Homologs of the Hh signalling network in C. elegans^*

The Drosophila hedgehog (hh) mutant was identified by Nüsslein-Volhard and Wieschaus in their classic genetic screen that led to the identification of key embryonic segment polarity genes (Nüsslein-Volhard and Wieschaus, 1980). Subsequently, the single Drosophila and the multiple vertebrate Hh proteins, Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh), have collectively proven to be key effectors in patterning the embryo, limb buds and neural tube, organ formation, cell proliferation, axon guidance and cell survival (for reviews, see Ingham and McMahon, 2001; Lum and Beachy, 2004). Mutations in genes constituting the Hh signalling pathway are also implicated in various human syndromes and cancers (Bale, 2002; Beachy et al., 2004; Pasca di Magliano and Hebrok, 2003; Taipale and Beachy, 2001). Details of the pathway are summarised in Figure 1 (Ingham and McMahon, 2001; Lum and Beachy, 2004). The various proteins involved in the Hh and Ptc signalling pathway, including those present in C. elegans, are listed in Table 1.

What role does the Hh/Ptc signalling pathway play in C. elegans development? To address this question, it is first reasonable to ask whether the major proteins of the pathway are encoded by the C. elegans genome, a task that is simplified by the availability of the complete genome sequence (Table 1; C. elegans Sequencing Consortium, 1998). Surprisingly, genes encoding recognisable Hh and Smo proteins are absent. In addition, homologs for Cos2, Fu and Su(fu), which transduce the Hh signal from Smo to Ci, are absent. The Ci homolog itself, TRA-1, is not essential for embryonic pattern formation, but continues to act at the transcriptional level to control sexual cell fate decisions and male gonadal development (Hodgkin, 1983; Mathies et al., 2004), although there is no evidence that the nuclear localisation or activity of TRA-1 is controlled in the same manner as Ci (Kuwabara et al., 2000; Zarkower and Hodgkin, 1992). It has been speculated that the C. elegans sex determination pathway could be evolutionarily related to the Hh/Ptc pathway because additional similarities have also been noted between the two pathways (Kuwabara et al., 1992).

The C. elegans genome encodes an abundance of what will be collectively referred to as Hh-related (Hh-r) proteins, three Patched proteins and 24 Patched-related proteins (PTR). Four Frizzled receptor homologues (see Wnt signaling), as well as a series of molecules participating in the early stages of Hh signalling are also present. Given our current understanding of the Hh/Ptc signal transduction pathway in other organisms, the presence of multiple C. elegans Hh-r, PTC and PTR proteins and the absence of Hh, Smo and other components is unexpected and raises a number of questions about the evolution and the function of these genes in C. elegans.

The Hh proteins are composed of an N-terminal signalling domain, Hh-N, and a C-terminal autoprocessing domain, Hh-C. Hh-C has autoproteolytic activity, which cleaves Hh and subsequently attaches a cholesterol moiety to the Hh-N C-terminus (see scheme Figure 2A; Lee et al., 1994; Porter et al., 1996). Hh-C shares sequence similarity with the self-splicing inteins; hence, this region was named Hint (Dalgaard et al., 1997; Hall et al., 1997; Koonin, 1995). The 60 terminal residues of Hh-C are named the sterol recognition region (SRR) because they are required for adding cholesterol to Hh-N (Figure 2A; Beachy et al., 1997). Here, we refer to the combined Hint-SRR domain as the Hog domain.

Surprisingly, early database searches uncovered eight distinct C. elegans ORFs sharing similarity to Hh (Bürglin, 1996; Porter et al., 1996). Further investigation revealed that the sequence similarity shared by Hh and these ORFs was confined only to the Hog domain. Analyses of the N-terminal regions revealed several new motifs (Bürglin, 1996; Porter et al., 1996), which were also found in other ORFs that lacked Hog domains. In mimicry of the bipartite “Hedge-hog” structure, the three new gene families were named, warthog (wrt), groundhog (grd) and ground-like (grl) and the protein domains found at their respective N-terminal regions were called Wart, Ground and Ground-like (Aspöck et al., 1999; Bürglin, 1996). The final additions to the nomenclature are two previously unnamed single copy genes, qua-1 for quahog (T05C12.10/M110, Hao et al., 2006b), and an ORF consisting only of the Hog domain, hog-1 (W06B11.4). The structural organization of Hh and the Hh-r genes is shown in Figure 2B.

