Pbx4 limits heart size and fosters arch artery formation by partitioning second heart field progenitors and restricting proliferation

Abstract

Vertebrate heart development requires the integration of temporally distinct differentiating progenitors. However, few signals are understood that restrict the size of the later-differentiating outflow tract (OFT). We show that improper specification and proliferation of second heart field (SHF) progenitors in zebrafish lazarus (lzr) mutants, which lack the transcription factor Pbx4, produces enlarged hearts owing to an increase in ventricular and smooth muscle cells. Specifically, Pbx4 initially promotes the partitioning of the SHF into anterior progenitors, which contribute to the OFT, and adjacent endothelial cell progenitors, which contribute to posterior pharyngeal arches. Subsequently, Pbx4 limits SHF progenitor (SHFP) proliferation. Single cell RNA sequencing of nkx2.5 + cells revealed previously unappreciated distinct differentiation states and progenitor subpopulations that normally reside within the SHF and arterial pole of the heart. Specifically, the transcriptional profiles of Pbx4-deficient nkx2.5 + SHFPs are less distinct and display characteristics of normally discrete proliferative progenitor and anterior, differentiated cardiomyocyte populations. Therefore, our data indicate that the generation of proper OFT size and arch arteries requires Pbx-dependent stratification of unique differentiation states to facilitate both homeotic-like transformations and limit progenitor production within the SHF.

From Stripes to a Beating Heart: Early Cardiac Development in Zebrafish

“…Nkx2.5 acts synergistically and at least in parts redundantly with its paralog Nkx2.7 in controlling arterial and venous pole SHF contribution, adding another level of fine-tuning [ 109 , 160 ]. Single cell RNA-sequencing of nkx2.5 -positive cells charted multiple subpopulations of SHF progenitors and differentiating ventricular cardiomyocytes at the arterial pole in zebrafish embryos [ 161 ], underscoring the broad involvement of nkx2.5 expression during multiple stages in cardiac development. More work is warranted to unlock the secrets of Nkx2.5’s ancient history of contributing to heart formation [ 47 , 162 , 163 ].…”

“…While various gene expression patterns and broader reporters have been used to map SHF progenitors within the ALPM, their specific location and dynamic migration remain to be fully determined. Single cell-based transcriptomics promises to reveal new details of SHF and earliest general heart field progenitors, as already approached in first studies [ 84 , 157 , 161 ]. Further, while broadly deemed SHF, to what extent its arterial versus venous pole-forming derivatives share a common progenitor pool in the early LPM is unclear, despite their regulatory differences [ 170 , 172 ].…”

Ex uno, plures–From One Tissue to Many Cells: A Review of Single-Cell Transcriptomics in Cardiovascular Biology

“…Genetic knockouts, lineage tracing, and scRNAseq have been used in combination to define the early stage of cardiovascular lineage segregation from Mesp1 + mesodermal precursors [ 33 ], the role of the Hippo pathway [ 34 ], Pitx2 [ 35 ], and pbx4 [ 36 ] in development, the effect of Hand2os1 lncRNA [ 37 ], and full microRNA KO [ 38 ]. The first study, similarly to what was described earlier [ 39 , 40 ], showed that Mesp1 + cells commit very early to different cardiovascular lineages, and the transcriptomic profiling at a single-cell level allowed for the identification of early pathways, determining the commitment to different cell fates, such as Notch1 for the endocardium.…”

“…A fourth study focused on Pbx4, a transcription factor similarly involved in SHF specification in zebrafish [ 36 ]. Single-cell transcriptomic analysis of Pbx4-depleted SHF progenitors showed a lack of heterogeneity and increased proliferation, which was in line with the cardiac dilation phenotype, and supporting its crucial role in the definition of the anterior and posterior progenitor fields that contribute to OFT and pharyngeal arches, respectively.…”

Cell – ECM interactions play distinct and essential roles at multiple stages during the development of the aortic arch

“…PAAs arise by vasculogenesis from endothelial precursors originating in the lateral plate mesoderm, also known as the second heart field (SHF) [7][8][9][10][11][12][13] . Experiments in zebrafish and mice have demonstrated that PAA formation is a multistage process that entails endothelial specification in the SHF, migration of SHF-derived endothelial progenitors into the pharyngeal region, differentiation into ECs, and the assembly of SHF-derived ECs into a plexus of small blood vessels 9,[13][14][15][16] . Thereafter, the pharyngeal endothelial plexus becomes connected with the ventral and dorsal aortae.…”