Zugasti O et al. (2005) Genome Res "The function and expansion of the Patched- and Hedgehog-related homologs in C. elegans."

Kuwabara PE, Zugasti O, Rajan J

[Genome Res, 2005]

The Hedgehog (Hh) signaling pathway promotes pattern formation and cell proliferation in Drosophila and vertebrates. Hh is a ligand that binds and represses the Patched (Ptc) receptor and thereby releases the latent activity of the multipass membrane protein Smoothened (Smo), which is essential for transducing the Hh signal. In Caenorhabditis elegans, the Hh signaling pathway has undergone considerable divergence. Surprisingly, obvious Smo and Hh homologs are absent whereas PTC, PTC-related (PTR), and a large family of nematode Hh-related (Hh-r) proteins are present. We find that the number of PTC-related and Hh-r proteins has expanded in C. elegans, and that this expansion occurred early in Nematoda. Moreover, the function of these proteins appears to be conserved in Caenorhabditis briggsae. Given our present understanding of the Hh signaling pathway, the absence of Hh and Smo raises many questions about the evolution and the function of the PTC, PTR, and Hh-r proteins in C. elegans. To gain insights into their roles, we performed a global survey of the phenotypes produced by RNA-mediated interference (RNAi). Our study reveals that these genes do not require Smo for activity and that they function in multiple aspects of C. elegans development, including molting, cytokinesis, growth, and pattern formation. Moreover, a subset of the PTC, PTR, and Hh-r proteins have the same RNAi phenotypes, indicating that they have the potential to participate in the same processes.

Burglin TR (2008) BMC Genomics "Evolution of hedgehog and hedgehog-related genes, their origin from Hog proteins in ancestral eukaryotes and discovery of a novel Hint motif."

Burglin TR

[BMC Genomics, 2008]

ABSTRACT: BACKGROUND: The Hedgehog (Hh) signaling pathway plays important roles in human and animal development as well as in carcinogenesis. Hh molecules have been found in both protostomes and deuterostomes, but curiously the nematode Caenorhabditis elegans lacks a bona-fide Hh. Instead a series of Hh-related proteins are found, which share the Hint/Hog domain with Hh, but have distinct N-termini. Results: We performed extensive genome searches such as the cnidarian Nematostella vectensis and several nematodes to gain further insights into Hh evolution. We found six genes in N. vectensis with a relationship to Hh: two Hh genes, one gene with a Hh N-terminal domain fused to a Willebrand factor type A domain (VWA), and three genes containing Hint/Hog domains with distinct novel N-termini. In the nematode Brugia malayi we find the same types of hh-related genes as in C. elegans. In the more distantly related Enoplea nematodes Xiphinema and Trichinella spiralis we find a bona-fide Hh. In addition, T. spiralis also has a quahog gene like C. elegans, and there are several additional hh-related genes, some of which have secreted N-terminal domains of only 15 to 25 residues. Examination of other Hh pathway components revealed that T. spiralis - like C. elegans - lacks some of these components. Extending our search to all eukaryotes, we recovered genes containing a Hog domain similar to Hh from many different groups of protists. In addition, we identified a novel Hint gene family present in many eukaryote groups that encodes a VWA domain fused to a distinct Hint domain we call Vint. Further members of a poorly characterized Hint family were also retrieved from bacteria. CONCLUSION: In Cnidaria and nematodes the evolution of hh genes occurred in parallel to the evolution of other genes that contain a Hog domain but have different N-termini. The fact that Hog genes comprising a secreted N-terminus and a Hog domain are also found in many protists suggests that this gene family must have arisen in very early eukaryotic evolution, which eventually gave rise to hh and hh-related genes in animals. The results indicate a hitherto unsuspected ability of Hog domain encoding genes to evolve new N-termini. In one instance in Cnidaria, the Hh N-terminal signaling domain is associated with a VWA domain and lacks a Hog domain, suggesting a modular mode of evolution also for the N-terminal domain. The Hog domain proteins, the inteins and VWA-Vint proteins represent three different families of Hint domain proteins that evolved in parallel in eukaryotesConclusion: In Cnidaria and nematodes the evolution of hh genes occurred in parallel to the evolution of other genes that contain a Hog domain but have different N-termini. The fact that Hog genes comprising a secreted N-terminus and a Hog domain are also found in many protists suggests that this gene family must have arisen in very early eukaryotic evolution, which eventually gave rise to hh and hh-related genes in animals. The results indicate a hitherto unsuspected ability of Hog domain encoding genes to evolve new N-termini. In one instance in Cnidaria, the Hh N-terminal signaling domain is associated with a VWA domain and lacks a Hog domain, suggesting a modular mode of evolution also for the N-terminal domain. The Hog domain proteins, the inteins and VWA-Vint proteins represent three different families of Hint domain proteins that evolved in parallel in eukaryotes.