Over 60 Hh-r genes have been identified in C. elegans, most of which are expressed, although a few appear to be pseudogenes (e.g., grd-17; Figure 2B). Many of these genes share a one to one orthology to C. briggsae genes, although several have arisen through recent duplications within the C. elegans lineage. Preliminary searches of the ongoing Brugia malayi genome project reveal that representatives of the different C. elegans families are also present (Figure 2B). This clearly demonstrates that these Hh-r gene families were already present in the last common ancestor of C. elegans and B. malayi more than 300 million years ago.

The Wart, Ground, Ground-like and Qua domain each have a characteristic pattern of conserved cysteine residues (Figure 2C). The Ground and Ground-like domains differ in their arrangement of these conserved cysteine residues (Aspöck et al., 1999). In some instances, it has been possible to predict likely disulfide bond arrangements because of pairwise cysteine substitutions (Figure 2C; Aspöck et al., 1999). The Wart domain is about twice the size of the Ground and Ground-like domain; however, the C-terminal half of the Wart domain has a cysteine arrangement similar to that of the Ground domain and both share a short conserved motif similarly positioned between cysteine residues (FDøI, FEøV, respectively, Figure 2C; Aspöck et al., 1999). Thus, it is likely that the C-terminal part of the Wart domain and the Ground domain was derived from the same ancestral domain. The short conserved FDøV “core” motif is also present in the Hh-N core, although its position relative to the cysteine residues is different (Figure 2C; Aspöck et al., 1999).

All Hh-r genes in C. elegans, apart from hog-1, are predicted to encode secreted molecules. Porter et al. (1996) have shown that WRT-1, similar to Hh, undergoes autoprocessing; thus it is expected that other Hh-r proteins with a Hog domain will also have autocatalytic properties. However, it remains unclear whether the Hh-r proteins can bind cholesterol or other sterols, because the region corresponding to the SRR of Hh is slightly different in the Hh-r proteins (see Figure 1A in Aspöck et al., 1999).

An expression survey of wrt and grd genes revealed that they are expressed primarily in ectodermal tissues, particularly the hypodermis and seam cells, but can also be found in neurons, neuron-associated cells and gland cells (Aspöck et al., 1999). qua-1 is also expressed in the hypodermis (Hao et al., 2006b). An internal deletion in qua-1 results in severe molting defects (Hao et al., 2006b). Functional studies of other wrt genes are in progress and preliminary results indicate that they also play important roles in development. For example, wrt-5 functions in the hypodermis during embryogenesis (Hao et al., 2006a). At present it is still unclear how Hh-r molecules function, and whether their activity could relate to the function of the PTC and PTR proteins.

In Drosophila, Hh is further modified by Skinny hedgehog (Ski), which palmitoylates the N-terminus of Hh-N, an essential process for Hh function (Chamoun et al., 2001). C. elegans has two co-orthologs, Y57G11C.17 and ZC101.3 (T.R. Bürglin, unpublished), but large-scale RNAi experiments have not uncovered any associated mutant phenotype. Modified Hh is secreted with the help of Dispatched; C. elegans has two Disp homologs, CHE-14 and PTD-2 (see below).

Secreted Hh forms a multimeric complex that moves through the extracellular matrix space. Heparan sulphate proteoglycans (HSPG) play an important role in Hh signal transduction. Three Drosophila genes, tout-velu (ttv), sister of tout-velu (sotv), and brother of tout-velu (botv) encode glycosyltransferases of the EXT family that are required for the biosynthesis of heparan sulfate and are essential for the movement of N-Hh (Han et al., 2004a; Takei et al., 2004). One target for these enzymes is Dally-like (Dlp), a glypican of the HSPG family that is also essential for Hh signalling (Desbordes and Sanson, 2003; Han et al., 2004b; Lum et al., 2003). C. elegans has two EXT family members, rib-1 and rib-2 (for review, see Berninsone and Hirschberg, 2002). rib-1 is orthologous to ttv and EXT1, and rib-2 is ortholgous to botv and EXTL3; however, orthologs of other EXT members are absent (Figure 3). The EXT proteins have two domains: the exostosin domain and a 300 amino acid C-terminal region that is highly conserved between members and probably corresponds to the catalytic transferase domain, which is missing in rib-1. Homozygous rib-2 animals show delayed larval growth and reduced life span, while their progeny display more than 90% early embryonic lethality (Morio et al., 2003). The C. elegans Dally-like gene ortholog, gpn-1, shows no phenotype in large-scale RNAi screens.

Two other molecules that bind and restrict the availability and range of N-Hh are Hip and Growth arrest specific 1 (GAS1; for review, see Cohen, 2003). Hip is absent in both Drosophila and C. elegans. GAS1 is a GPI-linked protein identified by its ability to arrest cell cycle progression when overexpressed. The C. elegans ortholog phg-1/phas-1 is expressed in the pharynx and may play a similar role in mitotic cell cycle regulation (Agostoni et al., 2002). Megalin, a member of the family of low-density lipoprotein receptors, is a third protein that binds to N-Hh (see Cohen, 2003). The C. elegans homolog lrp-1 is expressed in the hypodermis; mutations in lrp-1 produce molting defects (Yochem et al., 1999).

