Summer Studentship Reports 2017 – Emily Baker
“Hedgehog-like signalling in C. elegans?”
By Emily Baker
Hedgehog is one of a highly conserved handful of signal transduction pathways in metazoans with roles in both development and disease, ranging from the wings of fruit flies to the patterning of the vertebrate notochord to cancer. Aside from its clinical significance, Hedgehog has become of interest to evolutionary biologists too. Hedgehog has a common origin in the bilaterians and has diversified little since. Every bilaterian lineage has at least one orthologue, all except some enigmatic members of Nematoda. The model nematode species Caenorhabditis elegans is one such mysterious anomaly that lacks a bona fide Hedgehog pathway. So what does C. elegans have instead, if anything? What are the roles of these Hedgehog-like genes in the worm? And can an animal achieve developmental complexity in the absence of Hedgehog?
The answer to all of those questions lies with a gene family which is rife with duplication and diversification. Cue some key players: the Warthogs (Wrts). Discovered in the mid 90s by Thomas Bürglin, the Wrts as they were so wittily named were found by mining the C. elegans genome for the ‘Hog’ domain, that is the C-terminus of the Hedgehog protein in Drosophila. Through extensive bioinformatic searches, Bürglin found around 60 of what he called Hedgehog-related (Hh-r) genes. Of the 60 Hh-r genes, ten were identified as belonging to the Warthog family according to their novel class of N-terminus, the Wart domain. The Wart domain is characterised by a consensus sequence of eight cysteine residues and whilst all ten members of the family contain it, only five contain a C-terminal Hog domain as well, these are: WRT-1, WRT-4, WRT-6, WRT-7 and WRT-8. This leaves WRT-2, WRT-3, WRT-5, WRT-9 and WRT-10 which are all ‘Hog-less’. Sadly following their initial discovery, interest in the gene family dwindled because they were considered too divergent from the mammalian story to be of any relevance to humans. And anyway, why does a simple 1mm long worm need so many developmental patterning genes, considering most vertebrates suffice with just three Hedgehog paralogues? Frankly, the Wrts must all be pseudogenised rubbish, no?
Until my project, the conventional wisdom was that the majority of these genes didn’t possess any major developmental function. By building a phylogeny using the Wart domains, we could deduce where the gene duplication events occurred and which genes may be redundant with one another. Consequently, I set about scouring the genomes of a phylogenetically widespread sample of nematodes. I decided to use Trichinella spiralis as an outgroup because this was the species in which I could find only a single Warthog. Based on the results of this, we formed hypotheses that could be tested in the lab. We focussed on the wrt-2 clade, which also contains wrt-4 and the C. elegans -specific wrt-7 and wrt-8 (Figure 1).
A previous student noted that some wrt-2(ok2810) deletion mutants had a strange directional asymmetry defect whereby the natural left-right (LR) asymmetry of the middle body was disrupted (Figure 2). Therefore, I quantified these defects in various deletion mutants in the wrt-2 clade and I made double and triple mutants to evaluate the genetic redundancy between the paralogues (Figure 3). In short, wrt-4(tm1911) mutants had the greatest number of asymmetric defects with wrt-2(ok2810) mutants having only slightly fewer. The wrt-4(tm1911);wrt-2(ok2810) double mutant reveals that these two genes have partially overlapping functions. Meanwhile, wrt-7(ok3270) mutants did not display any defects. It has been previously suggested that wrt-7 is a pseudogene after work showing it did not have an expression pattern, a conclusion which this phenotypic dataset would agree with.
The role of wrt-8 in C. elegans development is less clearcut from this study. wrt-8(tm1585) mutants had some asymmetric defects but the defects present in the wrt-8(tm1585);wrt-4(tm1911) double mutant were not statistically significant from those present in the wrt-4(tm1911) single mutant, which suggests wrt-8 has not (yet) subfunctionalised from its progenitor (wrt-4), with respect to the development of LR asymmetry. However, it is entirely possible that wrt-8 has neofunctionalised and is involved in roles that have not been studied here such as cell fate determination, moulting or the regulation of body size or even innate immunity. If this were the case, only further research could tell us the answer.
Working at the interface of evolutionary and developmental biology is incredibly illuminating. Using phylogenetics as a platform for laboratory investigation, we have been able to delve into the roles of this mysterious gene family in C. elegans and study their possible duplication-degeneration-complementation (DDC) dynamics.
So where does this leave the possibility of Hedgehog signalling in C. elegans?
Critics of the cell biology will say that C. elegans does not have a Hedgehog signalling pathway, and of course they would be right. It is true that C. elegans does not have any traditional Hedgehog ligands and also lacks two components of the pathway, Smoothened (the G-protein coupled receptor) and Skinny hedgehog (the acyltransferase). (Although it is worth noting that the worm has sequence homologues for all other components of the pathway and it has not yet been investigated if it has structural homologues for those two which are absent). But this project has profound implications for how we think about the evolution of signals transduction pathways more generally. We are telling a story of evolutionary convergence. The role of the vertebrate Hedgehog ligand, Sonic Hedgehog (Shh), is well-elucidated in the development of LR asymmetry in Xenopus and other model species. It has long been suggested that it is the infrastructure of development that is highly conserved between taxa, but rather than a conserved developmental toolkit, here we have the recapitulated deployment of similar, yet independently-evolved toolkits. Indeed, with respect to Hedgehog at least, the worm boasts a far more complex toolkit than its tripartite vertebrate counterpart. In this way, C. elegans challenges the widely accepted notion that vertebrates always tell the most developmentally complex story with respect to gene duplication. Moreover, we have showed that a complex developmental phenomenon like LR asymmetry is capable of evolving multiple times but is broadly orchestrated by ‘Hedgehog’ signalling, just in different taxa… Not bad insights from a microscopic nematode worm, right?
Presenting my work at the Genes and Development Conference in Edinburgh was a brilliant experience, I certainly didn’t expect to win. I had never spoken in front of so many people before, but thankfully I get incredibly excited about Hedgehogs and worms so I managed to keep my audience engaged! Fundamentally, I think that is what is most important about giving a presentation: enthusiasm. If the speaker appears disinterested by their own topic, you can guarantee their audience is even more bored by the talk. That’s not to say the presenter should be speaking at a million miles per hour, clarity is equally as important. This applies to the slides too; nobody wants to see a chaotic PowerPoint slide packed full of text. If you can, use pictures and if you have to use text, be brief. Also it really won’t hurt to throw in a joke or two because even academics like to laugh sometimes! Have fun with it!
I’m so thankful for my time in the Woollard lab. Firstly, it would not have been possible without funding from the Genetics Society UK. Aside from buying worms over the internet (yes, that is a thing!), all the expenditure that is associated with performing experiments meant the 8 weeks was only possible with their financial help. Secondly, Alison and her lab in Oxford are a fantastic group of people. Their constant energy and enthusiasm for their, (read: everyone’s) favourite worm was infectious – it really was an amazing research environment to be in. I’ve come away from my time there wanting to be a scientist now more than ever and for that, I don’t know who I have to thank more for being so inspiring, Alison or the undulating enigma that is C. elegans.