Un nouveau modèle de poisson-zèbre pour l'étude de la reprogrammation de type embryonnaire lors de la régénération intestinale et sa régulation par le signalement redox // A novel zebrafish model for studying fetal-like reprogramming in intestinal regener

Introduction
Severe intestinal injury in humans affecting all tissue layers (epithelium, mesenchyme, muscle, and enteric neurons) can lead to life-threatening conditions requiring surgeries that risk complications like chronic inflammation and sepsis (1). In contrast, the zebrafish can functionally regenerate diverse tissues after severe injury (2). Our laboratory has developed a zebrafish model where a fully transected intestine regenerates, restoring structure and function, including peristalsis and nutrient processing. Unlike traditional models focused on epithelial healing, this system enables whole-gut regeneration studies.
A central process in tissue regeneration is reprogramming, where cells regain plasticity by reactivating embryonic gene programs (3). This process can be influenced by redox signaling, which regulates stemness, proliferation, and differentiation (4), and is often linked to metabolic shifts (from OXPHOS to glycolysis) (5). However, how cell proliferation, redox state, and potential metabolic changes coordinate reprogramming remains unclear.

In our model, we observed a transient proliferation peak at 6 hours post-injury (hpi), followed by epithelial reattachment and sox17 re-expression at 24 hpi. This early proliferation may prime the tissue for reattachment and reprogramming. sox17 is downregulated once gut lumen continuity and function are restored. Notably, pharmacological modulation of redox signaling affects both sox17 expression and successful regeneration, suggesting a role in proliferation and fetal-like reprogramming.
This model offers a powerful in vivo system to study cell reprogramming and the roles of proliferation and redox signaling, as well as their potential link to metabolic shifts, in regulating cell state transitions during regeneration.

Hypothesis
We hypothesize that cell reprogramming during gut regeneration following severe injury in zebrafish is regulated by early proliferation and redox signaling, with a potential role for metabolic shifts in modulating the regenerative process.

Aims
1. Characterize the cell reprogramming process during intestinal regeneration.
2. Determine the role of redox signaling in controlling proliferation and reprogramming during regeneration.
3. Explore potential links between redox signaling and metabolic shifts.

Methods
1. Characterize the reprogramming process during intestinal regeneration
Map the timeline of epithelial reattachment, proliferation, gut remodeling, lumen fusion, and reprogramming using time-lapse imaging with reporter lines (cldn15:GFP for epithelium, H2B:RFP for nuclei, and sox17:GFP for reprogramming).
Track proliferation dynamics using EdU/BrdU incorporation and cell cycle reporters (FUCCI) to align proliferation peaks with sox17 activation.
Modulate epithelial reattachment using ROCK inhibitors and actomyosin blockers to assess impacts on proliferation and reprogramming.
Trace the lineage of sox17⁺ cells using single-cell RNA sequencing (scRNA-seq) and a photoconvertible sox17-Kikume line.

2. Determine the role of redox signaling
Quantify tissue redox states using CellRox and the HyPer7 biosensor (6) at different time points.
Correlate redox levels with proliferation and sox17 dynamics.
Modulate redox balance with antioxidants (N-acetylcysteine, mitoTEMPO) and pro-oxidants (menadione, hydrogen peroxide) and evaluate impacts on proliferation, sox17 activation, cell fate transitions, and gut remodeling.

3. Explore potential links between redox signaling and metabolic shifts
Analyze metabolic shifts during reprogramming using single-cell transcriptomics, correlating changes with sox17 expression, cell fate, and redox state.
Inhibit glycolysis (2-deoxyglucose) and OXPHOS (UK5099) to assess effects on proliferation, sox17 activation, and regeneration, both independently and with redox modulators.
Investigate if redox signaling alters metabolic shifts by comparing metabolic profiles under different redox states
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Introduction
Severe intestinal injury in humans affecting all tissue layers (epithelium, mesenchyme, muscle, and enteric neurons) can lead to life-threatening conditions requiring surgeries that risk complications like chronic inflammation and sepsis (1). In contrast, the zebrafish can functionally regenerate diverse tissues after severe injury (2). Our laboratory has developed a zebrafish model where a fully transected intestine regenerates, restoring structure and function, including peristalsis and nutrient processing. Unlike traditional models focused on epithelial healing, this system enables whole-gut regeneration studies.
A central process in tissue regeneration is reprogramming, where cells regain plasticity by reactivating embryonic gene programs (3). This process can be influenced by redox signaling, which regulates stemness, proliferation, and differentiation (4), and is often linked to metabolic shifts (from OXPHOS to glycolysis) (5). However, how cell proliferation, redox state, and potential metabolic changes coordinate reprogramming remains unclear.

