Fibronectin and integrin alpha 5 play requisite roles in cardiac morphogenesis

Abstract

Fibronectin and its major receptor, integrin α5β1 are required for embryogenesis. These mutants have similar phenotypes, although, defects in integrin α5-deficient mice are milder. In this paper, we examined heart development in those mutants, in which the heart is formed, and discovered that both fibronectin and integrin α5 were required for cardiac morphogenesis, and in particular, for the formation of the cardiac outflow tract. We found that Isl1+ precursors are specified and migrate into the heart in fibronectin- or integrin α5-mutant embryos, however, the hearts in these mutants are of aberrant shape, and the cardiac outflow tracts are short and malformed. We show that these defects are likely due to the requirement for cell adhesion to fibronectin for proliferation of myocardial progenitors and for Fgf8 signaling in the pharyngeal region.

Keywords: Cardiac; Fibronectin; Heart; Integrin a5; Morphogenesis; Outflow tract.

Figure 1. FN and integrin α5 are required for cardiac morphogenesis

Scanning electron micrographs of control (A), FN-null (B) and integrin α5-null (C) embryos isolated at E9.5. All views are ventral. LV-left ventricle, RV-right ventricle, OFT – outflow tract. In B and C, arrow points toward the connection of the heart with the systemic circulation. Note dysmorphic and short RV and OFT in the mutants. Magnification is the same in all panels.

Figure 2. FN is not required for cardiac chamber specification

A–D. Expression of ANF mRNA in control (A, C) and FN-null (B, D) hearts. ANF is expressed in the atria, the left ventricle and to a limited extent, in the right ventricle. The expression patterns of ANF in control and FN-null embryos are comparable. Long arrow in A points to the proximal OFT, and short arrow – to the distal. E. Cited 1 mRNA is confined to the left ventricle in control embryos. Inset in E shows the right side view. F. Expression of Cited 1 mRNA in FN-null heart is limited to the lower region of the straight heart tube, the presumptive left ventricle. E9.5 embryos are shown. A- atria, other abbreviations are as in Fig. 1. Scale bars in all figures are 100 μm unless otherwise stated.

Figure 3. The outflow tract and the right ventricle are specified in the absence of FN

BMP4 mRNA is expressed in the outflow tract of control embryo, blue dotted line (A). In FN-null, BMP4 expression is limited to the short OFT connecting the LV to the embryo, blue dotted line. C–D. Expression of Wnt11 mRNA. Wnt11 is expressed in the OFT of control embryo, arrows (C) and in the short OFT of FN-null. All embryos were collected at E9.5.

Figure 4. FN is required for the morphogenesis of the cardiac outflow tract and the right ventricle

Fate mapping of Isl1⁺ cells and their descendants in control (A, C, E) and FN-null (B, D, F) embryos isolated at E9.5. Note the presence of blue cells (β-gal+) in the outflow tracts of the mutants. Dotted line is the plane of sections shown in E–F. Regions outlined by brackets in E–F show H&E stained sections of thickened myocardial wall in FN-null embryos, they are expanded in Fig. 6.

Figure 5. FN is required for proliferation of cells in splanchnic mesoderm

A–B. E8.75 control (A) and mutant (B) embryos were stained with anti-pHH3 antibody (green) and DAPI (blue). Representative sections are shown. Splanchnic mesoderm is underlined by the dotted lines and shown enlarged in A′–B′. C. Quantification of the fraction of pHH3+ labeled cells in splanchnic mesoderm. 4–5 sections from each embryo were analyzed, total of 413 control and 537 mutant nuclei were counted per genotype.

Figure 6. FN is required for normal ventricular morphogenesis

A–B. Expression of myosin heavy chain is visualized using MF20 antibody. Dotted lines show planes of sections in A′, B′ and B″ below. Blue nuclei show expression of Isl1 protein. Note thickened myocardial wall in the hearts of FN-null embryos (B′, B″, D) compared with controls (A′, C,). E. Quantification of myocardial thickness in serial sections from control and mutant embryos. Ventral ventricular walls in 4–6 sections of serialy-sectioned embryos were measured. In this figure, H&E panels C and D are magnified views of regions indicated by the brackets in Fig. 4E–F.

Figure 7. Integrin α5 is not required for specification of cardiac chambers

Control embryos-A, C, E, G, I; integrin α5-nulls – B, D, F, H, J. A–F. In situ hybridization. G–J. Fate mapping of Isl1⁺ cells. Abbreviations are as in Fig. 1. Scale bars are 100 μm.

Figure 8. Fibronectin and integrin α5 regulate Fgf8 signaling in vivo and in vitro

mRNA expression of downstream signaling reporters of Fgf8 signaling in E8.75 embryos. A–B. Control (top) and FN-null mutants (bottom). C–D. Control (top) and integrin α5-null mutants (bottom). E. Cell adhesion to FN but not to poly-d-Lys potentiates Fgf8 activation of Erk (double arrows). F. Cell adhesion to FN mediates Fgf8 signaling in a dose-dependent manner and does not lead to increases in Erk protein levels (bottom gel). Activation of Erk in response to cell adhesion to FN and increased doses of Fgf8 is quantified in G.