WITHDRAWN: Complementary functions of the mechanosensitive factorsegr1,klf2bandklf2ainstruct the endocardial program

Abstract

Mechanical forces are key modulators of valvulogenic developmental programs. However, the mechanosensitive gene network underlying this process remains unclear. It is well established that contractile and flow forces activate endocardial expression of the transcription factor klf2a during valve morphogenesis. We report two novel transcription factors with a function in heart valve formation in zebrafish: egr1 and klf2b. Genomewide analysis of gene expression reveals that the endocardial transcriptional programs modulated by klf2a, klf2b, and egr1 mainly contain non-redundant targets. Several of these targets have been implicated in endothelial-to-mesenchymal transition (EMT). VEGF receptor 1 (flt1) is a target of egr1 and klf2b during early valvulogenesis. These findings suggest that klf2a, klf2b, and egr1 cooperate for the activation of EMT program in response to mechanosensitive inputs. We propose that the combinatorial action of these factors mediates flow mechanotransduction to control the endocardial program, especially for valve development. Many of the deregulated genes exhibit changes in chromatin accessibility pointing to potential direct effects of these factors. Finally, in vivo phenotypic analyses show that

Multiple pkd and piezo gene family members are required for atrioventricular valve formation

“…In this context, it would be interesting to assess whether double and triple mutants of the channels described in our study would display calcium pulse phenotypes in addition to the enrichment defect. We also found that the pkd genes cooperate to repress the AV endocardial expression of the transcription factor genes klf2a and klf2b, which are essential for valve formation 7,19,22 . This observation suggests that the expression level of these transcription factors must be tightly regulated and that higher levels of their expression negatively affect AV valve formation.…”

“…Although Klf2a targets have been studied in the myocardium and endocardium during zebrafish valve development 7,[19][20][21][22] , the intermediates of Pkd and Klf2a remained unknown. Here, we identified the camk2g genes as upstream mediators of klf2a expression and downstream effectors of Pkd proteins during AV valve elongation.…”

“…We then analyzed the expression levels of valve-specific transcription factor genes in the heart. We selected klf2a and klf2b, whose loss is associated with AV valve defects, and which control a large set of endocardial and myocardial genes essential for valve formation 7,19,22 . We performed Fluorescence In Situ Hybridization (FISH) and observed apparent endocardial upregulation of klf2a (Fig.…”

“…specifically, membrane-localized mechanosensitive channels, which are in direct contact with the blood flow, control valve elongation. Studies in zebrafish have shown that these mechanosensors, Pkd2 and Trpv4 in all cardiac valves and Piezo1 and Piezo2a in the OFT valves, tightly regulate klf2a expression 10,11,18 , and that they subsequently control the endocardial and myocardial expression of other genes important for valvulogenesis 7,[19][20][21][22] . In addition, it was also recently reported that the adenosine triphosphate (ATP)-dependent purinergic receptor pathway works in parallel to these channels as a blood flow mechanosensor to mediate AV valve formation 12 .…”