ectopic expression of ptc in P cells blocks signaling, suppresses the production of Fu*, and redistributes Hh into Ptc-containing particles. It is not know whether the Hh protein in these punctate structures signals or has been sequestered for lysosomal degradation or whether these particles are heterogeneous and have different functions. The finding that P cells with a diffuse distribution of Hh produce Fu* while P cells with a particulate distribution of Hh do not shows that these particles do not correlate with signaling (Ramirez-Weber, 2000).

Perhaps the role of Ptc is in part to titrate Hh activity by targeting Hh to an endocytic pathway. This proposal places Ptc in a class of proteins that downregulates the signal that induces its own expression. Others in this class include Dad, an antagonist of Drosophila Dpp; Sprouty, an antagonist of Drosophila FGF; Argos, an antagonist of Drosophla EGF, and Naked, an antagonist of Wg. This model also suggests the presence of a Hh receptor other than Ptc that mediates signal transduction. The contrasting behavior of embryos and discs may reflect the use of different receptors, different regulatory components in the pathway, or the existence of compensating signaling systems in embryos that are not present in discs. Given the multiplicity of Hh binding proteins and the large and diverse group of organs in which Hh plays an instructive role, there may be significant heterogeneity in its downstream effectors (Ramirez-Weber, 2000).

Hh signaling in embryos and discs may also differ in the way they transport Hh to the target cells. The distances between Hh-producing cells and Hh-receiving cells does not exceed 2–3 cells in embryos, but may be significantly greater in discs. Different mechanisms may be used to move Hh over long distances or short, requiring distinct ways to engage the receptor. Further studies on the mechanisms that transport and bind Hh should resolve these issues (Ramirez-Weber, 2000).

Cell pattern in the ventral neural tube is organized by Sonic hedgehog (Shh) secreted by floor plate cells. To assay the range of direct Shh action, a general method was developed for blocking transduction of Hedgehog (Hh) signals through ectopic expression of a deleted form of the Hh receptor Patched (Ptc), termed PtcDeltaloop2. This deleted form of Ptc appears to lack the capacity to bind Hh but retains the ability to inhibit Smo or a downstream Smo effector in the presence of Hh. This method was validated in Drosophila and mouse Ptc1Deltaloop2 (mPtc1Deltaloop2) was used to block Shh transduction in the chick neural tube. This Ptc protein lacks most of the second large extracellular loop. When expressed in clones of cells in the developing Drosophila wing, PtcDeltaloop2 autonomously blocks the ability of cells to sequester and transduce Hh, generating a phenotype that is indistinguishable from that caused by loss of Smo activity. An equivalently modified vertebrate Ptc1 protein, mPtc1Deltaloop2, attenuates the response of chick neural cells to Shh, providing the means to test whether the long-range effects of Shh in neural tissues are direct (Briscoe, 2001).

mPtc1Deltaloop2 expression causes cell-autonomous ventral-to-dorsal switches in progenitor identity and neuronal fate throughout the ventral neural tube, supporting a gradient mechanism whereby Shh acts directly and at long range. mPtc1Deltaloop2 expression also causes the abnormal spread of Shh to more dorsal cells, indicating that Shh in the neural tube, like Hh in Drosophila, induces a feedback mechanism that limits its range of action (Briscoe, 2001).

In Drosophila, the upregulation of Ptc in response to Hh has a crucial role in restricting the range of Hh action. The results presented here suggest that Ptc1 acts similarly to restrict the action of Shh derived from the floor plate. Expression of mPtc1Deltaloop2 in ventral neural cells results in a cell nonautonomous dorsal-to-ventral shift in the identity of progenitor cells positioned dorsal to cells that express mPtc1Deltaloop2. This change in fate is most easily explained by the idea that cells dorsal to mPtc1Deltaloop2 cell clusters have been exposed to a higher level of Shh activity. Normally then, exposure of cells to Shh may limit the level of Shh activity available to cells positioned further from a source of Shh. Consistent with a net ventral-to-dorsal movement of Shh through the ventral neuroepithelium, only those cells positioned dorsally to the mPtc1Deltaloop2 cell clusters appear to be exposed to elevated Shh signaling. In Drosophila tissues, Ptc itself is responsible for the sequestration of Hh and thus for the restriction in Hh movement. By extension, it is likely that the inability of neural cells that express mPtc1Deltaloop2 to sequester Shh results from the reduced level of expression of endogenous Ptc1. However, Shh signaling also induces other proteins such as hedgehog interacting protein (HIP) and vitronectin, which can themselves bind to Shh and could potentially participate in restricting the movement of Shh. Independent of the respective contributions of these proteins to the sequestration of Shh, these data indicate that Shh signaling in the neural tube, as in Drosophila, initiates a feedback system that limits the range of Shh movement and signaling activity (Briscoe, 2001).

