This post is written by UW grad student Alexander Fodor
Figure 1. Photographs of the ascidian Molgula occidentalis taken in an isolation tank at the University of Washington’s Friday Harbor Labs. Photo by Alexander Fodor.
Searching through the lower intertidal and subtidal rocky beaches you notice a small strange creature attached to the underside of a rock in a small pool of water. It has a round body and two tubes pointing straight at you. You reach down and touch it, it feels leathery and almost plant like; you squeeze it and it shoots a stream of water right in your face. You have found a sea squirt (also called ascidians or tunicates) (Figure 1). These creatures appear very different from us and you may not even recognize it as an animal, but they are chordates, members of our own phylum that you can easily see in their tadpole larvae (Figure 2L). When it comes time to reproduce they are free spawners, spraying sperm and eggs into the water where the eggs are fertilized by another individual and develop into tadpoles. Ascidian development (Figure 2) is determinant and invariant across all solitary sea squirts where the cellular fate is set up with each division and each cell is always destined to become the same structures; so if you trace the cellular fate of a cell in one solitary ascidian, if you look in a different species, that same cell will become the exact same tissue. As they develop they form a tadpole that looks much like a tiny larval frog, complete with a notochord in the center of the tail and gravity sensing and light sensing structures in the head. They spend anywhere from a few hours to several days as a tailed larva with a notochord, searching for the appropriate substrate, using their light sensing and gravity sensing structures to swim underneath an object (like a dock or rock) and attach their head to the bottom. They then absorb their tail and completely change their anatomy, absorbing all of their larval structures, growing two feeding siphons, and surrounding themselves with a tough tunic, containing cellulose. The cellulose operon, consisting of three genes, was retro-virally spliced into the tunicate ancestor’s genome (Dehal et al. 2002).
Figure 2. Development of Boltenia villosa up to tadpole larva (L) then metamorphosis (M) though adulthood (N and O). The orange color in the developing embryos (A-N) is caused by myoplasm of cells destined to become muscle cells. Larva photos taken during an Undergraduate Apprenticeship at the University of Washington’s Friday Harbor Labs spring of 2001, adult photo by Alexander Fodor at Friday Harbor Labs.
Molgulids are a special family of sea squirts where the larval tail and notochord has been lost several times in species in the family completely independently of one another. The tailless species all have a similar phenotype where they only have fewer than 40 notochord cells, and they do not converge and extend into a notochord, but rather sit on the side of the embryo in what has been described as a “notoball”. In addition they have lost their gravity and light sensing organs (Figure 3C). These animals typically live in northern waters where there are very strong tidal currents so it is conceivable that animals could still disperse enough even without a swimming tail (Huber et al. 2000). We are uncovering the molecular mechanisms underlying this tail loss by studying two species, Molgula oculata and Molgula occulta, in the Swalla lab at the University of Washington’s Friday Harbor Labs. These two species are sister species, but they have very different looking larvae: M. oculata has a fully functioning tail with a 40 cell notochord inside and gravity and light sensors in the head, but M. occulta is lacking all of the larval structures. The species are still closely enough related that they can be hybridized them in the lab. If the egg of the tailed M. oculata is used, then the resulting larva always has a fully functioning tail and notochord; but if the egg of the tailless M. occulta is used, then some of the time the resulting hybrid has a half tail which is only composed of 20 notochord cells, but it still converges and extends out (Swalla et al. 1990), in addition, the paternal expression of the tyrosinase gene saves the gravity and light sensors (Raccicopi et al. 2017) (Figure 3).
In collaboration with the C. Titus Brown Lab (formally MSU, now UC Davis), our lab has recently sequenced the genomes and transcriptomes of M. oculata, M. occulta, the hybrid made with M. occulta eggs, and M. occidentalis (the outgroup for the Molgula family) (Stolfi et al. 2014). We are currently searching through the genomes and transcriptomes, looking for the molecular mechanisms responsible for this change in morphology. We have identified a number of genes that could be involved in this tail loss, and examining their sequences, and are testing their expression profiles. In the summer of 2018, Dr. Billie Swalla and I are going to go to Roscoff, France where M. oculata and M. occulta live to dissect gravid adults and obtain embryos for transgenic experiments. We will use gene-editing techniques to express tailed M. oculata genes in the tailless M. occulta and the hybrid made with the occulta eggs to see if we can recapitulate the tailed expression. It is very intriguing to think about how evolution can make a small number of changes to a gene network, which can in turn change the expression of a whole structure and the life history of the organism. It is nice to use sea squirts for such experimentation as they are very closely related to the vertebrates so can teach us much about how complex gene networks can be altered, and in turn change complex evolutionary traits. We are grateful for the BEACON funding that we have received for this project and are looking forward to making progress on the project in the coming years.
Figure 3. Pictures and cartoons of the larvae of A. M. oculata C. M. occulta and B. The hybrid made with M. occulta eggs and M. oculata sperm. Adapted from Swalla and Jeffery 1996
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