Periodic Reporting for period 1 - REGENECREST (Enhancing endogenous regenerative response in mammals by redeploying Cranial Neural Crest Cells pluripotency developmental programs and positional identity remodeling)
Berichtszeitraum: 2023-01-01 bis 2025-06-30
Cell differentiation is a unidirectional and irreversible process. During early development, pluripotent embryonic stem cells can generate every cell type of the organism. Yet, this ability is progressively restricted, especially during the formation of the three embryonic germ layers, the endoderm, the mesoderm and the ectoderm. Within these layers, cells see their differentiation potential reduced and can only give rise to certain cell types, specific to each layer. However, a cell population called cranial neural crest cells (CNCC) does not respect this rule. Originating from the ectoderm, CNCC not only give rise to neurons and glial cells, but they also generate cell types such as facial bones and cartilages, tissues normally formed by cells originating from the mesoderm.
CNCC remarkable plasticity is linked with the re-expression of pluripotency genes, which allows them to increase their differentiation potential. In addition, pre-migratory CNCC carry positional information reflective of their spatial origin in the neuroepithelium. However, this identity is subsequently erased with migratory CNCC forming a transcriptionally homogenous population, which later re-diversifies as CNCC undergo commitment events.
By combining sequencing approaches with in vivo and in vitro functional validations, our project intends to understand the molecular mechanisms controlling CNCC plasticity during development and post-natal regeneration which represent fundamental questions in the fields of developmental biology, stem cell research and regenerative medicine. This knowledge will be essential to establish prototype procedures aiming at enhancing endogenous regenerative responses during tissue repair for treatments of several congenital craniofacial syndromes – which represent a third of all congenital malformations within human populations – that require heavy maxillo-facial surgeries and reconstruction for humans suffering traumatic injuries.
CNCC remarkable plasticity is linked with the re-expression of pluripotency genes, which allows them to increase their differentiation potential. In addition, pre-migratory CNCC carry positional information reflective of their spatial origin in the neuroepithelium. However, this identity is subsequently erased with migratory CNCC forming a transcriptionally homogenous population, which later re-diversifies as CNCC undergo commitment events.
By combining sequencing approaches with in vivo and in vitro functional validations, our project intends to understand the molecular mechanisms controlling CNCC plasticity during development and post-natal regeneration which represent fundamental questions in the fields of developmental biology, stem cell research and regenerative medicine. This knowledge will be essential to establish prototype procedures aiming at enhancing endogenous regenerative responses during tissue repair for treatments of several congenital craniofacial syndromes – which represent a third of all congenital malformations within human populations – that require heavy maxillo-facial surgeries and reconstruction for humans suffering traumatic injuries.
Being a new lab, we spent some time initially setting up the lab and all the necessary protocols for the generation and evaluation of our data.
- Together with Mathilde Mularczyk (engineer hired through the ERC), we set up and validated our scRNA-seq pipeline. This encompassed (i) ordering and installing the robots necessary for the automation and miniaturization of the process; (ii) optimizing our cytometry to confidently sort single cell in 384-well plate; and (iii) validating the conditions to generate high quality sequencing library from each single cell sorted. We are now confident each of our protocols are optimized to generate top quality scRNAseq.
- For Aim1, Mary Daude-Pressé (postdoc hired through the ERC) generated embryos necessary for the project. She validated the expression pattern of the fluorescent reported at different embryo stages. Confirming Oct4 is dynamically expressed in the most anterior part of the embryo. She is now collecting cells for scRNAseq analysis.
- To test the function of the candidates scRNAseq analyses will identify, we adapted and optimized an in vitro culture protocol recapitulating CNCC differentiation from embryonic stem cells. We identified two culture protocols both generating CNCC at the end of differentiation. While in condition A cells recapitulate Oct4 dynamic expression as observed in vivo, in condition B they never re-express Oct4. We are now leveraging these two culture protocols to assess chromatin accessibility changes and transcriptional dynamics since comparing these two conditions will facilitate the identification of candidate genes and loci regulating pluripotency programs dynamic expression during CNCC differentiation. In addition, we are starting to map OCT4 genome-wide chromatin binding sites during CNCC differentiation in conditions A and B. This will allow to determine how OCT4 function to expand CNCC differentiation potential. This work is led by Rémi Coux (postdoc hired through the ERC) and Amélie Brun (engineer hired through the ERC). They are currently finishing generating Oct4-GFP and Oct4-Tdtomato cell lines to enrich Oct4 re-expressing cells using flow cytometry.
I believe we will have the first scRNAseq analyses done by mid-2025. Our Oct4 reporter lines are almost ready, we will be able to generate the ChIPseq data also by mid-2025. In the second half of 2025 we will cross reference these two datasets to pinpoint most promising candidates and start functional testing in vitro using siRNA or CRISPR.