HMGCoA reductase potentiates hedgehog signaling in Drosophila

Drosophila HMG Coenzyme A reductase (Hmgcr), also referred to as columbus (clb), catalyzes the biosynthesis of a mevalonate precursor for isoprenoids and has been implicated in the production of a signal by the somatic gonadal precursor cells (SGPs) that attracts migrating germ cells. Mevalonate is required for the biosynthesis of many different compounds such as ubiquinones, carotenoids, and isoprenoids and cholesterol. Hmgcr is the enzyme required for the conversion of 3-hydroxy-3-methylglutaryl coenzyme A into mevalonate. It has now been shown that hmgcr functions in the hedgehog (hh) signaling pathway. When hmgcr activity is reduced, high levels of Hh accumulate in hh-expressing cells in each parasegment, while the adjacent 'Hh-receiving' cells cannot sustain wg expression and fail to relocalize the Smoothened (Smo) receptor. Conversely, ectopic Hmgcr upregulates Hh signaling when it is produced in hh-expressing cells, but has no effect when produced in the receiving cells. These findings suggest that Hmgcr might orchestrate germ cell migration by promoting the release and/or transport of Hh from the SGPs. Consistent with this model, there are substantial germ cell migration defects in trans combinations between hmgcr and mutations in different components of the hh pathway (Deshpande, 2005).

hh is expressed exclusively in the posterior compartment of the wing disc and orchestrates wing development by signaling the expression of downstream target genes such as decapentaplegic (dpp) and ptc in the anterior compartment. In the absence of hh signaling, these target genes are not properly activated, resulting in defects in growth and patterning along the anterior/posterior axis. Conversely, when hh is inappropriately expressed in the anterior compartment, it activates dpp in a pattern that leads to overgrowth of anterior tissues and the partial duplication of distal wing structures. These gain-of-function phenotypes are associated with a dominant hh mutation, hhMoonrat (hhMrt), that causes a partial transformation of anterior wing to posterior (Felsenfeld, 1995). The anterior-to-posterior transformations induced by the Mrt allele can be dominantly suppressed by mutations in hh signaling pathway genes that are required to promote hh signaling in either the sending or responding cell. To assess if hmgcr influences hh signaling in the wing, interactions with Mrt were tested. As positive controls mutations in the hh signaling pathway gene dispatched (disp) were used; disp is thought to function in the sending cell (Deshpande, 2005).

The Mrt wing blades were assigned to five different classes (classes I-V based on the severity of the wing phenotype (Felsenfeld, 1995). Roughly 75% of the control Mrt wing blades (hhMrt/TM3) fall into classes III and IV, which represent moderate to relatively severe wing deformations. The phenotypic effects of Mrt can be dominantly suppressed by the hmgcr mutation, and 75% of the wing blades in hhMrt/hmgcr1 trans-heterozygotes belong to either classes I or II, which represent nearly normal wing morphology. Moreover the extent of suppression of the Mrt wing phenotypes by hmgcr is equivalent to that observed when a disp mutation is trans to hhMrt (Deshpande, 2005).

One model that could explain the suppression of the Mrt wing phenotypes is that hmgcr potentiates hh signaling. If this is correct, then hmgcr mutants might be expected to exhibit segmentation defects similar to those of known hh pathway genes. To explore this possibility, cuticles of hmgcr embryos were examined. Nearly 30% (14/49) of the hmgcr embryos showed fusions of one or more segments and/or the deletion of pattern elements characteristic of mutations in segment polarity genes. The most prevalent defects were the fusion of abdominal segments 7 and 8 (9/14); however, more severe disruptions in patterning were also evident. The same types and range of patterning defects were observed for another hmgcr allele. The frequency of such defects in control embryos was never more than 3%-5% (Deshpande, 2005).

