This MSCA project aimed at revealing the function of primary cilia during cardiovascular development and cardiovascular diseases, through an imaging approach based on expansion light microscopy.
Nearly every cell in the human body exhibits one or multiple cilia for which functions may vary depending on tissue specificity and developmental stage. Primary cilia (PC), defined as a single microtubule-based protrusion per cell, have been identified in vertebrates and are described as cellular antennae. Defects in PC are responsible for a disease group referred to as ciliopathies, affecting 1 in 1000 people, displaying a large spectrum of symptoms ranging from neurodevelopmental abnormalities to cardiovascular defects, polycystic kidneys, obesity, etc. However, while cilia composition seems to be conserved among cell types, it is unclear why ciliopathies affect specific tissues and not others.
Cardiovascular defects are common in patients diagnosed with ciliopathies. Genetic approaches used to study congenital heart diseases (CHDs) have highlighted a strong implication of genes coding for ciliary proteins, suggesting an important role for this organelle during cardiovascular development. Still, the role of PC in cardiovascular development is understudied and poorly understood. Essential for ciliogenesis, Dzip1 has recently been identified as a key gene leading to CHD in humans when mutated. To date, ciliary and DZIP1 functions in cardiovascular development are unknown.
We aimed to understand how cilia assembly, maintenance, and disassembly are correlated with essential regulatory functions during cardiogenesis. To reach a comprehensive description of ciliary implication in CHDs, we aimed to study ciliary features upon cardiac remodeling in a quantitative manner with nanometric resolution. Specifically, our objectives were:
- to use the previously developed U-ExM method in cultured cells and establish a precise molecular mapping of the CHD-related protein DZIP1 to reveal its structural function in cilium assembly and disassembly.
- to establish U-ExM in zebrafish and characterize PC remodeling upon cardiac maturation.
- to investigate to which extent cilia are blunted, stunted, or erased specifically in the heart of Dzip1 mutants.
While we rapidly chose to move from cultured human cells to develop tissue ultrastructure expansion microscopy (TissUExM) in whole zebrafish embryos, we extended the scope of this MSCA to additional models: Drosophila melanogaster wing discs and whole mouse embryos, in collaboration with the Mao’s and Norris’s labs (UK). This resulted in two first-author publications (Steib et al, Cell Rep Methods 2022; Steib et al, STAR Protocol 2023), and this work was acknowledged by cilia experts through the award for best talk for the Fellow at the 2022 EMBO Cilia Meeting.
In the meantime, we also set up a collaboration with the Roy Lab (Singapore), the leading expert of Dzip1 studies in zebrafish embryos. Current and future work consists in applying TissUExM and live imaging expertise from the Vermot Lab to different Dzip1 mutants, to provide a comprehensive structural and functional description of Dzip1 in zebrafish valve development and maturation.