Porter JA et al. (1996) Cell "Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain."

Ekker SC, Cotter RJ, Koonin EV, Ma Y, von Kessler DP, Beachy PA, Chen CH, Porter JA, Woods AS, Young KE, Park WJ

[Cell, 1996]

Autocatalytic processing mediated by the carboxyterminal domain of the hedgehog (hh) protein precursor (Hh) generates an amino-terminal product that accounts for all known signaling activity. The role of autoprocessing biogenesis of the hh signal has been unclear, since a truncated unprocessed protein lacking all carboxy-terminal domain sequences retains signaling activity. Here, we present evidence that the autoprocessing reaction proceeds via an internal thioester intermediate and results in a covalent modification that increases the hydrophobic character of the signaling domain and influences its spatial and subcellular distribution. We demonstrate that truncated unprocessed amino-terminal protein causes embryonic mispatterning, even when expression is localized to cells that normally express Hh, thus suggesting a role for autoprocessing in spatial regulation of hh signaling. This type of processing also appears to operate in the biogenesis of other novel secreted proteins.

Thomas R. Burglin (2006) WormBook "Homologs of the Hh signalling network in C. elegans."

Thomas R. Burglin

[WormBook, 2006]

In Drosophila and vertebrates, Hedgehog (Hh) signalling is mediated by a cascade of genes, which play essential roles in cell proliferation and survival, and in patterning of the embryo, limb buds and organs. In C. elegans, this pathway has undergone considerable evolutionary divergence; genes encoding homologues of key pathway members, including Hh, Smoothened, Cos2, Fused and Suppressor of Fused, are absent. Surprisingly, over sixty proteins (i.e. WRT, GRD, GRL, and QUA), encoded by a set of genes collectively referred to as the Hh-related genes, and two co-orthologs ( PTC-1 ,-3) of fly Patched, a Hh receptor, are present in C. elegans. Several of the Hh-related proteins are bipartite and all can potentially generate peptides with signalling activity, although none of these peptides shares obvious sequence similarity with Hh. In addition, the ptc -related ( ptr ) genes, which are present in a single copy in Drosophila and vertebrates and encode proteins closely related to Patched, have undergone an expansion in number in nematodes. A number of functions, including roles in molting, have been attributed to the C. elegans Hh-related, PTC and PTR proteins; most of these functions involve processes that are associated with the trafficking of proteins, sterols or sterol-modified proteins. Genes encoding other components of the Hh signalling pathway are also found in C. elegans, but their functions remain to be elucidated.

Wang X et al. (1998) East Coast Worm Meeting "A MOLECULE CONTAINING A HEDGEHOG-LIKE AUTOPROCESSING DOMAIN IS REQUIRED FOR MOLTING IN C. ELEGANS."

