Pettitt, Jonathan et al. (2013) International Worm Meeting "The evolution of nematode operons."

Goth, Henrike, Phillippe, Lucas, Sarkar, Debjani, Pettitt, Jonathan, Connolly, Bernadette, Muller, Berndt

[International Worm Meeting, 2013]

Operons are a means of organising multiple independent coding regions such that they are transcribed into a single, polycistronic RNA. The presence of operons in an organism's genome is strongly correlated with the ability to carry out spliced leader (SL) trans-splicing, consistent with the hypothesis that the processing of polycistronic RNAs in eukaryotes is dependent upon SL trans-splicing. Operons have been found in C. elegans and other nematodes that fall within the Chromodoria, one of the three major nematode clades. We have previously shown that the nematode, Trichinella spiralis, which lies in one of the other two main clades, the Dorylaimia, engages in SL trans-splicing, suggesting that it is a nematode-wide trait. An important question is whether the same is true of operons. We reasoned that if there is an intimate relationship between SL trans-splicing and polycistronic RNA processing, then we should also expect to be able to identify genes organised into operons in these nematodes. We have previously identified a set of T. spiralis genes whose mRNAs undergo SL trans-splicing, and using this dataset, we have demonstrated the existence of operons in this nematode. We have confirmed that they produce polycistronic RNAs and that they are present in the closely related Trichuris muris. At least two of the operons are conserved between nematodes in the Dorylaimia and the Chromodoria clades, suggesting that these represent operons likely present in the ancestor of the nematode phylum. We find that mRNAs derived from downstream genes in operons are SL trans-spliced, just as is found for other nematode operons, but there is no equivalent to the specialised SL2 found in C. elegans. We are currently expanding upon our limited set of data to build a more comprehensive picture of SL trans-splicing and operon organisation in the Dorylaimia, and thereby gain a better understanding of the influence these processes have had on the evolutionary dynamics of the nematode genome.

Pettitt, Jonathan et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "Nematode beta-catenin subfunctionalisation is correlated with gene loss and taxon- specific gene origination"

Korswagen, Hendrik C, Sarkar, Debjani, Pettitt, Jonathan

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Beta-catenin is an essential component of both the canonical Wnt signalling pathway and the cadherin-dependent adhesion complex. In most animals a single beta-catenin participates in both activities, but in C. elegans the conserved signalling and adhesion functions are distributed between two beta-catenin homologues, BAR-1 and HMP-2. Two further C. elegans beta-catenins, WRM-1 and SYS-1, are components of the highly-derived Wnt/beta-catenin asymmetry pathway, a divergent Wnt signalling mechanism that appears to be nematode specific. We are interested in understanding the evolutionary context and the molecular events that accompanied the subfunctionalisation of nematode beta-catenins. To understand when beta-catenin subfunctionalisation occurred, we characterised the beta-catenin and Wnt signalling components encoded by all available nematode genomes. The most striking finding of this analysis was that the beta-catenins of enoplean nematodes more closely resemble those from arthropods and vertebrates than they do the beta-catenins found in C. elegans and other chromadorean nematodes. Like their arthropod and vertebrate homologues, the enoplean beta-catenins are likely capable of functioning in both Wnt signalling and cadherin-mediated cell adhesion. Our data thus show that beta-catenin subfunctionalisation, which also correlates with loss of ancestral Wnt signalling components, occurred after the radiation of the major nematode clades.The remodelling of Wnt signalling pathways that accompanied beta-catenin subfunctionalisation likely involved the recruitment of new Wnt signalling components. To investigate this we searched for novel proteins that show differential interactions with C. elegans beta-catenins. From this screen we identified three related proteins, all of which interact specifically with BAR-1 and WRM-1, but not HMP-2. Homologues of the genes encoding these proteins can be identified in other chromadorean nematodes, but they appear to be absent from the available enoplean genomes, suggesting that they evolved in parallel with the diversification of beta-catenin function in the former nematodes. C. elegans mutants that lack these proteins display cell fate and spindle orientation defects, indicating that they function in the Wnt/beta-catenin asymmetry pathway. Thus the rewiring of the ancestral Wnt signalling machinery that gave rise to the Wnt/beta-catenin asymmetry pathway involved the incorporation of novel components that likely facilitated the diversification of beta-catenin function.