Although segment polarity defects are clearly evident in hmgcr embryos, the cuticle phenotypes are much less severe than those seen for genes like hh and wg (which give a lawn of denticles). One explanation for the relatively weak segment polarity defects is that maternally derived Hmgcr compensates for the lack of the zygotic gene product. To test this possibility, hmgcr germline clones were generated. While fertile females were not obtained for the strong hmgcr11.57 allele, fertile females were obtained for the hypomorphic allele hmgcr11522. These females were mated to either hmgcr11.57/TM3 Ubx-LacZ or wild-type males. The cuticle phenotypes observed when the germline clone females were mated to heterozygous hmgcr males were examined. The embryos could be divided into roughly four groups. Group I (15%) embryos arrested development without forming cuticle. In a subset of these embryos, abnormal mouth parts and/or filzkorper could be detected. Group II (27%) embryos formed at least some cuticle, but embryos had severe segmentation defects. Many of the embryos in this group had deletions/fusions of cuticle pattern elements. In others, cuticle structures like the denticle belts were incompletely formed. Much less pronounced developmental defects were observed in embryos in groups III and IV. Embryos in group III (22% embryos) had fusions of abdominal segments 7 and 8, but were otherwise normal. Embryos in group IV, which represents about 37% of the embryos, resembled wild-type; however, less than half of these animals hatched, suggesting that they may have other vital defects. Since group III or IV embryos were observed only when the hmgcr germline clone females were mated to wild-type males, it is presumed that embryos in groups I and II were fertilized by hmgcr mutant sperm. Three conclusions can be drawn from these data: (1) there is a substantial hmgcr maternal contribution; (2) the loss of this maternal product can be partially compensated by zygotic expression from the paternal gene; (3) while hmgcr seems to function in the wg-hh regulatory circuit, it must have additional roles that are critical for normal development that may be unrelated to the segment polarity pathway (Deshpande, 2005).

To provide additional evidence that hmgcr functions in segment polarity, the pattern of wg expression was examined in hmgcr mutant embryos. Up until stage 9/10, no defects were discerned in the pattern or level of wg stripe expression in the ectoderm of hmgcrz− embryos. However, beginning around stage 11, wg expression in hmgcrz− embyros is downregulated, and the level of Wg accumulation is reduced compared to wild-type. Further reductions in Wg protein accumulation are evident in older hmgcrz− embryos, though even in these older embryos, some residual Wg protein can still be seen in the ectoderm. These findings indicate that hmgcr resembles hh in that it is not required in the initial activation of wg stripe expression in the ectoderm, but is required to sustain wg expression. In contrast, the effects of reduced hmgcr activity on wg are considerably less severe than those seen in hh null mutant embryos. In the absence of hh, wg stripe expression in the ectoderm disappears almost completely by the end of stage 9, whereas small amounts of Wg protein are still clearly evident in stage 12 and older hmgcrz− embryos. This difference could indicate that hmgcr activity is not essential for maintaining wg expression. Another factor that could contribute to the difference is the substantial maternal contribution of hmgcr. To confirm this possibility, Wg expression was examined in progeny from hmgcr11522 germline clone females mated to hmgcr 11.57/TM3 Ubx:LacZ males. As expected, the effects on Wg expression were more pronounced when maternal hmgcr activity was compromised (Deshpande, 2005).

To confirm these findings, the expression of the Engrailed (En) protein was examined in hmgcr mutant embryos. hh signaling is required to maintain a high level of En expression in the stripes, and, in hh mutants, en expression begins to decay around stages 10-11. hmgcr is also required to maintain a high level of En expression, and, in embryos lacking zygotic hmgcr activity, En expression is reduced compared to wild-type by stage 11 (Deshpande, 2005).