Beachy P, Seydoux GC, Wang X

[East Coast Worm Meeting, 1998]

The Hedgehog family of signaling molecules controls many aspects of animal development, from embryonic patterning to limb formation. Recently, these proteins have been shown to rely on a novel mechanism to regulate their spatial distribution (1,2). Hedgehog proteins undergo an autoprocessing reaction to yield two distinct products, an amino-terminal fragment (Hh-N), and a carboxy-terminal fragment (Hh-C). Hh-N functions in signaling, whereas Hh-C mediates the processing reaction. This reaction proceeds via an internal thioester intermediate and results in the covalent linkage of cholesterol to Hh-N. This modification in turn causes Hh-N to remain tightly associated with the cell surface, thus effectively limiting its free diffusion and range of action. We are interested in determining whether similar mechanisms may be operating in the biogenesis of other secreted molecules. Towards this goal, we have started to characterize a set of eight C. elegans proteins with sequence similarity to the Hh-C domain of Hedgehog (2,3). In these proteins, as in the Hedgehog family, the Hh-C-like domain is located in the carboxy-teminal end, and is preceded by an amino-terminal domain bearing a signal sequence (but with otherwise no homology to Hh-N). This structure is consistent with the possibility that these proteins are secreted and undergo processing in a manner similar to that described for Hedgehog. To test this possibility we have fused the Hh-C-like domain of one of these proteins to a His tag, and purified the fusion protein from E. coli. We find that such a fusion can undergo the processing reaction in vitro, suggesting that the Hh-C-like domain is functionally active in the nematode proteins. We have begun to characterize the functions of Hh-C-related proteins using RNA-mediated interference. Our initial results suggest that one of these proteins, T05C12, is essential for molting. Injection of T05C12 double-stranded RNA results in 90% larval lethality. The larvae apparently die from a failure to shed old cuticles during molts. In many animals, a cuticular plug can be seen obstructing the mouth opening where it presumably interferes with feeding. The amino-terminal domain of T05C12 contains several sequence motifs similar to those found in collagens and other extracellular matrix proteins, raising the possibility that the T05C12 product is a cuticle component. Consistent with this possibility, a T05C12:GFP fusion is expressed in hypodermal cells during larval and adult stages. We are currently determining the effects of expressing mutant forms of T05C12 to test the function of its Hh-C-like domain. 1. Porter, JA. et al. (1996). Cell 86, 21-34. 2. Porter, JA. et al. (1996). Science 274, 255-259. 3. Burglin, TR. (1996). Current Biology 6, 1047-1050.

Aspock G et al. (1999) Genome Res "Caenorhabditis elegans has scores of hedgehog-related genes: sequence and expression analysis."

Kagoshima H, Burglin TR, Aspock G, Niklaus G

[Genome Res, 1999]

Previously, we have described novel families of genes, warthog (wrt) and groundhog (grd), in Caenorhabditis elegans. They are related to Hedgehog (Hh) through the carboxy-terminal autoprocessing domain (called Hog or Hint). A comprehensive survey revealed 10 genes with Hog/Hint modules in C. elegans. Five of these are associated with a Wart domain in wrt genes, and three with multiple copies of the Ground domain in grd genes. Both the Wart domain and the Ground domain occur also in genes encoding no Hog domain. Further, we define a new group of genes related to the grd genes, called ground-like (grl). Overall, C. elegans has more than 50 genes belonging to these gene families. Phylogenetic and sequence analysis shows that the wrt, grd, and grl genes are derived from each other. Further examination reveals a sequence motif with similarity to the core of the amino-terminal-signaling domain of Hh proteins. Our data suggest that the wrt, grd, grl, and hh genes are derived from a single ancestral gene. wrt, grd, and grl genes are also present in other nematodes, but so far not in any other phyla. Conversely, hh is not found presently in C. elegans nor other nematodes. Thus, the nematode genes could be the homologs of Hh molecules in other phyla. The membrane molecule Patched has been shown previously to be a receptor of Hh. Many Patched-related proteins are present in C. elegans, which may be targets of the hh-related genes. No Hedgehog-interacting protein (Hip) was found. We analyzed the expression patterns of eight wrt and eight grd genes. The results show that some closely related genes are expressed in the same tissues, but, overall, the expression patterns are diverse, comprising hypodermis, seam cells, the excretory cell, sheath and socket cells, and different types of neurons.

Kuwabara PE et al. (2000) Genes Dev "A C. elegans patched gene, ptc-1, functions in germ-line cytokinesis."

