Old tools reused for a new design: the making of the Oikopleura House

Oikopleura dioica is a tunicate of the Larvacea class found in all oceans. It owes its name because, like all larvaceans, it builds a “house.”  This complex extracellular structure allows it to filter its food composed of bacteria and unicellular algae. The house is replaced around 20 times during the animal’s short life, and discarded houses play an important role in the descent of organic matter to the ocean floors.

Eric Thompson, a former Group Leader at the Michael Sars Centre, characterized in detail the components of the house, made of cellulose and several dozen proteins. These are produced and secreted by the epithelium covering the animal’s trunk, a fascinating tissue that contains well-defined cellular territories that produce a specific cocktail of proteins. Some have small cells, while others, like the Fol territory, contain giant cells that produce the filter for concentrating food.

The house and the epithelium that produces it have no equivalent outside of larvaceans, either morphologically or at the molecular level. "Its appearance during evolution is therefore largely mysterious. How all this new machinery is put in place during development is a very open question that has interested the Chourrout group for several years," said Group Leader Daniel Chourrout.

Yana Mikhaleva previously showed that during embryogenesis, the territorialization of the epithelium was accompanied by the transient and spatially distinct expression of genes coding for well-known transcription factors, which in other animals are involved in forming the main tissues and organs. She demonstrated that the knockdown of two of these genes disrupted the formation of the Rosette, a central epithelium zone.

In a new Developmental Biology article, David Lagman, a former postdoc at the Centre, identified Pax37, another transcription factor, as having a major role in Fol territory development. “The results relied heavily on the use of CRISPR-Cas9, which was essential in making the mutant lines and for us to observe any phenotypes throughout the development of the epithelium,” David explained. Through CRISPR, true mutant lines were produced, where mutants lacked the giant cells that produce the food concentration filter, leading to the premature death of the animals.

The results relied heavily on the use of CRISPR-Cas9, which was essential in making the mutant lines and for us to observe any phenotypes throughout the development of the epithelium.
- David Lagman

These findings led to a close collaboration with Anthony Leon, Head Engineer of the Chourrout group. Anthony recently established the single-cell RNA sequencing method in Oikopleura, providing a detailed map of gene expression during development. Epithelial cells expressing the Pax37 gene were found to eventually express not only genes coding for certain house proteins but also genes typically active in neurons. Michael Sars Centre Group leader Marios Chatzigeorgiou contributed to identifying this “neuronal signature”. This was a surprising discovery for cells previously thought to be solely involved in building the house. Interestingly, this observation parallels findings in Ciona intestinalis, another tunicate species from another branch, where the trunk epithelium contains sensory neurons called ATENs that also express the Pax37 gene.

“This work adds additional information on the mechanisms that are involved in the development of novel features as well as the specialization of gene paralogs during evolution,” David concluded. Overall, the study shed light on the origin of the house, which may involve the addition of new functions to cells from a distant common ancestor. The persistence of ancestral neuronal characteristics is intriguing, suggesting that the epithelium of Oikopleura may be potentially reactive to external stimuli, allowing for various types of regulation, including the production of the house itself.