Sema3D, Sema3F, and Sema5A are expressed in overlapping and distinct patterns in chick embryonic heart

Abstract

An increasing number of axon guidance cues have been shown recently to play important roles in the development of non-neural tissues. Semaphorins comprise one of the largest conserved families of axon guidance factors. We analyzed the expression patterns of Sema3D, Sema3F, and Sema5A genes in the chick embryonic heart by in situ hybridization. All three genes are expressed in the cardiac cushion regions, both in the mesenchymal cells, and epithelial cells in the endocardial layer, during the period of cardiac remodeling. In addition to the overlapping expression patterns in the cardiac cushion regions, these genes also exhibit distinct expression patterns in the developing heart: Sema3D is additionally expressed in the tips of the ventricular trabeculae; Sema3F is expressed in a subset of cells scattered throughout the ventricles; and Sema5A is expressed in the newly formed atrioventricular valves. The overlapping and distinct expression patterns of these genes suggest that they may play important roles in heart development.

Fig. 1

Expression of Sema3D transcripts in embryonic chick heart at embryonic day (E) 6. In situ hybridization experiments were carried out on cryosections of chick heart at E6. Results from the sections of two different hearts are shown. A: Expression of Sema3D was found at the atrioventricular junction (avj) and the tip of the forming interventricular septum (ivs). Higher magnification images of the avj and ivs areas are shown in B and D, and C and E, respectively. B,D: At the atrioventricular junction regions, expression of Sema3D was found both in the epithelial cells in the endocardial layer (arrows) and in the mesenchymal cells inside the cardiac jelly (arrowheads). C,E: In the interventricular septum regions, Sema3D expression was detected at the tip of the interventricular septum, both in the endocardial (arrows) and myocardial layers (arrowheads). F: A Sema3D sense probe was used for the control experiments. Note that no signal was detected with the sense probe. Scale bars = 250 μm in A, 50 μm in F (applies to B–F).

Fig. 2

A–C,E–G: Expression of Sema3D transcripts in embryonic chick hearts at embryonic day (E) 9 (A–C) and E12 (E–G). In situ hybridizations were carried out on cryosections of chick hearts. Higher magnification images of the boxed areas in A are shown in B and C. Note that the cells at the tip and peripheral margins of the interventricular septum (ivs) and the leading edges of some trabeculae in the ventricles are positive for the expression of Sema3D transcripts (arrows). D: E9 heart sections hybridized with a control Sema3D sense probe did not show any signal. Similar patterns at the peripheral margins of the interventricular septum were found at E12 (E–G). However, most trabeculae and the tip of the interventricular septum are negative for Sema3D expression at this stage. Higher magnification image of the boxed area in E is shown in F. G: The ivs area of another heart sample hybridized with the antisense Sema3D probe is shown. H: E12 heart section hybridized with a control Sema3D sense probe did not show any signal. a, atrium; v, ventricle; ivs, interventricular septum. Scale bars = 500 μm in A,E, 50 μm in H (applies to B–D,F–H).

Fig. 3

A–F: Expression of Sema3F transcripts in the embryonic chick heart at embryonic day (E) 6 (A,B), E9 (C,D), and E12 (E,F). In situ hybridizations were performed on cryosections of the hearts. A: At E6, Sema3F expression was detected in the cardiac cushion (marked with an asterisk) at the atrioventricular junction (avj) and at the leading edge of the growing interventricular septum (ivs). B: The boxed area in A is shown at higher magnification. Note both the epithelial (arrowheads) and mesenchymal cells (arrow) in the cardiac cushion (cc) express Sema3F transcripts. C: At E9, Sema3F is similarly expressed at the atrioventricular junction and at the tip of the interventricular septum. D: A higher magnification image of the boxed area in C; cells positive for Sema3F are visible throughout the heart, scattered inside the myocardium (arrows in D), but are more concentrated in the area outside of the trabeculae. E,F: At E12, scattered Sema3F-positive cells (arrows) are still present inside the heart, now more in the trabeculae than in the compact zone (arrows). Scale bars = 250 μm in A,C, 50 μm in F (applies to B,D–F).

Fig. 4

Expression of Sema5A transcripts in embryonic chick heart at embryonic day (E) 6 (A,B), E9 (C,D), and E12 (E,F). A,B: At E6, Sema5A expression was only detected in the cardiac cushion region (cc), shown at lower (A) and higher (B) magnifications. Note that the expression of Sema5A was detected both in the epithelial (arrow) and mesenchymal cells (arrowhead) in the cardiac cushion. C,D: At E9, Sema5A transcripts are expressed in the conotruncus cardiac cushion (ccc), the forming tricuspid valve (tcv), and mitral valve (mv). E,F: Similar expression patterns were observed in the E12 heart. rv, right ventricle; la, left atrium; lv, left ventricle. Scale bar = 250 μm in A, 50 μm in B (applies to C–F).