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.
