XB-ART-53092Dev Biol. April 1, 2017; 424 (1): 28-39.
The CapZ interacting protein Rcsd1 is required for cardiogenesis downstream of Wnt11a in Xenopus laevis.
Wnt proteins are critical for embryonic cardiogenesis and cardiomyogenesis by regulating different intracellular signalling pathways. Whereas canonical Wnt/β-catenin signalling is required for mesoderm induction and proliferation of cardiac progenitor cells, β-catenin independent, non-canonical Wnt signalling regulates cardiac specification and terminal differentiation. Although the diverse cardiac malformations associated with the loss of non-canonical Wnt11 in mice such as outflow tract (OFT) defects, reduced ventricular trabeculation, myofibrillar disorganization and reduced cardiac marker gene expression are well described, the underlying molecular mechanisms are still not completely understood. Here we aimed to further characterize Wnt11 mediated signal transduction during vertebrate cardiogenesis. Using Xenopus as a model system, we show by loss of function and corresponding rescue experiments that the non-canonical Wnt signalling mediator Rcsd1 is required downstream of Wnt11 for ventricular trabeculation, terminal differentiation of cardiomyocytes and cardiac morphogenesis. We here place Rcsd1 downstream of Wnt11 during cardiac development thereby providing a novel mechanism for how non-canonical Wnt signalling regulates vertebrate cardiogenesis.
PubMed ID: 28237811
Article link: Dev Biol.
Genes referenced: actc1 alcam capza1 gapdh isl1 myh6 nkx2-5 pes1 ppan rcsd1 tbx20 tnni3 tnnt2 wnt11 wnt11b
Morpholinos referenced: rcsd1 MO rcsd1 MO2 rcsd1 MO3
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|Fig. 2. : Expression of rcsd1 during embryogenesis. A. Temporal expression of rcsd1 at different stages of Xenopus development. Gapdh was used as loading control. Negative controls (-RT) were performed without reverse transcriptase (RT). Rcsd1 is weakly expressed maternally. Zygotic rcsd1 expression starts at stage 13. B-N. Spatial expression of rcsd1 at different developmental stages as indicated in Xenopus. Rcsd1 expression is first detected in the common cardiac progenitor at stage 20 (black arrowhead). Cardiac expression persists until later stages (black arrowheads). In addition, rcsd1 transcripts were detected in the somites (white arrowheads) and myeloid cells (red arrowheads). KN. Cross section through the cardiogenic region of Xenopus embryos as indicated by the black dashed lines in C, E, G and I, respectively. Expression in cardiac tissue is detected at stage 20 (K, sagittal section), 24 (L, transversal section), 30 (M, transversal section) and 38 (N, transversal section). e, endocardium; m, myocardium; me, mesocardium; p, pericardium; r, pericardial roof. O-Q Expression of Rcsd1 during mouse embryogenesis. Rcsd1 is expressed in the developing murine heart at embryonic stages E7.7 (O), E8.25 (P) and E9.0 (Q).|
|Fig. 5. : Rcsd1 acts downstream of Wnt11a. A. Unilateral injection of Wnt11a MO leads to inhibition of cardiac marker gene expression (red arrowheads), which is restored by the co-injection of Xenopus full-length rcsd1 RNA (black arrowheads). B. Quantitative presentation of data shown in A. C. Unilateral injection of Wnt11a MO leads to inhibition of rcsd1 expression (red arrowheads) D. Quantitative presentation of data shown in C. n, number of independent experiments; N, number of embryos analysed. Error bars indicate standard error of the means (s.e.m.). *, p≤0.05, **; p≤0.01; ***; p≤0.001; ****, p≤0.0001; calculated by a non-parametric Mann-Whitney rank sum test.|
|Supplement Fig. 1. Serial sections through a heart of a Control MO injected embryo derived from OPT image stacks. a, atrium; oft, outflow tract; v, ventricle.|
|Supplement Fig. 2. Serial sections through a heart of a Wnt11 MO injected embryo derived from OPT image stacks. a, atrium; oft, outflow tract; v, ventricle.|
|Supplement Fig. 3. Xenopus laevisRcsd1. The Rcsd1 variant found during early Xenopus embryogenesis is shorter at the C-terminus due to alternative splicing at the boundary between intron 6 and exon 7. Small letters indicate intron sequences, capital letters indicate exon sequences.|
|Supplement Fig. 5. Serial sections through a heart of a Rcsd1 MO injected embryo derived from OPT image stacks. a, atrium; oft, outflow tract; v, ventricle.|
|Supplement Fig. 6. Negative controls for the YFP interaction assay. HEK293 cells were transiently transfected with the different murine Rcsd1 and CapZ constructs used in Fig. 6 together with unrelated genes for negative controls. Neither CapZ (CapZa and CapZb) do interact with Xenopus Pes1 nor the full length or different deletion constructs of Rcsd1 with Xenopus Ppan.|
|Supplement Fig. 7. Rcsd1 gene and protein analyses in various species. A. Phylogenetic tree of the human, mouse, rat, chicken, Xenopus and zebrafish Rcsd1 shows that Rcsd1 is closely related across species. Branch lengths present real lengths. B. Schematic representation of X. laevis Rcsd1 containing a capping protein interaction (CPI) and NLS motif. C. Homology analysis of the amino acid sequences of the full length (overall), full-length and subunits of the CPI domain (highlighted in yellow) and the NLS domain (red) of the Rcsd1 proteins among different species. Numbers represent similarities of the indicated species in percentage compared to Homo sapiens. Amino acid (aa) length is given in numbers. D. Sequence alignment of the CPI and NLS motif of the Rcsd1 protein among different species. Asterisks mark conserved nucleotides.|
|rcsd1 (RCSD domain containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, anterior view, dorsal up.|
|rcsd1 (RCSD domain containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view anterior up.|
|rcsd1 (RCSD domain containing 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior right, dorsal up.|