Three ptc genes, ptc-1,-2,-3 have been identified in C. elegans, although ptc-2 is absent in C. briggsae and is likely to be a pseudogene arising from a recent duplication of ptc-1. In addition, 24 genes have been identified that encode proteins sharing both sequence and topological similarities to PTC; these proteins have been named PTR (for Patched-related; Figure 4).

Drosophila, human and mouse are each predicted to encode a single PTR protein; thus the PTR family appears to have undergone an expansion in C. elegans (Kuwabara et al., 2000). Moreover, all 24 ptr genes are present in C. briggsae and share a one to one orthology with C. elegans.

The C. elegans PTC and PTR proteins share characteristics common to all known Ptc proteins. The PTC and PTR proteins are predicted to have 12-membrane spanning domains with cytoplasmic N- and C-termini. The membrane domains can be further sub-divided into two cassettes of 1+5, which are separated by a large intracellular loop. Each cassette contains a large extracellular loop predicted to facilitate Hh binding (Figure 5). Carried within the first set of TM domains is a sterol-sensing domain (SSD), which was first identified in SCAP, a protein involved in cholesterol homeostasis (Radhakrishnan et al., 2004).

ptc-1 is the first member of the ptc and ptr family to be analysed in detail (Kuwabara et al., 2000). Surprisingly, it was found that ptc-1 plays a crucial role in germline cytokinesis. Elimination of ptc-1 activity either by RNAi or mutation produces sterile adults that carry multinucleate oocytes and sperm. In other respects, development of a ptc-1 mutant is essentially wild type. In situ hybridization reveals that ptc-1 mRNA is maternally provided, and becomes enriched in P4, the germ line precursor. Immunocytochemistry performed using a polyclonal antibody capable of detecting PTC-1 protein in wild type adult hermaphrodites reveals that PTC-1 protein is enriched in the plasma membrane of mitotic germ cells undergoing proliferation, or in membranes undergoing rapid expansions, such as oocytes. PTC-1 protein is particularly enriched in vesicles at the apex of the membrane furrows separating individual germ nuclei.

An analysis of ptc-1 mutants further revealed that the absence of cytokinesis generated membrane furrows leads to a loss of cell autonomous development in the germline syncytium, as evidenced by the presence of clusters of nuclei in M-phase (Kuwabara et al., 2000). The involvement of ptc-1 in cytokinesis has led to the hypothesis that PTC-1 could play a role in the transport of lipids, proteins or lipoproteins to the cleavage furrow. Importantly, a proteomic survey of proteins involved in mammalian cytokinesis reveals that Ptc-1 is a component of the mid-body (Skop et al., 2004). Hence, the involvement of PTC-1 in cytokinesis is likely to highlight an ancestral function for Ptc proteins, which is apparently independent of Smoothened.

A mutation has also been obtained for ptc-3. Preliminary phenotypic characterisation indicates that ptc-3 is an essential gene; mutants arrest in late embryogenesis (P.E.K., unpublished; Zugasti et al., 2005). A survey of the other ptr genes by RNAi reveals that many have roles in molting, but also affect growth and morphogenesis (Zugasti et al., 2005). It has also been demonstrated that C. elegans daf-6 encodes a Patched-related protein required for lumen formation (Perens and Shaham, 2005).

Another clue to the function of PTC-1 in worms is provided by the presence of an SSD and its connections to sterols and protein transport. The SSD of SCAP, the first identified SSD protein, is capable of binding directly to cholesterol (Radhakrishnan et al., 2004; Figure 4). Other SSD protein families that are closely related to PTC are the Dispatched family, which includes CHE-14 and PTD-2, and the Niemann-Pick Type C family, which includes the NCR-1, -2 proteins in C. elegans (Kuwabara and Labouesse, 2002). che-14 was first identified in a screen for animals that were defective in dye-filling (Perkins et al., 1986). Subsequent work demonstrated that che-14 promotes apical sorting in epithelial cells. che-14 mutants accumulate vesicles that are trapped near the apical surface, indicative of problems in protein secretion (Michaux et al., 2000). The phenotype of che-14 mutants reveals that the problem is more likely to be associated with defects in exocytosis, rather than in endocytosis. The presence of an SSD in CHE-14 led the authors to speculate that CHE-14 might be involved in the secretion of proteins containing sterol or GPI adducts. Similarly, Disp promotes the exocytosis of Hh away from the cells from which it was synthesised (Burke et al., 1999). A protein transported by CHE-14 might be WRT-6, which is expressed in sensory socket cells (Aspöck et al., 1999).

