Inactivation of the Sema5a gene results in embryonic lethality and defective remodeling of the cranial vascular system

Abstract

The semaphorins are a large family of proteins involved in the patterning of both the vascular and the nervous systems. In order to analyze the function of the membrane-bound semaphorin 5A (Sema5A), we generated mice homozygous for a null mutation in the Sema5a gene. Homozygous null mutants die between embryonic development days 11.5 (E11.5) and E12.5, indicating an essential role of Sema5A during embryonic development. Mutant embryos did not show any morphological defects that could account for the lethality of the mutation. A detailed analysis of the vascular system uncovered a role of Sema5A in the remodeling of the cranial blood vessels. In Sema5A null mutants, the complexity of the hierarchically organized branches of the cranial cardinal veins was decreased. Our results represent the first genetic analysis of the function of a class 5 semaphorin during embryonic development and identify a role of Sema5A in the regional patterning of the vasculature.

FIG. 1.

Generation of Sema5a knockout mice. (A) Exons 4 and 5 of the Sema5a locus were replaced by the PGK-neomycin resistance cassette (PGKneo; gray box). Schematic representations of the wild-type allele (top), the targeting vector (middle), and the mutant allele (bottom) are shown. The probes (5′ probe, neo probe) used for screening ES cells are indicated by black bars. Arrowheads represent the primers used to genotype Sema5a mutant mice. Arrows indicate the restriction fragments detected by the 5′ probe after digestion with SacI. Essential restriction enzyme sites are indicated by vertical bars. (B) Genomic DNA was isolated from the ES cell clone used to generate mutant mice, digested with SacI, and analyzed by Southern blotting with the 5′ probe. The wild-type (wt) and targeted alleles are represented by restriction fragments of 2.6 and 9.2 kb, respectively. (C) Western blot analysis of membrane fractions prepared from wild-type, heterozygous, and homozygous mutant embryos. No Sema5A protein was detected in extracts from homozygous mice. Detection of β-actin showed that comparable amounts of protein were loaded in each lane. K, KpnI; N, NotI; X, XhoI; S, SacI.

FIG. 2.

Development of the nervous system in Sema5a null mutants. Wild-type and Sema5a^−/− E10.5 (A and B) and E11.5 (C to F) embryos were stained with an antineurofilament antibody. In wild-type (A and C) and homozygous mutant (B and D) embryos, the ophthalmic (op), maxillary (mx), facial (f), and mandibular (md) nerves extended normally and were tightly fasiculated. At E11.5, motor nerve bundles (mn) began to innervate the limb buds (E and F) normally in both wild-type and homozygous mutant embryos. a+v, accessory and vagal nerve.

FIG. 3.

Development of extraembryonic tissues in Sema5a null mutants. Bright-field images of freshly dissected yolk sacs from E11 wild-type (A) and mutant (B) embryos showed pervasive vascularization by highly branched vessels in both cases. Hematoxylin-eosin-stained sections of E11 placentas from wild-type (C) and mutant (D) embryos showed a normal organization of the spongiotrophoblast (sp) and labyrinthine layers (ll) and the chorionic plate (cc).

FIG. 4.

Expression of Sema5a in the embryonic heart. Twelve-micrometer paraffin sections of E10.5 wild-type embryos were hybridized with digoxigenin-labeled RNA probes specific for Sema5a (A). A higher magnification of the areas marked by boxes is shown in panels B and C. The highest level of Sema5a mRNA was detected in the atrial septum (as) and endocardial (ec) cushion (A and B). Sema5a was also expressed in the atrial and ventricular endocardium, as indicated by arrows (C).

FIG. 5.

Cardiac development in Sema5a mutants. Twelve-micrometer paraffin sections of E10.5 wild-type (A, C, E, G, I, and M) and mutant (B, D, F, H, L, and N) embryos were stained with hematoxylin and eosin (H/E) (A and B) to analyze the morphology of the outflow tract or hybridized with digoxigenin-labeled RNA probes specific for Bmp4 (C and D), Bmp10 (E and F), Nkx2.5 (G and H), Cx40 (I and L), and Anf (M and N) as markers for the differentiation and growth of endocardial cushions (C and D) and ventricles and atria (E to N). The morphology of the outflow tract and the expression pattern of all markers were similar in mutant and wild-type embryos.

FIG. 6.

Sema5a null mutants have a largely normal vascular system. The vascular system of E10.5 (A and B) and E11.5 (C and D) wild-type (A and C) and homozygous Sema5a mutant (B and D) embryos was analyzed by whole-mount immunohistochemistry with an anti-PECAM antibody. No obvious morphological abnormalities could be detected in the vascular system.

FIG. 7.

Branching of large vessels is impaired in the cranial region of Sema5a null mutants. The cranial vasculature of E10.5 (A to H) and E11.5 (I to R) wild-type (A to D and I to M) and homozygous Sema5a mutant (E to H and O to R) embryos was analyzed by whole-mount immunohistochemistry with an anti-PECAM antibody. Panels B, C, F, G, L, M, P, and Q show a higher magnification of the marked areas. While the capillary network in the peripheral regions of the head (arrowheads in B, F, L, and P) was not affected in mutant embryos, the branching pattern of the large vessels (B, F, L, and P) was less complex in Sema5a^−/− embryos (D, H, N, and R). The branching pattern of the large vessels is outlined in panels C, D, G, H, M, N, Q, and R. Sema5a mutants showed a decrease in the number of secondary (green lines) and tertiary (blue lines) branches. The cardinal veins and the primary branches are represented by black and red lines, respectively.

FIG. 8.

Sema5A is expressed in the mesoderm surrounding the cranial vessels, and their morphology is normal in the null mutants. Cryosections (A and B) and paraffin sections (C, D, E and F) of E10.5 wild-type (A, C, and F) and homozygous Sema5a mutant (B, D and E) embryos were stained with an anti-PECAM antibody (red in A and B) to visualize vascular endothelial cells and with a digoxigenin-labeled probes specific for Cx40 (C and D) and Sema5a (E and F) mRNAs by in situ hybridization. No differences were detectable between the PECAM and Cx40 stainings in wild-type and homozygous mutant embryos. Strong expression of Sema5a was detected in the mesoderm surrounding the vessels in wild-type embryos (E), while no signal was detectable in homozygous mutants (F). The nuclei (blue in panels A and B) were stained with Hoechst-33258. Arrowheads indicate the erythrocytes in the vessels; n, neuroepithelium.