Highlights d Neonatal cardiomyocytes (CMs) in mice are heterogeneous d Immature CMs enriched in regenerative hearts enter the cell cycle upon injury d Defined transcriptome changes occur in regenerating CMs in response to injury d NFYa and NFE2L1 exert proliferative and protective functions, respectively, in CMs

The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. To uncover the molecular mechanisms underlying neonatal heart regeneration, we compared the transcriptomes and epigenomes of regenerative and nonregenerative mouse hearts over a 7-d time period following myocardial infarction injury. By integrating gene expression profiles with histone marks associated with active or repressed chromatin, we identified transcriptional programs underlying neonatal heart regeneration, and the blockade to regeneration in later life. Our results reveal a unique immune response in regenerative hearts and a retained embryonic cardiogenic gene program that is active during neonatal heart regeneration. Among the unique immune factors and embryonic genes associated with cardiac regeneration, we identified Ccl24, which encodes a cytokine, and Igf2bp3, which encodes an RNA-binding protein, as previously unrecognized regulators of cardiomyocyte proliferation. Our data provide insights into the molecular basis of neonatal heart regeneration and identify genes that can be modulated to promote heart regeneration.

SUMMARY The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. Neonatal heart regeneration is orchestrated by multiple cell types intrinsic to the heart, as well as immune cells that infiltrate the heart after injury. To elucidate the transcriptional responses of the different cellular components of the mouse heart following injury, we perform single-cell RNA sequencing on neonatal hearts at various time points following myocardial infarction and couple the results with bulk tissue RNA-sequencing data collected at the same time points. Concomitant single-cell ATAC sequencing exposes underlying dynamics of open chromatin landscapes and regenerative gene regulatory networks of diverse cardiac cell types and reveals extracellular mediators of cardiomyocyte proliferation, angiogenesis, and fibroblast activation. Together, our data provide a transcriptional basis for neonatal heart regeneration at single-cell resolution and suggest strategies for enhancing cardiac function after injury.

By gastrulation the ectodermal territories of the sea urchin embryo have developed an unexpectedly complex spatial pattern of sharply bounded regulatory states, organized orthogonally with respect to the animal/vegetal and oral/aboral axes of the embryo. Although much is known of the gene regulatory network (GRN) linkages that generate these regulatory states, the principles by which the boundaries between them are positioned and maintained have remained undiscovered. Here we determine the encoded genomic logic responsible for the boundaries of the oral aspect of the embryo that separate endoderm from ectoderm and ectoderm from neurogenic apical plate and that delineate the several further subdivisions into which the oral ectoderm per se is partitioned. Comprehensive regulatory state maps, including all spatially expressed oral ectoderm regulatory genes, were established. The circuitry at each boundary deploys specific repressors of regulatory states across the boundary, identified in this work, plus activation by broadly expressed positive regulators. These network linkages are integrated with previously established interactions on the oral/aboral axis to generate a GRN model encompassing the 2D organization of the regulatory state pattern in the pregastrular oral ectoderm of the embryo.regulatory state boundaries | pattern formation | repression circuitry B y the onset of gastrulation, bilaterian embryos consist of a complex mosaic of sharply bounded regulatory state domains, where "regulatory state" refers to the sum of specifically expressed mRNAs encoding DNA sequence-recognizing transcription factors in each nucleus. The regulatory state domains or territories are organized spatially in respect to the two major axes of the embryo, and they constitute informational specifications that determine the subsequent embryonic fates and functions of the cells descendant from these domains. Although in different modes of pregastrular embryogenesis regional specification functions are accomplished in somewhat different ways (1, 2), the end result is always the same: subdivision of the embryo into (transient) spatial regulatory states. These progressively specified regulatory states, and the boundaries between them, are the output of networks of genomically ordained interactions among regulatory genes. Gene regulatory networks (GRNs) encompass the heritable code for the embryonic development of each species. At present, the best known, experimentally determined, large-scale GRN drives the specification of endoderm and mesoderm in the embryo of the sea urchin Strongylocentrotus purpuratus up to gastrulation (3-6). This GRN model encompasses about half of the embryo, covers 30 h of development (18 h in the nonskeletogenic mesoderm), and all or almost all relevant regionally expressed regulatory genes. A recent study (7) shows that the endomesoderm GRN model contains sufficient regulatory relationships to generate a computational automaton that successfully predicts almost all spatial and temporal regulatory gene expre...