Jonathan Pettitt et al. (2005) International Worm Meeting "-catenin: actin' at cadherin junctions"

Jonathan Pettitt, Laura Manning

[International Worm Meeting, 2005]

It has been known for many years that -catenin is an essential component of the cadherin-catenin complex; being required to link the cadherin adhesion interface to the actin cytoskeleton. Biochemical studies indicate that it is able to bind directly to F-actin and to a variety of proteins that have actin-binding, bundling and remodelling activities, however the mechanistic basis of alpha-catenin function is still unclear. In order to better understand the role of alpha-catenin we have been exploiting a viable, hypomorphic mutation (fe4) that affects the C. elegans alpha-catenin HMP-1. We have previously shown that loss of function mutations affecting the Ena/VASP homologue UNC-34 are synthetic lethal in combination with hmp-1(fe4). Using a GFP-tagged version of UNC-34 we have shown that it localises to epithelial junctions. Surprisingly, junctional localisation of UNC-34-GFP is independent of the cadherin-catenin complex, in contrast to the situation in cultured keratinocytes, indicating that other molecules recruit UNC-34 to the junctions. Studies in other organisms have implicated members of the zyxin family in the recruitment of Ena/VASP proteins, and we thus examined the role of the C. elegans zyxin ZYX-1. Both zyx-1 loss-of-function mutations are synthetic lethal in a hmp-1(fe4) background, but have no observable phenotype in a wild-type hmp-1 background. However, the zyx-1; hmp-1(fe4) double mutants display a less severe phenotype compared to unc-34 hmp-1(fe4) double mutants. We are currently examining the effect of zyx-1 mutations on UNC-34-GFP localisation. In addition to screening for enhancer mutations, we have identified mutations that behave as dominant suppressors of the hmp-1(fe4) phenotype. These mutations completely suppress the embryonic lethality and morphological defects associated with hmp-1(fe4), and all are intragenic suppressors. Three are substitution mutations that map to a region predicted by homology modelling to lie near the serine residue affected by the fe4 mutation. Two of the mutations were recovered together: one being a substitution mutation affecting a residue in the so-called M-domain, and the other a nonsense mutation that truncates the C-terminus. This latter mutation removes a conserved region required for the in vitro interaction between alpha-catenin and F-actin, indicating that such experiments do not necessarily reflect in vivo functions. These mutations thus offer a unique opportunity to begin a dissection of alpha-catenin function in its normal physiological context.

Jonathan Pettitt et al. (2001) International C. elegans Meeting "A family of beta-catenin interacting proteins involved in hypodermal morphogenesis"

Hendrik C Korswagen, Jonathan Pettitt

[International C. elegans Meeting, 2001]

In both vertebrates and Drosophila a single protein, beta-catenin/Armadillo, is a key component of both Wnt signalling and cadherin-mediated cell adhesion. In C. elegans the signalling and adhesion functions of beta-catenin appear to have been distributed between separate proteins. HMP-2 appears to function exclusively in cadherin adhesion complexes, while BAR-1 and WRM-1 act in Wnt signalling pathways. We have identified three genes whose products interact with both BAR-1 and HMP-2 in yeast two hybrid screens. These genes have been designated bin-1 , -2 and -3 ( b eta-catenin in teracting). The three genes encode related gene products that do not show any significant similarities to other non-worm proteins in the publicly available sequence databases. In order to determine the role of these genes we have examined the effect of depleting their function using RNA interference. bin-1(RNAi) resulted in variable defects in hypodermal cell positioning, particularly in the seam and ventral hypodermis, often resulting in ventral enclosure, or elongation defects. Neither bin-2(RNAi) nor bin-3(RNAi) resulted in an obvious mutant phenotype, and neither did the double RNAi of both bin-2 and bin-3 . However, double RNAi of both bin-1 and bin-2 resulted in an enhancement in the penetrance and severity of the defects observed with bin-1(RNAi) alone. RNAi of bin-1 and bin-3 resulted in a synthetic phenotype not seen in bin-1(RNAi) animals. These embryos all arrested with very pronounced defects in the pattern of hypodermal cell junctions, and never initiated ventral enclosure. These results demonstrate that bin-1 shares functions in regulating hypodermal patterning and morphogenesis with bin-2 and bin-3 . We do not know how these gene products act to regulate hypodermal development. They do not appear to regulate the activities of HMP-2 or BAR-1, since the mutant phenotypes of these genes do not cause the same phenotypes. We are currently attempting to determine whether the products of the bin genes can interact with other Armadillo repeat containing proteins, such as WRM-1, APR-1 and the C. elegans homologue of p120 catenin, as well as examining where they are expressed.