The failure to maintain high levels of wg expression in older embryos would be consistent with the idea that Hmgcr is required for sending and/or receiving the Hh ligand. To test this hypothesis, the distribution of the Smo protein was compared in wild-type, hmgcrz− (hmgcr11.57), and hh embryos. Previous studies have shown that reception of the Hh signal stabilizes Smo protein and induces it to relocalize from intracellular membrane vesicles to membranes on the cell surface. In wild-type embryos, the effects of Hh signaling on Smo stability and localization can be visualized as a series of stripes that are about five cells wide. In these stripes, Smo is concentrated predominantly at the surface of the cell, giving a ring around the edge of each cell in the stripe in confocal cross-sections. The stripes are separated by a band of about five cells that have a lower level of localized Smo. In hmgcrz− embryos, the stripe pattern is much less well defined. Moreover, unlike wild-type, the Smo protein is not tightly localized to the cell surface in many of the cells in the stripe, but, instead, it is distributed in the cytoplasm. Though the Smo localization pattern across each segment in hmgcrz− embryos is disrupted, the effects on Smo are not as severe as those seen in hh null embryos (Deshpande, 2005).

The defect in Smo relocalization in hmgcrz− embryos supports the idea that Hmgcr activity is required for the production and/or activity of the Hh ligand. To test this possibility further, the pattern of Hh accumulation was compared in wild-type and hmgcrz− (hmgcr11.57) embryos. In wild-type embryos, Hh is expressed in each parasegment in a two cell wide stripe, and the protein in these cells is distributed around the membrane in a punctate pattern. Extending outward in either direction from the stripe is a relatively sharp gradient of Hh protein. Like the cells in the stripe, the Hh protein associated with the interstripe cells is generally distributed in a punctate pattern around the membrane. No defects in Hh protein expression are apparent in hmgcrz− embryos, and, as seen in wild-type, there is a two cell wide stripe of Hh-expressing cells in each parasegment. Moreover, like wild-type, the protein is concentrated in a punctate pattern around the cell membrane. In contrast, the amount of Hh protein in the hmgcrz− stripes is considerably higher than wild-type. Concomitant with the increase in the level of Hh in cells in each stripe, the amount of protein in interstripes is greatly reduced in hmgcrz− embryos relative to that seen in wild-type. Similar results were obtained for hmgcrm−z− (Deshpande, 2005).

The abnormal pattern of accumulation of Hh seen in hmgcr mutant embryos suggests that hmgcr is required for the efficient release of Hh from the two cells that express this ligand and/or in the transport of Hh from these cells to the adjacent receiving cells. To test this idea further, the effects of ectopically produced Hmgcr were examined on expression of the Hh target gene wg. It was reasoned that if Hmgcr functions primarily in Hh-producing cells to promote the efficient release or dispersal of the Hh ligand, then overexpression of Hmgcr in these cells might be expected to have a more pronounced effect on wg than overexpression in the neighboring Hh-receiving cells. To direct Hmgcr expression in cells that normally produce the Hh ligand, an hh-Gal4 driver was used, while, for the control, either a ptc or a wg driver was used to direct Hmgcr expression in cells that normally respond to the Hh ligand (Deshpande, 2005).

These expectations were met. In embryos in which Hmgcr is expressed in Hh-receiving cells by using the ptc or wg driver, the pattern of Wg accumulation resembles that of wild-type. Wg is expressed in a single cell wide stripe in each parasegment, and it localizes in these cells in a punctate pattern near the cell membrane. A low level of Wg associated with the membranes of cells in the interstripe region can also be detected. In contrast, when Hmgcr is expressed in Hh-producing cells, the level of Wg accumulation cells in the wg stripe is substantially upregulated. Moreover, an expansion of the stripe from a single cell to a two cell wide stripe was sometimes observed. In addition, high amounts of Wg could be seen extending through much of the interstripe region (Deshpande, 2005).

The driver-dependent effects of Hmgcr on Wg accumulation would be consistent with the idea that Hmgcr is most effective in enhancing Hh signaling when it is expressed in Hh-producing cells. To test this idea further, the distribution of Hh protein was examined in embryos in which Hmgcr was expressed under the direction of the hh, wg, and ptc drivers. When Hmgcr expression is directed by the wg or ptc drivers, the distribution of Hh protein resembles that seen in wild-type embryos. Hh accumulates in a two cell wide stripe in each parasegment, while there is only a relatively low level of Hh protein to either side of this stripe. A different result is obtained with the hh driver. Though the Hh parasegmental stripes are still discernable, the stripes are much broader than in wild-type (or when Hmgcr expression is controlled by wg or ptc-Gal4), and there are high levels of Hh extending to almost the middle of the interstripe region. The effect of ectopic hmgcr was also examined by using a paired-Gal4 driver that drives expression in alternate segments. As expected, it was found that the breadth of the Hh stripe was increased in alternate segments (Deshpande, 2005).

