Periodic Reporting for period 1 - ZEBREXPANSION (Expansion microscopy in zebrafish embryo to study primary cilia function during cardiovascular development and diseases)
Período documentado: 2021-06-01 hasta 2023-05-31
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.
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.
Most of the work covered in this MSCA was published in peer-reviewed open access articles.
In Steib et al, Cell Rep Methods 2022, we described the development and strength of TissUExM, a method that enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy. We addressed the fact that there was no prior versatile super-resolution approach for ultrastructural analysis compatible with whole vertebrate embryos. TissUExM is a method to expand millimeter-scale and mechanically heterogeneous whole embryonic tissues, including Drosophila wing discs, whole zebrafish, and mouse embryos. It is designed for the observation of endogenous proteins and permits quantitative characterization of protein complexes in various organelles at super-resolution in a range of ∼3 mm-sized tissues using conventional microscopes.
Importantly, we demonstrated its strength by investigating tissue-specific ciliary architecture heterogeneity and ultrastructural defects observed upon ciliary protein overexpression. Overall, TissUExM is ideal for performing ultrastructural studies and molecular mapping in situ in whole embryos.
Unpublished TissUExM data were also generated from attempts to apply the method to sea urchin embryos and Arabidopsis leaves and roots, in collaboration with the Minc’s (FR) and Nakayama’s (UK) labs. Further work will be needed for optimal application of TissUExM in these models.
An important part of this MSCA consisted in disseminating the method to multiple international developmental biology labs. In addition to training students and collaborators, we published a detailed and video-recorded version of the TissUExM protocol (Steib et al, STAR protocol 2023) in which we describe steps for embedding and denaturing zebrafish larvae or mouse embryos. We then detail procedures for hydrogel handling and mounting. This tool will undoubtedly allow for researchers to transfer the method easily to their lab and push its applications beyond the field of cilia biology.
In Steib et al, Cell Rep Methods 2022, we described the development and strength of TissUExM, a method that enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy. We addressed the fact that there was no prior versatile super-resolution approach for ultrastructural analysis compatible with whole vertebrate embryos. TissUExM is a method to expand millimeter-scale and mechanically heterogeneous whole embryonic tissues, including Drosophila wing discs, whole zebrafish, and mouse embryos. It is designed for the observation of endogenous proteins and permits quantitative characterization of protein complexes in various organelles at super-resolution in a range of ∼3 mm-sized tissues using conventional microscopes.
Importantly, we demonstrated its strength by investigating tissue-specific ciliary architecture heterogeneity and ultrastructural defects observed upon ciliary protein overexpression. Overall, TissUExM is ideal for performing ultrastructural studies and molecular mapping in situ in whole embryos.
Unpublished TissUExM data were also generated from attempts to apply the method to sea urchin embryos and Arabidopsis leaves and roots, in collaboration with the Minc’s (FR) and Nakayama’s (UK) labs. Further work will be needed for optimal application of TissUExM in these models.
An important part of this MSCA consisted in disseminating the method to multiple international developmental biology labs. In addition to training students and collaborators, we published a detailed and video-recorded version of the TissUExM protocol (Steib et al, STAR protocol 2023) in which we describe steps for embedding and denaturing zebrafish larvae or mouse embryos. We then detail procedures for hydrogel handling and mounting. This tool will undoubtedly allow for researchers to transfer the method easily to their lab and push its applications beyond the field of cilia biology.
This MSCA allowed the fellow to gain experience in developmental biology and fluorescence microscopy, in addition to project management and student supervision experience. This experience led to secure an Advanced Workflow Specialist position in one of the world-leading microscopy company.
This MSCA further allowed the Vermot Lab to establish new collaborations and to maintain its established reputation as a productive environment to produce excellent scientific data and researchers training.
Regarding the benefits for European society, the development and publication of TissUExM as an imaging tool provide insights that could lead to novel medical diagnosis approaches, in addition to inputs for the leading imaging companies in Europe and interdisciplinary collaborations.
This MSCA further allowed the Vermot Lab to establish new collaborations and to maintain its established reputation as a productive environment to produce excellent scientific data and researchers training.
Regarding the benefits for European society, the development and publication of TissUExM as an imaging tool provide insights that could lead to novel medical diagnosis approaches, in addition to inputs for the leading imaging companies in Europe and interdisciplinary collaborations.