Alternative splicing of jnk1a in zebrafish determines first heart field ventricular cardiomyocyte numbers through modulation of hand2 expression

Abstract

The planar cell polarity pathway is required for heart development and whilst the functions of most pathway members are known, the roles of the jnk genes in cardiac morphogenesis remain unknown as mouse mutants exhibit functional redundancy, with early embryonic lethality of compound mutants. In this study zebrafish were used to overcome early embryonic lethality in mouse models and establish the requirement for Jnk in heart development. Whole mount in-situ hybridisation and RT-PCR demonstrated that evolutionarily conserved alternative spliced jnk1a and jnk1b transcripts were expressed in the early developing heart. Maternal zygotic null mutant zebrafish lines for jnk1a and jnk1b, generated using CRISPR-Cas9, revealed a requirement for jnk1a in formation of the proximal, first heart field (FHF)derived portion of the cardiac ventricular chamber. Rescue of the jnk1a mutant cardiac phenotype was only possible by injection of the jnk1a EX7 Lg alternatively spliced transcript. Analysis of mutants indicated that there was a reduction in the size of the hand2 expression field in jnk1a mutants which led to a specific reduction in FHF ventricular cardiomyocytes within the anterior lateral plate mesoderm. Moreover, the jnk1a mutant ventricular defect could be rescued by injection of hand2 mRNA. This study reveals a novel and critical requirement for Jnk1 in heart development and highlights the importance of alternative splicing in vertebrate cardiac morphogenesis. Genetic pathways functioning through jnk1 may be important in human heart malformations with left ventricular hypoplasia.

Ventricular, atrial, and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak

“…Our analysis will also help identify the initial mesodermal population that when dysregulated leads to specific malformations in the heart. For example, left ventricle hypoplasia results from a reduction in the number of specified cardiomyocytes within the mesoderm, and this is due to a reduction in the expression of key cardiac transcription factors [ 82 ]. Whether such cardiac defects can arise from mutations affecting initial primitive streak cell populations and mesoderm remains to be addressed.…”

Ventricular, atrial and outflow tract heart progenitors arise from spatially and molecularly distinct regions of the primitive streak

“…Our analysis will also help to identify the initial mesodermal population that when dysregulated lead to specific malformations in the heart. For example, left ventricle hypoplasia results from a reduction in the number of specified cardiomyocytes within the mesoderm and this is due to a reduction in the expression of key cardiac transcription factors (Santos-Ledo et al, 2020). Whether such cardiac defects can arise from mutations affecting initial primitive streak cell populations and mesoderm remains to be addressed.…”

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

“…At the molecular level, how the different SHF regulators interact transcriptionally with each other remains to be charted. Beyond Islet factors and Nkx2.5/7, examples include how the AP-1 factor Fosl2 connects to other SHF-regulating activities to promote the addition of these cells to the heart tube [ 217 ], or how jnk1a splice forms influence the ventricular myocardium upstream of hand2 [ 218 ]. Notably, IFT formation from SHF progenitors has received considerably less attention than the development of the distal ventricle and OFT portion of the zebrafish heart, in part due to its at times disparate definitions and nomenclature [ 67 , 72 , 98 , 219 ].…”