An ancient protein mediates tube formation during early chordate development

In a new article published in PLOS Biology, authors Zonglai Liang, PhD graduate from the Michael Sars Centre, Daniel Dondorp, former staff engineer, and group leader Marios Chatzigeorgiou have delved into the molecular regulation of morphogenesis. Using as a model the developing notochord of the tunicate Ciona intestinalis, a tubular organ present in chordate embryos, they discovered that a single protein from the evolutionarily ancient Anoctamin/TMEM16 family orchestrates the complex process of tubulogenesis.

Biological tubes are fundamental structures that are extremely abundant in the animal kingdom. From lungs to kidneys and blood vessels, tubular structures are crucial for gas exchanges and nutrient transport, provide structural support and act as signalling centres. The building of these tubes during development is an elaborate process involving multiple, carefully choreographed cell behaviours. Using genome editing, pharmacological approaches and quantitative imaging, the team discovered that the protein Anoctamin 10 (Ano10) is required for several of these coordinated cell movements, including convergent extension, lumen expansion and connection during notochord development. “Researchers always try to map more molecules which determine the notochord development”, Zonglai explained. “Now it is a surprise and exciting for us to find Anoctamin family member involved in this process”

“Researchers always try to map more molecules which determine the notochord development. Now it is a surprise and exciting for us to find Anoctamin family member involved in this process.” – Zonglai Liang

Having established a role for Ano10 in notochord morphogenesis, the group switched their focus to the mechanism by which the protein acts at the cellular level. They found that Ano10 works in concert with the plasma membrane localized Na⁺/Ca²⁺ exchanger (NCX) and the endoplasmic reticulum residing SERCA, RyR and IP₃R proteins to establish specific patterns of calcium signaling activity that coordinate the different steps of notochord development. In addition, they discovered that Ano10 is able to influence notochord morphogenesis by controlling the subcellular localization of the evolutionarily conserved Ca²⁺ sensor proteins Calmodulin (CaM) and Ca²⁺/Calmodulin-dependent protein kinase (CaMK).

Using drugs to block the activity of these molecules, the researchers discovered that the calcium activity in the notochord cells was ‘scrambled’, and that embryonic notochord cells were unable to carry out successfully the processes of convergent extension and tubulogenesis. “The key experiment is the Ciona embryo development experiment, the morphology change after we knocked out Ano10 is the fundamental evidence for this project which I spent a lot of time to confirm this phenotype”, Zonglai said. The findings indicate that Ano10 is involved in both the ‘encoding’ and ‘decoding’ of Ca²⁺ signals, which are critical for normal development.   

Curious to further explore the role of Ano10 during development, the team used two additional approaches. Leveraging quantitative imaging of two key cytoskeletal regulators, the small GTPase Ras homolog family member A (RhoA) and the actin binding protein Cofilin, they showed that Ano10 is also required for the cytoskeletal reorganization of notochord cells. Finally, they performed heterologous expression experiments in combination with electrophysiological recordings and scramblase assays to demonstrate that Ano10/Tmem16k acts as an ion channel and not as a phospholipid scramblase.

With this work, the authors demonstrate for the first time the role of Anoctamins in embryonic development and the underlying mechanism has been at least in part elucidated. The impact of the study is expected to reach way beyond marine invertebrate developmental biology. “Zonglai’s study will have a broad impact on multiple fields: biological tubes are extremely abundant across plants and animals”, Marios Chatzigeorgiou explained. “On the other hand, Anoctamins have been implicated in numerous diseases and pathological conditions including cancer thus obtaining new knowledge about members of this important protein family will be of interest to the biomedical community.”