Periodic Reporting for period 1 - HEART_STATES (Mechanisms and consequences of cell state transitions during heart regeneration)
Okres sprawozdawczy: 2023-03-01 do 2025-08-31
Organs consist of cells with a large diversity of specialized roles. A fundamental question is how these cells mount a coordinated response in space and time to maintain or restore organ function after perturbation. Recent progress in single-cell genomics has generated the opportunity to understand this process on a system-wide scale. We will use the adult zebrafish heart as a powerful model system to dissect how regeneration after injury is orchestrated by the activation response of multiple different cell types. To understand how activated cell states are generated and how they interact to drive the regenerative process, we will:
1) Define which cell types react to injury and measure their activation profiles. We will develop new experimental and computational strategies for measuring cell states, including a “molecular time machine” that records the past transcriptome of single cells based on RNA labeling.
2) Discover the mechanisms that induce cell state activation upon injury. We will combine single-cell transcriptomics and open chromatin profiling to infer gene regulatory networks, and we will use functional experiments to validate the identified pathways.
3) Reveal pro-regenerative cell types and understand their role in the regenerative process. We will combine spatial transcriptomics and computational analysis to identify putative cellular interactions, and we will use targeted cell type depletion and signaling inhibition to confirm our findings.
In this manner, we will provide the first comprehensive view of how cell type activation leads to a synergistic response in organ regeneration. Furthermore, the approaches and concepts developed in this project will be applicable to other systems in regeneration and beyond. Finally, understanding the underlying mechanisms in zebrafish, the preeminent model for heart regeneration, will open up exciting avenues for awakening the dormant regenerative potential of the human heart.
1) Define which cell types react to injury and measure their activation profiles. We will develop new experimental and computational strategies for measuring cell states, including a “molecular time machine” that records the past transcriptome of single cells based on RNA labeling.
2) Discover the mechanisms that induce cell state activation upon injury. We will combine single-cell transcriptomics and open chromatin profiling to infer gene regulatory networks, and we will use functional experiments to validate the identified pathways.
3) Reveal pro-regenerative cell types and understand their role in the regenerative process. We will combine spatial transcriptomics and computational analysis to identify putative cellular interactions, and we will use targeted cell type depletion and signaling inhibition to confirm our findings.
In this manner, we will provide the first comprehensive view of how cell type activation leads to a synergistic response in organ regeneration. Furthermore, the approaches and concepts developed in this project will be applicable to other systems in regeneration and beyond. Finally, understanding the underlying mechanisms in zebrafish, the preeminent model for heart regeneration, will open up exciting avenues for awakening the dormant regenerative potential of the human heart.
WP1: Activation states in the regenerating heart
Work package 1 was the main focus of our research activities in the initial stages of the project, and this WP progressed exceptionally well:
Following up on the preliminary data presented in the research proposal, we investigated the transcriptomics diversity and lineage origin of cardiac fibroblasts. Specifically, we found that fibroblast expression profiles change drastically upon heart injury, with three new populations of transient fibroblasts emerging after injury. For one of these fibroblast populations, which is characterized by expression of col12a1a, we could show together with our collaborator Daniela Panakova (University of Kiel) that they have a pro-regenerative function (Hu et al., Nat Genet, 2022). This work was also the first application of our LINNAEUS method for CRISPR/Cas9-based high-throughput single-cell lineage tracing (Spanjaard et al., Nat Biotech, 2018) in an adult disease model.
In the second part of WP1, we want to establish a single-cell metabolic RNA labeling technique (scSLAM-seq) in the adult zebrafish heart, in order to directly measure the first response of the zebrafish heart to injury. We reasoned that, if we labeled all new mRNA molecules that are made after heart injury, we would be able to increase the signal-to-noise ratio and measure which cells act as the sentinels of the heart that kickstart the regenerative cascade. We now successfully established and characterized scSLAM-seq in the zebrafish heart, and we developed a computational pipeline as well as a mathematical model for analysis and interpretation of the data. These experiments revealed that activation of damage response pathways in macrophages is the first response to injury. In collaboration with the lab of Didier Stainier (Max Planck Institute for Heart and Lung Research, Bad Nauheim), we generated functional data using macrophage-specific overexpression of myd88, a key component of the damage response pathways, which revealed a significant effect on hallmarks of regeneration after injury. In summary, with these results WP1 is approaching successful completion.
WP2: Gene-regulatory networks in cell type activation
In WP2 we want to combine scRNA-seq, scATAC-seq, computational modeling, and functional perturbation experiments to understand gene-regulatory networks in cell type activation during heart regeneration. Specifically, we focus on activation of cardiomyocytes and fibroblasts. Our initial efforts in this work package were concentrated on generating scATAC-seq data of sufficiently high quality. This proved to be harder than expected due to experimental challenges specific to the zebrafish heart, but in the meantime we have managed to establish a protocol that yields high quality open chromatin data. We have finished the data acquisition phase and are currently integrating and processing the datasets for further analysis. In parallel, we have set up the computational pipelines for reconstruction of gene-regulatory networks. After identification of candidate transcription factors, we will proceed to functional validation and phenotypic characterization with collaborators.