In our model, we observed a transient proliferation peak at 6 hours post-injury (hpi), followed by epithelial reattachment and sox17 re-expression at 24 hpi. This early proliferation may prime the tissue for reattachment and reprogramming. sox17 is downregulated once gut lumen continuity and function are restored. Notably, pharmacological modulation of redox signaling affects both sox17 expression and successful regeneration, suggesting a role in proliferation and fetal-like reprogramming.
This model offers a powerful in vivo system to study cell reprogramming and the roles of proliferation and redox signaling, as well as their potential link to metabolic shifts, in regulating cell state transitions during regeneration.

Hypothesis
We hypothesize that cell reprogramming during gut regeneration following severe injury in zebrafish is regulated by early proliferation and redox signaling, with a potential role for metabolic shifts in modulating the regenerative process.

Aims
1. Characterize the cell reprogramming process during intestinal regeneration.
2. Determine the role of redox signaling in controlling proliferation and reprogramming during regeneration.
3. Explore potential links between redox signaling and metabolic shifts.

Methods
1. Characterize the reprogramming process during intestinal regeneration
Map the timeline of epithelial reattachment, proliferation, gut remodeling, lumen fusion, and reprogramming using time-lapse imaging with reporter lines (cldn15:GFP for epithelium, H2B:RFP for nuclei, and sox17:GFP for reprogramming).
Track proliferation dynamics using EdU/BrdU incorporation and cell cycle reporters (FUCCI) to align proliferation peaks with sox17 activation.
Modulate epithelial reattachment using ROCK inhibitors and actomyosin blockers to assess impacts on proliferation and reprogramming.
Trace the lineage of sox17⁺ cells using single-cell RNA sequencing (scRNA-seq) and a photoconvertible sox17-Kikume line.

2. Determine the role of redox signaling
Quantify tissue redox states using CellRox and the HyPer7 biosensor (6) at different time points.
Correlate redox levels with proliferation and sox17 dynamics.
Modulate redox balance with antioxidants (N-acetylcysteine, mitoTEMPO) and pro-oxidants (menadione, hydrogen peroxide) and evaluate impacts on proliferation, sox17 activation, cell fate transitions, and gut remodeling.

3. Explore potential links between redox signaling and metabolic shifts
Analyze metabolic shifts during reprogramming using single-cell transcriptomics, correlating changes with sox17 expression, cell fate, and redox state.
Inhibit glycolysis (2-deoxyglucose) and OXPHOS (UK5099) to assess effects on proliferation, sox17 activation, and regeneration, both independently and with redox modulators.
Investigate if redox signaling alters metabolic shifts by comparing metabolic profiles under different redox states
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Début de la thèse : 01/10/2025

Contrat doctoral

Concours pour un contrat doctoral

Institut Curie - PSL

515 Complexité du vivant

The ideal candidate for this PhD project will have a strong background in developmental biology, cell biology, or regenerative biology, with practical experience in genetic manipulation techniques, preferably in zebrafish or similar model organisms. Proficiency in live imaging, microscopy, and molecular biology techniques (such as in situ hybridization or gene expression analysis) is essential. Experience with redox biology, cell proliferation assays, or single-cell transcriptomics would be highly beneficial, though not mandatory. The candidate should demonstrate a solid understanding of cell signaling pathways involved in tissue regeneration and an interest in exploring complex mechanisms such as redox signaling and metabolic regulation. Familiarity with bioinformatics tools or a strong willingness to acquire these skills is desirable, given the data analysis component of the project. Key personal qualities include the ability to work independently while collaborating within a multidisciplinary team, strong problem-solving skills, and a high level of motivation for scientific discovery. A genuine interest in tissue regeneration, cell reprogramming, and the application of zebrafish models in biomedical research will be considered strong assets.
The ideal candidate for this PhD project will have a strong background in developmental biology, cell biology, or regenerative biology, with practical experience in genetic manipulation techniques, preferably in zebrafish or similar model organisms. Proficiency in live imaging, microscopy, and molecular biology techniques (such as in situ hybridization or gene expression analysis) is essential. Experience with redox biology, cell proliferation assays, or single-cell transcriptomics would be highly beneficial, though not mandatory. The candidate should demonstrate a solid understanding of cell signaling pathways involved in tissue regeneration and an interest in exploring complex mechanisms such as redox signaling and metabolic regulation. Familiarity with bioinformatics tools or a strong willingness to acquire these skills is desirable, given the data analysis component of the project. Key personal qualities include the ability to work independently while collaborating within a multidisciplinary team, strong problem-solving skills, and a high level of motivation for scientific discovery. A genuine interest in tissue regeneration, cell reprogramming, and the application of zebrafish models in biomedical research will be considered strong assets.