Single-Cell Transcriptomics Reveals Chemotaxis-Mediated Intraorgan Crosstalk During Cardiogenesis

Abstract

Rationale: We hypothesized that the differentiation processes of cardiac progenitor cell (CP) from first and second heart fields (FHF and SHF) may undergo the unique instructive gene regulatory networks or signaling pathways, and the precise SHF progression is contingent on the FHF signaling developmental cues. Objective: We investigated how the intraorgan communications control sequential building of discrete anatomic regions of the heart at single-cell resolution. Methods and Results: By single-cell transcriptomic analysis of Nkx2-5 (NK2 homeobox 5) and Isl1 (ISL LIM homeobox 1) lineages at embryonic day 7.75, embryonic day 8.25, embryonic day 8.75, and embryonic day 9.25, we present a panoramic view of distinct CP differentiation hierarchies. Computational identifications of FHF- and SHF-CP descendants revealed that SHF differentiation toward cardiomyocytes underwent numerous step-like transitions, whereas earlier FHF progressed toward cardiomyocytes in a wave-like manner. Importantly, single-cell pairing analysis demonstrated that SHF-CPs were attracted to and expanded FHF-populated heart tube region through interlineage communications mediated by the chemotactic guidance (MIF [macrophage migration inhibitory factor]-CXCR2 [C-X-C motif chemokine receptor 2]). This finding was verified by pharmacological blockade of this chemotaxis in embryos manifesting limited SHF cell migration and contribution to the growth of the outflow tract and right ventricle but undetectable effects on the left ventricle or heart tube initiation. Genetic loss-of-function assay of Cxcr2 showed that the expression domain of CXCR4 was expanded predominantly at SHF. Furthermore, double knockout of Cxcr2/Cxcr4 exhibited defective SHF development, corroborating the redundant function. Mechanistically, NKX2-5 directly bound the Cxcr2 and Cxcr4 genomic loci and activated their transcription in SHF. Conclusions: Collectively, we propose a model in which the chemotaxis-mediated intraorgan crosstalk spatiotemporally guides the successive process of positioning SHF-CP and promoting primary heart expansion and patterning upon FHF-derived heart tube initiation.

Single-Cell RNA Sequencing of the Cardiovascular System: New Looks for Old Diseases

“…There has been tremendous effort to study the developing embryonic cardiovascular system to gain insight into the factors associated with cardiac regeneration. Single-cell resolution has allowed us to obtain a far more detailed and more complete picture of cellular transitions during development (66,67). scRNA-seq has been utilized to better understand the complex genetic regulatory and epigenetic networking of various stages of multipotent stem cell differentiation during heart development.…”

Primate heart regenerationviamigration and fibroblast repulsion by human heart progenitors

“…Collectively, our data support the model that HVPs expressing CXCR4 sense CXCL12 secreted by CFs at the injury site as a chemoattracting signal to repopulate the damaged myocardial compartment. A similar, chemokine-controlled deployment of SHF cells has been identified as intra-organ crosstalk between progenitors and FHF CMs during mouse cardiogenesis 36 , suggesting that migration programs that are functional during cardiac development are re-activated in HVPs during organ regeneration.…”

Review of Single-Cell RNA Sequencing in the Heart

“…They revealed spatiotemporal transcriptomic changes during heart development and showed that Nkx2-5 is an important factor in CM maturation. Xiong et al utilized FACS and revealed that Nkx2-5 directly regulates spatial expression of the chemokine receptors Cxcr2 and Cxcr4 in the second heart field, which directs second heart field cardiac progenitor migration with spatiotemporal precision [9]. In addition to Nkx2-5, Mesp1 is also essential for CM maturation.…”

“…Velocyto is a package for the analysis of expression dynamics from the estimation of RNA velocities by distinguishing unspliced and spliced mRNAs [56]. They have been used in studies of cardiac development and disease progression [9,11,14,[21][22][23]32,36,38].…”