Functional coordination of non‐myocytes plays a key role in adult zebrafish heart regeneration

Abstract

Cardiac regeneration occurs primarily through proliferation of existing cardiomyocytes, but also involves complex interactions between distinct cardiac cell types including non-cardiomyocytes (non-CMs). However, the subpopulations, distinguishing molecular features, cellular functions, and intercellular interactions of non-CMs in heart regeneration remain largely unexplored. Using the LIGER algorithm, we assemble an atlas of cell states from 61,977 individual non-CM scRNA-seq profiles isolated at multiple time points during regeneration. This analysis reveals extensive non-CM cell diversity, including multiple macrophage (MC), fibroblast (FB), and endothelial cell (EC) subpopulations with unique spatiotemporal distributions, and suggests an important role for MC in inducing the activated FB and EC subpopulations. Indeed, pharmacological perturbation of MC function compromises the induction of the unique FB and EC subpopulations. Furthermore, we developed computational algorithm Topologizer to map the topological relationships and dynamic transitions between functional states. We uncover dynamic transitions between MC functional states and identify factors involved in mRNA processing and transcriptional regulation associated with the transition. Together, our single-cell transcriptomic analysis of non-CMs during cardiac regeneration provides a blueprint for interrogating the molecular and cellular basis of this process.

Origin and function of activated fibroblast states during zebrafish heart regeneration

“…Altogether, these data suggest that cell types or transient activated cell states residing in the regenerative niche may be important cellular regulators of regeneration. A number of recent single-cell RNA sequencing (scRNA-seq) studies have determined cell type diversity in the mammalian heart [18][19][20][21] , as well as in the regenerating zebrafish heart 22,23 . However, to our knowledge, there has been no systematic data-driven attempt to identify proregenerative cell states and their cell type of origin.…”

Activation of a transient progenitor state in the epicardium is required for zebrafish heart regeneration

“…CM proliferation is aided by the cellular and molecular environment provided by non-muscle tissues, such as the epicardium, a mesothelial layer of vertebrate hearts [5][6][7] . The epicardium is a heterogeneous population containing stem cells or progenitors that convert into other supporting cell types, such as mural cells (i.e., smooth muscle cells and pericytes) and fibroblasts, during development and regeneration 5,[8][9][10][11] . Following cardiac injury in adult zebrafish, epicardial cells are activated to turn on embryonic genes, proliferate, and migrate to repopulate the wound site.…”

leptin b and its regeneration enhancer illustrate the regenerative features of zebrafish hearts

“…To determine cell types composing these clusters, we annotated each cluster with known marker genes (Fig. 1A, C and S1A, C): CMs with tnnt2a and myl7 23,24 ; epicardial cells (Epi) and cardiac fibroblasts (cFB) with tcf21, fn1b, col1a1a, tagln, and col5a1 12,[25][26][27] ; endocardial/endothelial cells (ECs) with cdh5, kdrl, and flt1 28,29 ; coronary endothelial cells (cEC) composing blood vessels in hearts with cdh5, kdrl, flt1, and aplnra 12 ; mesenchyme-like cells (Mes) with mgp, angptl7, and rspo1 30,31 ; thrombocytes (throm) with itga2b and gp1bb 32,33 ; neutrophils (Neu) with mpx and lyz 34,35 ; macrophages (MC) with mfap4, c1qa, and mpeg1.1 [36][37][38] ; leukocytes (leu) with mhc2a, coro1a, and cxcr4b [39][40][41] (Table 1).…”

“…For instance, cardiac injury-activated endocardium and Epi produces paracrine factors to stimulate CM proliferation 5,[7][8][9] and extracellular matrix proteins (ECMs) to construct the regenerative niche/environment 10,11 . Activated endocardium and Epi are thought to be heterogenous [12][13][14][15] , but molecular identity representing diverse subgroups of these cardiac tissues in regenerating hearts is relatively unexplored. Tissue regeneration enhancer elements (TREEs) are key regulatory elements that relay injury signals to direct gene expression 3,[16][17][18][19][20] .…”