Kcp is essential for heart development and survival in X. laevis.
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(A) PRM quantification of peptides specific for Kcp confirms that Kcp is enriched in Xenopus. (B) Human and Xenopus show chromosomal synteny as revealed by Metazome. KCP is indicated in black. Upstream and downstream genes are colored as indicated. (C) Schematic of Kcp showing relative position of TALEN-induced mutations and bottom predicted protein generated by TALEN mutagenesis. (D) Stiol images of ultrasound Doppler of living wild type and KcpΔexon2/ Δexon2 at Stage 64 positioned with dorsal top, ventral bottom of image. Blood flow shown in red. (E) Contrast-enhanced CT imaging of wild-type and KcpΔexon2/ Δexon2 hearts (Stage 64) with AVV highlighted in white, bottom panels show lateral and posterior views showing thicker and misshapen AVV in KcpΔexon2/ Δexon2 hearts verses control. (F) The AVV are detached from the muscular walls in Kcp mutants. Transverse sections through Masson trichrome stained Stage 64 control hearts (heterozygous) at low (upper left panel) and high (upper right panel) magnification focused on the AVV. Similar sections through a Kcp null froglet in at low (lower left panel) and high (lower right panel) magnification. Note in lower left panel an enlarged AVV in the outer valve leaflet detaching from the muscle wall. (G) Histology of wild-type and KcpΔexon2/ Δexon2 hearts with Alcian Blue staining shows massive accumulation of collagen (blue) in AVV. a* Denotes position of AVV. (H) AVVs in kcp null hearts show a decrease in Filamin A and a concomitant increase in Fibrilin in the outer valve leaflet. (1–8) Immunochemistry of Cardiac-Actin:GFP (marking cardiomyocytes with GFP) froglets stained for Filamin (red), and DAPI (blue) in (1 and 2) heterozygous controls and (3 and 4) Kcp null. (5 and 6) Immunochemistry of Cardiac-Actin:GFP (marking cardiomyocytes with GFP) froglets stained for Fibrillin (red) and DAPI (blue) in (5 and 6) heterozygous controls and (7 and 8) Kcp null. See S16 Table for numerical data underlying figure. a, atria; avv, atrioventricular valve; GFP, green fluorescent protein; Kcp, Kielin/chordin-like protein; PRM, parallel reaction monitoring; TALEN, transcription activator-like effector nuclease; v, ventricle.