Jonathan Pettitt et al. (2003) International Worm Meeting "C. elegans Disabled is a modulator of cadherin function during epithelial morphogenesis"

Alex Holmes, Jonathan Pettitt

[International Worm Meeting, 2003]

The cadherin-catenin complex is a conserved adhesion system required for the maintenance of epithelial integrity. However, adhesion between cells within an epithelium must be dyanmic to allow for the various cell migrations and movements that accompany epithelial morphogenesis. A number of mechanisms have been implicated in the modulation of cadherin-catenin complex function, but their details and physiological relevance remain uncertain. We have previously identified a weak hypomorphic allele (fe4) of the hmp-1/alpha-catenin gene that we have used as a sensitised genetic background to identify molecules involved in the regulation of the cadherin-catenin complex. Using this approach we have shown that the C. elegans Disabled homologue, M110.5, is involved in the cadherin-mediated morphogenesis of the embryonic epidermis. Members of the Disabled family of PTB-domain-containing proteins are found in insects and vetebrates, and are involved in a variety of signalling pathways, as well as endocytic trafficking of lipoprotein receptors. They have not previously been implicated in the regulation of cadherin-catenin function, although a murine Disabled homologue is associated with the vertebrate-specific cadherin-related neuronal receptors. In a wild-type background, depletion of M110.5 function by RNAi leads to a variety of developmental defects, but does not obviously affect embryonic epidermal morphogenesis. However, M110.5(RNAi) enhances the penetrance of the Hmp phenotype displayed by hmp-1(fe4) homozygotes from approximately 10% to 100%, indicating that C. elegans Disabled acts to positively modulate cadherin-catenin complex function. GFP-tagged versions of the two M110.5 isoforms expressed under the control of their endogenous promoter localise to the membranes of most, if not all cells, with strong expression in the epidermis, pharynx and intestine. We are currently attempting to identify the molecular basis of the interaction between M110.5 and the cadherin-catenin complex.

Jonathan Pettitt et al. (2002) European Worm Meeting "Multiple regulators of actin dynamics modulate cadherin function during hypodermal morphogenesis"

Jonathan Pettitt, Ian D Broadbent

[European Worm Meeting, 2002]

Recent work in a variety of model systems has revealed that changes in actin cytoskeleton organisation are pivotal to the stabilisation and maturation of cadherin-based adherens junctions. However the mechanisms that co-ordinate changes in cell adhesion with cytoskeletal organisation are only just beginning to emerge, and little information has been obtained in the context of a developing animal. We have recently identified a novel maternal-effect Hmp mutation, fe4, which is a weak hypomorphic allele of hmp-1 (the C. elegans alpha-catenin gene). Homozygous mutant progeny of fe4 heterozygotes display only minor defects in the shape of the tail, whereas the progeny of these homozygotes display more severe morphological phenotypes. The molecular nature of the fe4 mutation, coupled with its relatively weak phenotype, suggests that HMP-1 function is compromised, but not abolished in fe4 homozygotes, and is supported by the fact HMP-1 expression and localisation is not obviously affected in these animals. The variable nature of the phenotype suggests that the hypodermal cells of fe4 mutants are sensitive to stochastic fluctuations in cadherin-catenin function. We have used RNAi, and previously identified mutations to identify genes whose loss of function enhances the fe4 mutant phenotype, assuming that such genes will encode molecules that have roles in the formation and function of cadherin adhesion junctions. In cultured keratinocytes members of the Ena/VASP family, which regulate actin remodelling, localise to nascent cadherin junctions, where they regulate changes in the actin cytoskeleton that occur during adherens junction formation. The unc-34 gene encodes the sole C. elegans member of the Ena/VASP family, and we therefore sought to examine its role in cadherin function. None of the available alleles of unc-34 display any obvious defects in epithelial morphogenesis. However unc-34 fe4 double mutant combinations result in 100% maternal-effect embryonic lethality, with all embryos displaying either ventral enclosure, or elongation defects. This indicates that despite not being essential for hypodermal morphogenesis in animals with a wild- type cadherin-catenin complex, UNC-34 nevertheless contributes to the modulation of cadherin adhesion. The other group of candidate genes that we have examined are the Rho family of small GTPases, which have previously been shown to regulate cadherin adhesion in cell culture, as well as a variety of morphogenetic events in vivo. Loss-of-function mutations in the Rac genes mig-2 and ced-10, as well their putative guanine-nucleotide exchange factor, unc-73, enhance the penetrance of ventral enclosure defects in combination with fe4, indicating that Rac signalling is involved in the regulation of the formation and/or function of cadherin-catenin complexes in C. elegans. Taken together these results suggest that UNC-34 and MIG-2/CED-10 act in parallel to regulate the cadherin-catenin complex activity required for ventral enclosure. In support of this we find that ced-10/mig-2(RNAi) in unc-34 mutants results in ventral enclosure defects. We are currently exploring the molecular basis of these observations, to attempt to identify the targets of MIG- 2/CED-10.