WP3: Pro-regenerative cellular interactions
In WP3 we seek to established sequencing-based spatial transcriptomics in order to identify cell-cell interactions in heart regeneration. We successfully set up the OpenST technique for spatial transcriptomics in the zebrafish. However, similar to the scATAC-seq in WP2, it turned out that the adult zebrafish heart presented with particular challenges compared to the mammalian heart or other zebrafish samples (such as embryos, brain, tumors), which are potentially exacerbated by the requirement to use unfixed samples in OpenST. Specifically, we noticed poor preservation of morphology and spreading of mRNA beyond their cell of origin when performing OpenST under standard conditions in zebrafish heart. Building on promising preliminary data, we are currently optimizing the experimental conditions for OpenST in the zebrafish heart by adapting protocols for tissue embedding, fixation and permeabilization. Furthermore, we have set up a pipeline for cost-efficient validation the results by hybridization chain reaction.
Work package 1 was the main focus of our research activities in the initial stages of the project, and this WP progressed exceptionally well:
Following up on the preliminary data presented in the research proposal, we investigated the transcriptomics diversity and lineage origin of cardiac fibroblasts. Specifically, we found that fibroblast expression profiles change drastically upon heart injury, with three new populations of transient fibroblasts emerging after injury. For one of these fibroblast populations, which is characterized by expression of col12a1a, we could show together with our collaborator Daniela Panakova (University of Kiel) that they have a pro-regenerative function (Hu et al., Nat Genet, 2022). This work was also the first application of our LINNAEUS method for CRISPR/Cas9-based high-throughput single-cell lineage tracing (Spanjaard et al., Nat Biotech, 2018) in an adult disease model.
In the second part of WP1, we want to establish a single-cell metabolic RNA labeling technique (scSLAM-seq) in the adult zebrafish heart, in order to directly measure the first response of the zebrafish heart to injury. We reasoned that, if we labeled all new mRNA molecules that are made after heart injury, we would be able to increase the signal-to-noise ratio and measure which cells act as the sentinels of the heart that kickstart the regenerative cascade. We now successfully established and characterized scSLAM-seq in the zebrafish heart, and we developed a computational pipeline as well as a mathematical model for analysis and interpretation of the data. These experiments revealed that activation of damage response pathways in macrophages is the first response to injury. In collaboration with the lab of Didier Stainier (Max Planck Institute for Heart and Lung Research, Bad Nauheim), we generated functional data using macrophage-specific overexpression of myd88, a key component of the damage response pathways, which revealed a significant effect on hallmarks of regeneration after injury. In summary, with these results WP1 is approaching successful completion.
WP2: Gene-regulatory networks in cell type activation
In WP2 we want to combine scRNA-seq, scATAC-seq, computational modeling, and functional perturbation experiments to understand gene-regulatory networks in cell type activation during heart regeneration. Specifically, we focus on activation of cardiomyocytes and fibroblasts. Our initial efforts in this work package were concentrated on generating scATAC-seq data of sufficiently high quality. This proved to be harder than expected due to experimental challenges specific to the zebrafish heart, but in the meantime we have managed to establish a protocol that yields high quality open chromatin data. We have finished the data acquisition phase and are currently integrating and processing the datasets for further analysis. In parallel, we have set up the computational pipelines for reconstruction of gene-regulatory networks. After identification of candidate transcription factors, we will proceed to functional validation and phenotypic characterization with collaborators.
WP3: Pro-regenerative cellular interactions
In WP3 we seek to established sequencing-based spatial transcriptomics in order to identify cell-cell interactions in heart regeneration. We successfully set up the OpenST technique for spatial transcriptomics in the zebrafish. However, similar to the scATAC-seq in WP2, it turned out that the adult zebrafish heart presented with particular challenges compared to the mammalian heart or other zebrafish samples (such as embryos, brain, tumors), which are potentially exacerbated by the requirement to use unfixed samples in OpenST. Specifically, we noticed poor preservation of morphology and spreading of mRNA beyond their cell of origin when performing OpenST under standard conditions in zebrafish heart. Building on promising preliminary data, we are currently optimizing the experimental conditions for OpenST in the zebrafish heart by adapting protocols for tissue embedding, fixation and permeabilization. Furthermore, we have set up a pipeline for cost-efficient validation the results by hybridization chain reaction.
We identified a pro-regenerative fibroblast population in zebrafish heart regeneration (Hu et al., Nat Genet, 2022). This work gained widespread interest since it changes the perspective from fibroblasts being an obstacle to regeneration to having an actual pro-regenerative function, with important possibilities for further studies. Furthermore, the combination of novel single-cell genomics methods (in particular high-throughput lineage tracing) and functional perturbation experiments (e.g. targeted cell type ablation) can serve as a blueprint for future research. Furthermore, our finding that macrophages act as sentinel cells that kickstart the cascade of heart regeneration, and the role of damage response pathways in these processes also has the potential to become a significant advancement of the field. Finally, our establishment of single-cell RNA metabolic labeling in a live adult vertebrate, in the future to be combined with spatial resolution via OpenST, is a major technological advance with far-reaching implications.