Cadm4 Restricts the Production of Cardiac Outflow Tract Progenitor Cells

Abstract

SUMMARY Heart assembly requires input from two populations of progenitor cells – the first and second heart fields – that differentiate at distinct times and create different cardiac components. The cardiac outflow tract (OFT) is built through recruitment of late-differentiating, second heart field (SHF)-derived cardiomyocytes to the arterial pole of the heart. Mechanisms responsible for selection of an appropriate number of OFT cells from the SHF remain unclear. Here, we find that cell adhesion molecule 4 (cadm4) is essential for restricting the size of the zebrafish OFT. Knockdown of cadm4 causes dramatic OFT expansion, and overexpression of cadm4 results in a greatly diminished OFT. Moreover, cadm4 activity limits the production of OFT progenitor cells and the duration of their accumulation at the arterial pole. Together, our data are the first to suggest a role for cell adhesion in restraining SHF deployment to the OFT, perturbation of which could cause congenital OFT defects.

FGF signaling enforces cardiac chamber identity in the developing ventricle

“…Numerous studies have focused on the signaling pathways that regulate the production, proliferation or differentiation of SHF-derived progenitor cells (e.g. de Pater et al, 2009;Dyer and Kirby, 2009;Ilagan et al, 2006;Lazic and Scott, 2011;Park et al, 2006;Tirosh-Finkel et al, 2010;Zeng and Yelon, 2014;Zhou et al, 2011), but the signals that confer the appropriate chamber-specific characteristics to these late-differentiating cells have not been characterized. Strikingly, we find that reduction of FGF signaling causes late-differentiating cells to initiate amhc expression inappropriately, suggesting either misassignment to an atrial identity or failure to maintain the ventricular differentiation program.…”

“…For early-differentiating cells, FGF signaling is required to maintain the appropriate number of cardiomyocytes in the ventricle, as well as to preserve the ventricular characteristics of these cells. For late-differentiating cells, FGF signaling is required to promote their accretion to the arterial pole (de Pater et al, 2009;Lazic and Scott, 2011;Marques et al, 2008;Zeng and Yelon, 2014), as well as to regulate their presentation of appropriate ventricular traits. Thus, sustained FGF signaling suppresses myocardial plasticity and preserves the integrity of the ventricular chamber.…”

“…Additionally, inhibiting FGF signaling during gastrulation in zebrafish reduces the number of cells in both cardiac chambers, with a stronger impact on the ventricle (Marques et al, 2008). This early influence of FGF signaling is echoed at later stages, when FGF signaling promotes the accretion of late-differentiating cells to the arterial pole of the ventricle (de Pater et al, 2009;Lazic and Scott, 2011;Marques et al, 2008;Zeng and Yelon, 2014). Similarly, in mouse, a hypomorphic allele of Fgf8 or tissue-specific deletion of Fgf8 results in formation of a hypoplastic right ventricle and outflow tract (Abu-Issa et al, 2002;Ilagan et al, 2006;Park et al, 2006).…”

“…To begin investigating whether FGF signaling influences ventricular characteristics in latedifferentiating cells, we first examined whether SHF progenitor cells were present in embryos subjected to our experimental conditions. Prior studies have shown that strong inhibition of FGF signaling with a high dose of SU5402 (10 µM) beginning at 24 hpf can nearly eliminate the formation of mef2cb-expressing SHF progenitor cells (Lazic and Scott, 2011;Zeng and Yelon, 2014). However, our strategies for reduction of FGF signaling at 18 hpf, either by heat shock of Tg(hsp70:dnfgfr1) embryos or by exposure (E-L) SU5402-treated embryos exhibit Dendra + cells in the ventricle, in distinct clusters at the arterial pole and in the inner curvature (arrowheads).…”

“…Previously described protocols were also used for in situ hybridization (Thomas et al, 2008) and fluorescence in situ hybridization in combination with immunofluorescence (Zeng and Yelon, 2014), using established probes for amhc (myh6; ZDB-GENE-031112-1), vmhc (ZDB-GENE-991123-5), myl7 (ZDB-GENE-991019-3), nkx2.5 (ZDB-GENE-980526-321), nkx2.7 (ZDB-GENE-990415-179) and mef2cb (ZDB-GENE-04901-7). All data represent at least two technical replicates of each experiment.…”