Jonathan Pettitt et al. (2007) International Worm Meeting "Investigating the subfunctionalisation of b-catenin during nematode evolution."

Judith Reichmann, Jonathan Pettitt, Aileen Flett

[International Worm Meeting, 2007]

In most animals <font face=symbol>b</font>-catenin is a critical component of both the cadherin-catenin complex and the Wnt signalling pathway. This situation means that potentially the two functions of <font face=symbol>b</font>-catenin: cell adhesion and regulation of gene expression, can be integrated by modulating the interaction of <font face=symbol>b</font>-catenin with its binding partners. In contrast, in C. elegans, <font face=symbol>b</font>-catenins''s roles are subfunctionalised such that the cell adhesion and transcriptional activation functions are carried out by separate proteins: HMP-2 and BAR-1, respectively. We are interested in understanding how this apparently unique situation might have arisen by examining the distribution of <font face=symbol>b</font>-catenin homologues and their interaction partners in other nematodes. The C. elegans <font face=symbol>b</font>-catenins are conserved in closely related Caenorhabditis species, so to obtain a broader picture of <font face=symbol>b</font>-catenin conservation we searched the genome sequences of Brugia malayi and Trichinella spiralis. We were able to identify a clear homologue of HMP-2 in Brugia, and two closely related genes encoding apparent BAR-1-like molecules. In Trichinella we found two <font face=symbol>b</font>-catenin homologues, which are likely the product of a relatively recent gene duplication. Strikingly, based upon sequence similarity, both are predicted to encode dual function <font face=symbol>b</font>-catenins, as found in insects and vertebrates. Given the phylogenetic position of Trichinella, this provides the first evidence that the <font face=symbol>b</font>-catenin subfunctionalisation, exemplified in C. elegans, arose after the divergence of the major nematode clades. Recent work from a number of labs has shown that the transcriptional activity of <font face=symbol>b</font>-catenin is dependent upon the Legless/Bcl9 adaptor and its associated nuclear protein, Pygopus. A model has been proposed whereby the phosphorylation of Tyr-142 in <font face=symbol>b</font>-catenin leads to its dissociation from the cadherin-catenin complex, and sequestration into the nucleus by Legless/Bcl9. Pygopus and Legless/Bcl9 are conserved in mammals and insects, but are absent from C. elegans, suggesting that they are not essential for the transcriptional activity of BAR-1. In contrast, in Trichinella we can identify clear homologues of both Pygopus and Legless/Bcl9. Surveying all available nematode <font face=symbol>b</font>-catenins enables us to propose a mechanism that explains why subfunctionalisation of <font face=symbol>b</font>-catenin might lead to loss of Legless/Bcl9 and Pygopus. We are currently testing this hypothesis through functional studies in C. elegans, together with protein interaction assays.

