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PlexinA2 and semaphorin signaling during cardiac neural crest development

¹ Cardiovascular Division, Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
² Department of Neurobiology, University of Pennsylvania, Philadelphia, PA 19104, USA
^* Author for correspondence (e-mail: epsteinj{at}mail.med.upenn.edu )

View larger version (142K): [in a new window]   Fig. 1. Expression of potential Sema3C receptor components in mouse embryos at E12.5. Radioactive in situ hybridization analysis of transverse section through the neural tube (nt, panels A,D,G,J,M), aortic arches (B,E,H,K,N) and cardiac outflow tract (oft, panels C,F,I,L,O) is shown. The aortic arch arteries are numbered 4 and 6. (A-C) PlexinA2 is expressed at high levels in the roof plate of the neural tube (arrow, A) and throughout the ventral neural tube excluding the ventral horns. Expression is also observed in the dorsal root ganglia (drg, A) and adjacent to the pulmonary trunk (PT) and aortic arch arteries (B). (C) A column of expressing cells is seen extending into the outflow tract (oft) endocardial cushions consistent with expression by migrating cardiac neural crest (arrow). (D-F) PlexinA1 is expressed diffusely at levels barely above background. (G-I) PlexinA3 is expressed in the medial region of the neural tube and dorsal root ganglia (G) and broadly throughout the pharyngeal mesenchyme (H) and the mesenchyme surrounding the aorta (Ao) and pulmonary trunk (I). (J-L) Nrp1 is expressed at high levels in the motor columns (mc) of the neural tube and in the dorsal root ganglia (drgs) (J). Nrp1 expression overlaps PlexinA2 expression in the aortic arch mesenchyme (K) and adjacent to the aorta and pulmonary artery (L), but is also seen in the endothelial cells (e) of developing vessels (K). (M-O) Nrp2 expression is observed in the roof plate (arrow, M) and in the neural tube and drgs. Nrp2 is expressed by the mesenchyme surrounding the aorta (N), and extending into the outflow tract (arrow, O).

View larger version (72K): [in a new window]   Fig. 2. PlexinA2 (E11.5) and Sema3C (E12) expression in the cardiac outflow tract. Whole-mount in situ hybridization was performed to examine PlexinA2 (A) and Sema3C (B) expression. PlexinA2 can be seen in two prongs of cells extending into the septating outflow tract (A, black arrows) rostral to the right ventricle (RV). Sema3C is expressed in the myocardium of the outflow tract (white arrow, B).

View larger version (95K): [in a new window]   Fig. 3. Comparison of PlexinA2 expression to fate-mapping and transgenic markers of cardiac neural crest at E12.5. PlexinA2 expression was analyzed by whole-mount (A) or section in situ hybridization (E,I). For comparison, cardiac neural crest cells were fate mapped using the Pax3 promoter to drive expression of Cre recombinase and crossing with Cre-reporter mice (B,F,J) or using the Wnt1 promoter to direct Cre expression (C,G,K). Cardiac neural crest was also labeled with the Cx43-lacZ transgene (D,H,L). PlexinA2 is expressed by two prongs of cells invading the cardiac outflow tract (large arrow, A) in a pattern that resembles that of cardiac neural crest (large arrows, B-D). In frontal sections (E-H), PlexinA2 and cardiac neural crest are located surrounding the aorta (Ao) and pulmonary trunk (PT) and form a `saddle' within the outflow endocardial cushions (arrows, E-H). In cross section, PlexinA2 expression is localized to two clusters of cells within the endocardial cushions (arrows, I). Similar populations of cells are observed within the conotruncal cushions of Pax3promoter-Cre (J), Wnt1-cre (K), and Cx43-lacZ (L) hearts (black arrows). The extent of labeling within the conotruncal mesenchyme is variable in the different mouse lines; Pax3promoter-Cre expression (J) is most similar to PlexinA2 (I). Note that variable numbers of labeled cells are observed within the ventricles of all of these mouse lines (A-D small arrows).