- For Aim2, Mary Daude-Pressé is generating the first embryo necessary for the project and validate the expression pattern of the lines purchased confirming they can be used to separately enriched for pre- and post-migratory CNCC. Saverio Fortunato (PhD student who obtained a government PhD followship) studies how CNCC establish their initial positional identity and investigates how they remodel this identity during delamination and how it influences CNCC plasticity. Focusing on our in vitro system, Saverio showed CNCC generated in vitro recapitulate the establishment of positional identity (with a clear separation between anterior and posterior domain in the neurosphere) and then the erasure of this positional information at the start of delamination. Saverio is now generating a double reporter line to isolate anterior and posterior cells to compare their transcriptome and chromatin accessibility. In addition, we determined different culture conditions to modulate the axial identities of the generated CNCC which will be a valuable tool to further test how candidates identified in the future are regulating establishment of this positional identity. Finally, we designed a protocol to grow single neurosphere in a single well in a 96-well plate format. We have tested several imaging equipment and are now able to perform live imaging to track neurosphère growth and cell fate change (using reporter cell line).
I expect this project to be completed within a year or two.
Aim3 is more demanding and requires additional animal training to perform surgery to induce regeneration event. This project is led by Jean Christophe Deschemin (engineer with a Inserm permanent position) and myself. Since it requires generating double mutant mice to start the investigation, we have made fewer advances and this project. This project was not plan to start before Year 3 originally. Yet I expect first preliminary should be generated in early 2025.
- Together with Mathilde Mularczyk (engineer hired through the ERC), we set up and validated our scRNA-seq pipeline. This encompassed (i) ordering and installing the robots necessary for the automation and miniaturization of the process; (ii) optimizing our cytometry to confidently sort single cell in 384-well plate; and (iii) validating the conditions to generate high quality sequencing library from each single cell sorted. We are now confident each of our protocols are optimized to generate top quality scRNAseq.
- For Aim1, Mary Daude-Pressé (postdoc hired through the ERC) generated embryos necessary for the project. She validated the expression pattern of the fluorescent reported at different embryo stages. Confirming Oct4 is dynamically expressed in the most anterior part of the embryo. She is now collecting cells for scRNAseq analysis.
- To test the function of the candidates scRNAseq analyses will identify, we adapted and optimized an in vitro culture protocol recapitulating CNCC differentiation from embryonic stem cells. We identified two culture protocols both generating CNCC at the end of differentiation. While in condition A cells recapitulate Oct4 dynamic expression as observed in vivo, in condition B they never re-express Oct4. We are now leveraging these two culture protocols to assess chromatin accessibility changes and transcriptional dynamics since comparing these two conditions will facilitate the identification of candidate genes and loci regulating pluripotency programs dynamic expression during CNCC differentiation. In addition, we are starting to map OCT4 genome-wide chromatin binding sites during CNCC differentiation in conditions A and B. This will allow to determine how OCT4 function to expand CNCC differentiation potential. This work is led by Rémi Coux (postdoc hired through the ERC) and Amélie Brun (engineer hired through the ERC). They are currently finishing generating Oct4-GFP and Oct4-Tdtomato cell lines to enrich Oct4 re-expressing cells using flow cytometry.
I believe we will have the first scRNAseq analyses done by mid-2025. Our Oct4 reporter lines are almost ready, we will be able to generate the ChIPseq data also by mid-2025. In the second half of 2025 we will cross reference these two datasets to pinpoint most promising candidates and start functional testing in vitro using siRNA or CRISPR.
- For Aim2, Mary Daude-Pressé is generating the first embryo necessary for the project and validate the expression pattern of the lines purchased confirming they can be used to separately enriched for pre- and post-migratory CNCC. Saverio Fortunato (PhD student who obtained a government PhD followship) studies how CNCC establish their initial positional identity and investigates how they remodel this identity during delamination and how it influences CNCC plasticity. Focusing on our in vitro system, Saverio showed CNCC generated in vitro recapitulate the establishment of positional identity (with a clear separation between anterior and posterior domain in the neurosphere) and then the erasure of this positional information at the start of delamination. Saverio is now generating a double reporter line to isolate anterior and posterior cells to compare their transcriptome and chromatin accessibility. In addition, we determined different culture conditions to modulate the axial identities of the generated CNCC which will be a valuable tool to further test how candidates identified in the future are regulating establishment of this positional identity. Finally, we designed a protocol to grow single neurosphere in a single well in a 96-well plate format. We have tested several imaging equipment and are now able to perform live imaging to track neurosphère growth and cell fate change (using reporter cell line).
I expect this project to be completed within a year or two.
Aim3 is more demanding and requires additional animal training to perform surgery to induce regeneration event. This project is led by Jean Christophe Deschemin (engineer with a Inserm permanent position) and myself. Since it requires generating double mutant mice to start the investigation, we have made fewer advances and this project. This project was not plan to start before Year 3 originally. Yet I expect first preliminary should be generated in early 2025.
Developing a protocol to grow single neurosphere in 96 well plate, could potentially allow us to develop future projects to perform siRNA or CRISPR based screen and monitor how it affects CNCC development. This could have an impact for identifying biomarkers to predict craniofacial syndromes or the effect of environmental pollutants of neural crest cells development.