Annabelle Couthier et al. (2005) International Worm Meeting "Making the HuMPback bridge: a genome-wide screen for molecules that contribute to cadherin-catenin complex function"

Pettitt Jonathan, Annabelle Couthier, Aileen Flett, Jeff Hardin, Theresa Grana

[International Worm Meeting, 2005]

The morphogenesis of the epidermis is responsible for the change in shape of the C. elegans embryo from an ovoid into a worm. Epidermal morphogenesis involves two cadherin-dependent events: ventral enclosure and elongation and alpha-catenin is essential for both processes. A non-lethal hypomorphic hmp-1 (alpha-catenin) mutant (fe4) has previously been isolated (Pettitt et al., 2003), which shows a spectrum of morphological defects. Work in our lab and the Hardin lab exploiting this mutation has shown the existence of a class of genes which positively modulate cadherin-catenin complex activity, but are not absolutely required for its function. Together, these primary results have provided the basis for a genome-wide RNAi screen for C. elegans genes whose loss of function enhances the fe4 mutant phenotype. The outcome of such a screen should provide insight into cadherin-catenin function and uncover novel pathways involved in regulating epithelial morphogenesis. Pettitt, J., Cox, E. A., Broadbent, I. D., Flett, A. and Hardin, J. (2003). The C. elegans p120 catenin homologue, JAC-1, modulates cadherin-catenin function during epidermal morphogenesis. J. Cell Biol. 162, 15-22.

Bai, Keliya et al. (2013) International Worm Meeting "The molecular genetics of epithelial cell morphogenesis- functional dissection of C. elegans homologue zyxin."

Bai, Keliya, Pettitt, Jonathan

[International Worm Meeting, 2013]

The zyxin family has been implicated in a wide range of cell behaviours, including cell adhesion and motility, and more recently in the regulation of Hippo signalling. All family members consist of three well-conserved LIM domains and an N-terminal, "pre-LIM" domain. Although highly conserved across animal phyla, the precise mechanisms by which these proteins function are not well understood. We are investigating the function of the sole C. elegans family member, ZYX-1. Genetic analysis indicates that ZYX-1 contributes to, but is not required for cadherin-mediated morphogenesis in C. elegans. Our data shows that ZYX-1 localises to adhesion junctions and becomes more strongly recruited as those junctions undergo actomysin-dependent tension. We are investigating the roles of these domains in the function and localisation of ZYX-1, as well as determining the mechanism by which ZYX-1 is recruited to adhesion junctions. In addition to its role in epithelia morphogenesis, we have also shown that it collaborates with other regulators of actin dynamics to coordinate the changes in cell shape and adhesion that accompany gastrulation.

Sarkar, Debjani et al. (2013) International Worm Meeting "Evolution of Nematode Spliced Leader trans-splicing."

Muller, Berndt, Connolly, Bernadette, Sarkar, Debjani, Pettitt, Jonathan

[International Worm Meeting, 2013]

Spliced-leader (SL) trans-splicing is a pre-mRNA maturation event that has a pivotal role in processing polycistronic RNA transcripts from operons into mature monocistronic mRNAs. It is known to occur in multiple, widely distributed eukaryotic groups, including nematodes. Most of our knowledge on SL trans-splicing in nematodes has come from investigations in Caenorhabditis elegans and other nematodes from the Chromadorean clades. The identification of SL trans-splicing in nematodes that lie outside of the Chromadorea has led us to conclude that SL trans-splicing is likely to be a phylum-wide process and thus a trait found in the last common ancestor of the nematodes. In this project we investigate the nature of SL trans-splicing in nematodes outside of the Chromadorean clades. Previous studies have shown that the Dorylaimid Trichinella spiralis uses a range of highly polymorphic SL sequences that have only limited similarity to C. elegans SL1 and SL2 (Pettitt et al, 2008). In contrast, initial searches for SLs in Prionchulus punctatus have shown that it possesses clear SL2-like sequences (Harrison et al, 2010). In this study we carried out searches for putative SL sequences in Trichuris muris to address differences between SL sequences in T. spiralis and P. punctatus. Searches indicate that SLs in T. muris are similar to the SLs found in P. punctatus and C. elegans, which is unexpected given that T. muris shares a "recent" common ancestor with T. spiralis. This in turn highlights that the lineage leading to T. spiralis is derived in relation to SL trans-splicing. Our results provide us with a valuable insight into the likely nature of SL trans-splicing in the ancestral nematode and how it has evolved within the nematode phylum.