Niemann-Pick disease type C1 (NPC1) is a neurodegenerative disease associated with defects in cholesterol storage/transport. In C. elegans, ncr-1; ncr-2 double mutants are hypersensitive to cholesterol deprivation and inappropriately form dauers under favourable food conditions, a dauer-constitutive (Daf-c) phenotype (Sym et al., 2000). Recent studies further show that ncr-1 is hypersensitive to progesterone, a known inhibitor of cholesterol trafficking (Li et al., 2004). Hence, in C. elegans there is a strong link between SSD proteins and the transport of sterols, sterol modified proteins or in the case of dauer formation, a sterol modified hormone.

C. elegans are cholesterol auxotrophs, requiring exogenous cholesterol for growth and survival (Chitwood, 1999; Hieb and Rothstein, 1968). Sterols are involved in at least two processes in the worm: the decision to enter diapause, which is mediated by a sterol-derived hormone, and molting, which is impaired by sterol depletion (Matyash et al., 2004). Animals depleted for cholesterol arrest in the second generation during early larval stages (Matyash et al., 2001; Merris et al., 2003). These L2 arrested larvae became dauer-like and fail to complete molting (Matyash et al., 2004).

Because worms require so relatively little cholesterol, it has been postulated that cholesterol is unlikely to be an essential architectural component of membranes and could play a more important role in signalling (Matyash et al., 2001). Mutations in lrp-1, qua-1 and RNAi inhibition of most ptr genes, and post-embryonic RNAi of ptc gene activity all lead to defects in molting (Yochem et al., 1999; Zugasti et al., 2005; Hao et al., 2006b). Thus, taken together, it is speculated that the PTC and PTR proteins could promote the transport of sterols or sterol-modified proteins, possibly including sterol-modified Hh-r proteins.

C. elegans lacks the typical Hh signal transduction cascade found in flies and vertebrates that is composed of Smo, Fu, Su(fu), and Cos2. Since homologs of Fu and Su(fu) are found in plants and bacteria (T.R.B., unpublished), it follows that this part of the pathway was lost in nematodes. Concomitantly, we have observed a large increase in the number of C. elegans Hh-r and ptr genes. Several lines of evidence point towards the Hh-r expansion being nematode specific: 1) Homologs of the Hh-r genes have so far not been identified outside of the nematodes. 2) Conversely, Hh is present in both arthropods and vertebrates and only missing in nematodes. 3) Phylogenetic analyses of the Hog domain and motif similarity shared by the Wrt and Grd domains indicate that the wrt, grd and grl gene families are derived from a single ancestral gene (Aspöck et al., 1999); qua-1 may also be derived from the same ancestral gene, as its Hog domain is also more similar to those of the Wrt and Grd proteins than to that of Hh. 4) C. briggsae has fewer Hh-r genes than C. elegans, and B. malayi even fewer, which underscores the capacity of these genes to expand. The expansion of the ptr genes is likewise nematode specific, since Drosophila and humans have only a single copy.

There is a running debate whether or not nematodes should be classified as ecdysozoa. If nematodes are indeed ecdysozoa, or group at least within the protostome branch of ecdysozoa and lophotrochozoa (see chapter on evolution), then the loss, expansion and diversification of various Hh pathway components, which have been described here, are most parsimoniously explained by postulating that a C. elegans Hh signalling pathway did exist, but was disrupted at some unknown time in early nematode evolution. It also implies that the Hh-r genes were derived from Hh. On the other hand, if nematodes are an outgroup to coelomata, then one of several alternatives is that a complete Hh signalling pathway was never acquired.

It is clear that the Hh and Ptc homologs in C. elegans have undergone considerable evolutionary divergence when compared to their Drosophila and vertebrate counterparts. Studies in C. elegans are providing insights into the roles and mechanisms underlying the control of the Hh-r, PTC and PTC-related proteins and have established that these proteins can function independently of Smo. The involvement of Hh-r and ptr genes in molting suggests that at least some of the Hh-r and ptr genes can participate in similar pathways. Thus, it will be of particular interest to determine whether the Hh-r proteins and the PTC and PTR proteins have maintained a ligand/receptor relationship that could potentially mediate cell-cell signalling.

TRB would like to thank the Swedish Foundation for Strategic Research (SSF) for support. PEK thanks the MRC (UK) for funding. The authors would like to thank David Fitch for constructive discussions related to nematode phylogeny.