In the embryo, hh and wg establish an autoregulatory circuit in which signaling by one ligand potentiates signaling by the other. Thus, it is formally possible that the upregulation of hh signaling evident when hmgcr is overexpressed by using the hh driver is the indirect consequence of augmenting the reception of the wg signal in hh-expressing cells. To exclude this possibility, whether hh signaling can be potentiated by ectopic expression of hmgcr in hh-sending cells was tested in the wing disc, in which there is no autoregulatory circuit between hh and wg. In the wing disc, the Hh ligand is expressed in the posterior compartment, and it promotes Ptc protein accumulation in the anterior compartment along the compartment boundary. When Hmgcr is expressed in the receiving cells by using the ptc-Gal4 driver, there is little effect on Ptc accumulation, and it resembles that in wild-type. By contrast, when Hmgcr is ectopically expressed in hh-sending cells by using the hh-Gal4 driver, Ptc accumulation is upregulated. These findings indicate that Hmgcr can function in hh sending cells in the wing disc to potentiate hh signaling (Deshpande, 2005).

Although neither hh nor disp is haplo-insufficient with respect to germ cell migration, synergistic genetic interactions are observed when mutations in these two genes are combined in trans. Germ cell migration is essentially indistinguishable from wild-type in embryos heterozygous for an hh mutation, and fewer than 20% of the stage 13 to stage 16 embryos have four or more mispositioned germ cells. This is also true for embryos heterozygous for a mutation in disp. In contrast, germ cell migration defects are readily apparent in the trans combination, and nearly 90% of the stage 13 to stage 16 embryos have ten or more mispositioned germ cells (Deshpande, 2005).

If the requirement for hmgcr function in germ cell migration is related to its role in promoting the transmission or movement of the Hh ligand, then equivalent synergistic genetic interactions between hmgcr and either hh or disp should be found. This is the case. There are minor germ cell migration defects in hmgcr/+ embryos, and about 35% of the stage 13 to stage 16 embryos have four or more mispositioned germ cells. These minor defects are substantially exacerbated when hmgcr is combined with mutations in hh or disp. In hh/hmgcr trans-heterozygotes, more than 95% of the stage 13 to stage 16 embryos have ten or more mispositioned germ cells. Similarly, like the disp/hh combination, a high frequency of germ cell migration defects are evident in the disp/hmgcr trans combination (Deshpande, 2005).

In embryos heterozygous for the hh gain-of-function allele hhMrt, there are defects in germ cell migration, and about 60% of the stage 13 to stage 16 embryos have four or more mispositioned germ cells. Like the wing abnormalities in hhMrt flies, this weak germ cell migration phenotype is presumed to arise from the misexpression of Hh protein. However, the mechanism is likely to be different from that involved in the mispecification of anterior compartment cells by ectopic Hh. In this case, the ectopic Hh expressed by the Mrt allele probably competes with the protein produced by the somatic gonadal precursor cells as an attractant and misdirects the migrating germ cells. If the effects of Mrt on migration are due to competition, it was reasoned that it should be possible to enhance the germ cell migration phenotype of hhMrt by reducing the potency of the Hh signal emanating from the somatic gonadal precursor cells. Consistent with this expectation, mutations in both hh and disp significantly increase the severity of the migration defects seen in hhMrt, and, in each case, almost all of the embryos had ten or more mispositioned germ cells. An hmgcr mutation also substantially enhances the hhMrt migration defects, and its effects are equivalent to mutations in either hh or disp (Deshpande, 2005).