Schedl TB, Kuwabara PE, Jefferis GSXE, Lee MH

[Genes Dev, 2000]

Patched (Ptc), initially identified in Drosophila, defines a class of multipass membrane proteins that control cell fate and cell proliferation. Biochemical studies in vertebrates indicate that the membrane proteins Ptc and Smoothened (Smo) form a receptor complex that binds Hedgehog (Hh) morphogens. Smo transduces the Hh signal to downstream effectors. The Caenorhabditis elegans genome encodes two Ptc homologs and one related pseudogene but does not encode obvious Hh or Smo homologs. We have analyzed ptc-1 by RNAi and mutational deletion and find that it is an essential gene, although the absence of ptc-1 has no detectable effect on body patterning or proliferation. Therefore, the C. elegans ptc-1 gene is functional despite the lack of Hh and Smo homologs. We find that the activity and expression of ptc-1 is essentially confined to the germ line and its progenitors. ptc-1 null mutants are sterile with multinucleate germ cells arising from a probable cytokinesis defect. We have also identified a surprisingly large family of PTC-related proteins containing sterol-sensing domains, including homologs of Drosophila dispatched, in C. elegans and other phyla. These results suggest that the PTC superfamily has multiple functions in animal development.

X Wang et al. (1999) International C. elegans Meeting "A molecule containing a Hedgehog-intein (HINT) domain is required for molting in C. elegans."

G Seydoux, P Beachy, X Wang

[International C. elegans Meeting, 1999]

Hedgehog proteins undergo an autoprocessing reaction to yield an amino-terminal fragment (Hh-N) and a carboxy-terminal fragment (Hh-C). Hh-N functions in signaling, whereas Hh-C, which consists of a Hint domain and a sterol recognition region, mediates the processing reaction. This reaction proceeds via an internal thioester intermediate and results in the covalent linkage of cholesterol to Hh-N. This modification in turn causes Hh-N to remain tightly associated with the cell surface, thus effectively limiting its free diffusion and range of action. We are interested in determining whether similar mechanisms might be operating in the biogenesis of other secreted molecules. Towards this goal, we have started to characterize a C. elegans protein (T05C12.10) with sequence similarity to the Hint domain of Hedgehog (Porter, JA. et al.,1996a,b; Burglin, TR., 1996). In this protein, as in the Hedgehog family, the Hint domain is located near the carboxy-terminus, and is preceded by an amino-terminal domain bearing a signal sequence (but with otherwise no homology to Hh-N). We have shown that the Hint domain of T05C12.10 can lead to the formation of an internal thioester intermediate in vitro . We have begun to characterize the functions of the T05C12.10 using RNA-mediated interference. Our results suggest that T05C12.10 is essential for molting. Injection of T05C12.10 double-stranded RNA results in 100% larval lethality. The larvae apparently die from a failure to shed old cuticles during molts. This phenotype resembles the phenotype of worms starved for cholesterol and of mutants lacking the megalin homologue, lrp-1 (Yochem, J. et al. 1999). The amino-terminal domain of T05C12.10 contains several sequence motifs similar to certain extracellular matrix proteins. Consistent with this, T05C12.10 protein colocalizes with megalin in the apical surface of hypodermal cells during larval and adult stages. Western analysis indicates that there are two forms of T05C12.10 in vivo , and that the relative abundance of the smaller form increases during each molt. These observations suggest that processing of T05C12.10 coincides with, and may be regulated by, molting. We are currently determining the identity of the smaller form as well as testing its role in molting.

Hao L et al. (2006) BMC Genomics "Comprehensive analysis of gene expression patterns of hedgehog-related genes."