View larger version (103K): [in a new window]   Fig. 4. PlexinA2 expression during cardiac neural crest migration. PlexinA2 can be detected by radioactive in situ hybridization in the neural tube as early as day E8.5 (A, white arrows) before neural tube closure. High levels of PlexinA2 expression are noted in the roof plate of the neural tube at E9.5 (arrow, B) and at E10.5 (arrow, C). Extensive expression is observed throughout the pharyngeal arches (arrows D), and surrounding the aortic arch arteries at E10.5. A large population of PlexinA2-expressing cells is seen extending into the conotruncus (arrow, E). At E11.5, PlexinA2-expressing cells can be seen surrounding the aorta (Ao) and pulmonary trunk (PT), and extending into the outflow tract mesenchyme (arrow, F). PlexinA2 expression in the left ventricle (LV) is first seen at E11.5 (F). d, dorsal aorta. S, aortic sac. cv, cardinal vein. 3, aortic arch artery 3.

View larger version (108K): [in a new window]   Fig. 5. Complementary and overlapping expression domains of PlexinA2 and Sema3C. PlexinA2 and Sema3C expression patterns were determined by radioactive in situ hybridization (A-H) in E12.5 embryos, and by double label non-radioactive in situ hybridization at E11.5 (I, J). In the neural tube at E12.5, PlexinA2 (A) and Sema3C (G) are both expressed in the roof plate (arrows, A,G) and the dorsal root ganglia (drg, in A,G). In the outflow region of the heart, both genes are expressed around the aorta (Ao, in A,G). Both PlexinA2 (B) and Sema3C (H) are expressed in an overlapping population of cells in the condensed mesenchyme of the outflow tract where neural crest cells reside (B,H white arrows). However, complementary expression is observed elsewhere. PlexinA2 is expressed in the ductus arteriosus (DA, in A) while Sema3C is expressed in the myocardial cuff of the right ventricular outflow tract (oft, in G) and in the proximal pulmonary trunk. In the neural tube (nt), PlexinA2 expression excludes the ventral horns (A), where Sema3C is expressed (small arrows, G). Reciprocal expression domains are observed in the intestines (arrows, C (PlexinA2), D (Sema3C)) and in the lung (arrows, E (PlexinA2), F (Sema3C)). Double lable in situ hybridization of E11.5 embryos (I,J) reveals overlapping but not identical expression of Sema3C (red), and PlexinA2 (purple) in the aortic arch mesenchyme (white arrow, I) and outflow tract (boxed region, I). Sema3C is expressed in the inner curvature of the myocardial wall of the outflow tract (I, black arrow). A higher magnification (J) of the outflow tract (boxed region in I) demonstrates cells that express both Sema3C and PlexinA2 (black arrow), as well as cells that only express Sema3C (white arrow). LV, left ventricle.

View larger version (80K): [in a new window]   Fig. 6. Altered PlexinA2 expression in Sema3C null embryos. PlexinA2 expression was compared by whole-mount (A,B) in situ hybridization at E12.5. Prongs of PlexinA2-expressing cells observed in wild-type hearts (arrow, A) are reduced in Sema3C null embryos (arrow, B). PlexinA2 expression surrounding the aorta and pulmonary trunk is decreased in Sema3C null embryos compared to control.

View larger version (90K): [in a new window]   Fig. 7. Expression of PlexinA2 in Splotch embryos. Wholemount in situ hybridization (A,C,D,F) and radioactive in situ hybridization (B,E) was performed with E12.5 wild-type (A-C) and Splotch (D-F) littermates. PlexinA2 expression is noted in the outflow tract of both wild-type (arrow, A) and Splotch (arrow, D) embryos. However, analysis of transverse frontal sections reveals strong expression by a cluster of cells in the condensed mesenchyme of the conotruncal endocardial cushions in wild-type embryos (arrow, B) that is not seen in Splotch embryos (arrow, E). PlexinA2 is normally expressed only weakly in the left ventricular (LV) myocardium at this stage (arrowheads, A,B). In Splotch embryo, PlexinA2 expression is significantly up-regulated in the left ventricle as seen with both whole-mount (arrowhead, D) and radioactive (arrowhead, E) techniques. PlexinA2 is also expressed in hypaxial muscles of wild-type embryos (C, asterisks), while this domain of expression is absent in Splotch embryos (F, asterisks), consistent with the established lack of limb musculature in Splotch embryos.

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