A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway

Members of the Hedgehog (Hh) family of signaling proteins are powerful regulators of developmental processes in many organisms and have been implicated in many human disease states. This study reports the results of a genome-wide RNA interference screen in Drosophila cells for new components of the Hh signaling pathway. The screen identified hundreds of potential new regulators of Hh signaling, including many large protein complexes with pleiotropic effects, such as the coat protein complex I (COPI), the ribosome and the proteasome. The multimeric protein phosphatase 2A (PP2A) and two new kinases, the D. melanogaster orthologs of the vertebrate PITSLRE and cyclin-dependent kinase-9 (CDK9) kinases, were identified as Hh regulators. A large group of constitutive and alternative splicing factors, two nucleoporins involved in mRNA export and several RNA-regulatory proteins were identified as potent regulators of Hh signal transduction, indicating that splicing regulation and mRNA transport have a previously unrecognized role in Hh signaling. Finally, it was shown that several of these genes have conserved roles in mammalian Hh signaling (Nybakken, 2005).

Phosphorylation is associated with the activities of at least five components of the Hh pathway: Fu, Cos, Smo, Su(fu) and Ci. Little is known about the kinases that phosphorylate Su(fu) and Fu, but at least two sites in Cos are phosphorylated by Fu, and several kinases are involved in phosphorylating Ci and Smo, including PKA-C1, CkIalpha and Sgg. But no phosphatase has been implicated in Hh signaling, and a previous RNAi screen did not identify any phosphatases involved in Hh signaling. The screen identified microtubule star (mts), which encodes the D. melanogaster PP2A catalytic subunit, as a gene that substantially reduced Hh signaling when targeted by RNAi. PP2A is a multimeric enzyme that consists at minimum of the catalytic subunit, a regulatory A subunit (encoded by CG33297 in D. melanogaster) and a B subunit principally involved in substrate selection. The B-subunit family in D. melanogaster is represented by the gene twins (tws), the B' family by the genes widerborst (wdb) and PP2A-B', and the B" family by CG4733. All the PP2A component dsRNAs were obtained and tested from a dsRNA library and additional, distinct dsRNAs to these components were generated and tested. In addition to confirming the mts result, it was found that both the original-library dsRNA and three new, unique dsRNAs targeting wdb all reduced Hh signaling. This indicates that Wdb is likely to be the B subunit that targets Mts to its substrate in the Hh signaling pathway. This hypothesis is in agreement with recent findings from Xenopus laevis, where the wdb ortholog encoding B56e has been found to regulate Hh signaling. In addition, some PP2A-B' amplicons cause a reduction in reporter activity averaging ~30%, indicating that they may have a partially redundant role in targeting PP2A to its Hh pathway substrate (Nybakken, 2005).

To determine whether PP2A acts on Cos, whether overexpression of cos and mts results in similar phenotypes was examined. When overexpressed in Hh-stimulated clone 8 cells, cos completely abrogates Hh signaling, reducing it to near uninduced levels, whereas overexpression of mts reduces Hh signaling by 40%. Thus, Mts and Cos have different overexpression profiles and do not seem to regulate Hh signaling in the same way. The overexpression phenotype of mts was compared with those of cos and 14 other hits from the screen, including the fu, Cdk9 and Pka-C1 kinases. Overexpressing cos in uninduced cells further reduces background signaling, whereas mts overexpression doubled reporter activity, although these levels are still very low compared with the Hh-activated state. Of the 18 other genes tested, only Pka-C1 overexpression had an effect on Hh reporter activity similar to that of mts: doubling of reporter activity in the Hh-uninduced state and a 50% reduction of activity in the Hh-stimulated state. It is therefore possible that PKA-C1 and Mts act on similar substrates. Because several studies have identified Ci as a substrate of PKA-C1, Mts could also be acting on Ci, perhaps removing inhibitory phosphates in response to Hh stimulation (Nybakken, 2005).

This screen allowed the grouping of the ribosome, proteasome, COPI complex and PP2A phosphatase as important regulators of Hh signaling, none of which had been identified as Hh regulators in vivo. Notably, some of the components identified in the screen had already been implicated in aspects of Hh signaling. For instance, the gene encoding eRF1, a translational regulator, was identified in a screen for modifiers of a gain-of-function smo allele, and polyhomeotic and additional sex combs have both been shown to modify ectopic hh expression phenotypes. These results open many new avenues for investigation of Hh signaling. In particular, elucidation of the Hh pathway substrates affected by PP2A will be important in defining the role of dephosphorylation in Hh signaling. Finally, the paradigm of Hh signaling would change substantially if further investigation determines that alternative splicing and mRNA regulation do have vital roles in Hh signaling (Nybakken, 2005).


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

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