Burglin TR, Baillie D, Lauter G, Hao L, Johnsen R

[BMC Genomics, 2006]

ABSTRACT: BACKGROUND: The Caenorhabditis elegans genome encodes ten proteins that share sequence similarity with the Hedgehog signaling molecule through their C-terminal autoprocessing Hint/Hog domain. These proteins contain novel N-terminal domains, and C. elegans encodes dozens of additional proteins containing only these N-terminal domains. These gene families are called warthog, groundhog, ground-like and quahog, collectively called hedgehog (hh)-related genes. Previously, the expression pattern of seventeen genes was examined which showed that they are primarily expressed in the ectoderm. RESULTS: With the completion of the C. elegans genome sequence in November 2002, we reexamined and identified 61 hh-related ORFs. Further, we identified 49 hh-related ORFs in C. briggsae. ORF analysis revealed that 30% of the genes still had errors in their predictions and we improved these predictions here. We performed a comprehensive expression analysis using GFP fusions of the putative intergenic regulatory sequence with one or two transgenic lines for most genes. The hh-related genes are expressed in one or a few of the following tissues: hypodermis, seam cells, excretory duct and pore cells, vulval epithelial cells, rectal epithelial cells, pharyngeal muscle or marginal cells, arcade cells, support cells of sensory organs, and neuronal cells. Using time-lapse recordings, we discovered that some hh-related genes are expressed in a cyclical fashion in phase with molting during larval development. We also generated several translational GFP fusions, but they did not show any subcellular localization. In addition, we also studied the expression patterns of two genes with similarity to Drosophila frizzled, T23D8.1 and F27E11.3A, and the ortholog of the Drosophila gene dally-like, gpn-1, which is a heparan sulfate proteoglycan. The two frizzled homologs are expressed in a few neurons in the head, and gpn-1 is expressed in the pharynx. Finally, we compare the efficacy of our GFP expression effort with EST, OST and SAGE data. CONCLUSIONS: No bona-fide Hh signaling pathway is present in C. elegans. Given that the hh-related gene products have a predicted signal peptide for secretion, it is possible that they constitute components of the extracellular matrix (ECM). They might be associated with the cuticle or be present in soluble form in the body cavity. They might interact with the Patched or the Patched-related proteins in a manner similar to the interaction of Hedgehog with its receptor Patched.

Limin Hao et al. (2006) European Worm Meeting "Comprehensive Analysis of Gene Expression Patterns of Hedgehog-Related Genes"

Robert Johnsen, Limin Hao, Thomas R. Burglin, Gilbert Lauter, David Baillie

[European Worm Meeting, 2006]

Limin Hao1, Robert Johnsen3, Gilbert Lauter, David Baillie3, Thomas R. Brglin The Caenorhabditis elegans genome encodes ten proteins that share homology with the Hedgehog signaling molecule through their C-terminal autoprocessing Hint/Hog domain. These proteins contain novel N-terminal domains, and C. elegans encodes dozens of additional proteins containing only these N-terminal domains. These gene families are called warthog, groundhog, ground-like and quahog, collectively called hedgehog (hh)-related genes. Previously, the expression pattern of sixteen genes was examined which showed that they are primarily expressed in the ectoderm. With the completion of the C. elegans genome sequence in October 2002, we examined and identified all 61 hh-related ORFs. ORF analysis revealed that 30% of the genes still had errors in their predictions that we corrected here. We performed a comprehensive expression analysis using GFP reporters. The hh-related genes are expressed in one or a few of the following tissues: hypodermis, seam cells, excretory duct and pore cells, vulval epithelial cells, rectal epithelial cells, pharyngeal muscle or marginal cells, arcade cells, support cells of sensory organs, and neuronal cells. Using time-lapse recordings, we discovered that some hh-related genes are expressed in a cyclical fashion in phase with molting during larval development. We also generated several translational GFP fusions, but they did not show any subcellular localization. In addition, we also studied the expression patterns of two genes with similarity to Drosophila frizzled, T23D8.1 and F27E11.3A, and the ortholog of the Drosophila gene dally-like, gpn-1, which is a heparan sulfate proteoglycan. The two frizzled homologs are expressed in a few neurons in the head, and gpn-1 is expressed in the pharynx. No bona-fide Hh signaling pathway is present in C. elegans. Given that the hh-related gene products have a predicted signal peptide for secretion, it is possible that they constitute components of the extracellular matrix (ECM). They might be associated with the cuticle or be present in soluble form in the body cavity. They may interact with the Patched or the Patched-related proteins in a manner similar to the interaction of Hedgehog with its receptor Patched.