Ephrin-B2 forward signaling regulates somite patterning and neural crest cell development

Abstract

Genetic studies in the mouse have implicated ephrin-B2 (encoded by the gene Efnb2) in blood vessel formation, cardiac development and remodeling of the lymphatic vasculature. Here we report that loss of ephrin-B2 leads to defects in populations of cranial and trunk neural crest cells (NCC) and to defective somite development. In addition, we show that Efnb1/Efnb2 double heterozygous embryos exhibit phenotypes in a number of NCC derivatives. Expression of one copy of a mutant version of Efnb2 that lacks tyrosine phosphorylation sites was sufficient to rescue the embryonic phenotypes associated with loss of Efnb2. Our results uncover an important role for ephrin-B2 in NCC and somites during embryogenesis and suggest that ephrin-B2 exerts many of its embryonic function via activation of forward signaling.

Figure 1. Generation of an Efnb2 null mouse line

A. Efnb2 wild type locus (a), targeting vector (b) and targeted locus (c). B. Left: Southern blot analysis of ES clones showing a wild type clone (+/+) and a targeted clone (+/GFP) that was used for blastocysts injection. The probe is indicated in grey in Aa. Right: RT-PCR analysis on RNA isolated from E10.5 embryos of various genotypes, as indicated. Efnb2 expression is absent in Efnb2^GFP/GFP embryos. Expression of βActin is used as a control. C. Epifluorescence images of E10.5 embryos showing expression of H2B-GFP in Efnb2^+/GFP embryos (a) and Efnb2^GFP/GFP embryos (b). D. Epifluorescence images of Efnb2^+/GFP embryos at E9.5 (a, d, e), E12.5 (b) and E14.5 (c). Expression of H2B-GFP in endothelial cells (d) epithelial lining of branchial arches (e).

Figure 2. Neural crest cell defects in Efnb2 null embryos

A. Efnb2^+/GFP embryos (a, c, e) and Efnb2^GFP/GFP embryos (b, d, f) were stained with the following markers: Sox10 (a, b), Wnt1Cre/R26R (c, d) and neurofilament (e, f). A decrease in the cranial NCC population migrating to the first (arrow) and second (arrowhead) BA is visible and all markers show a reduced size of the fifth cranial ganglion (asterisk) in Efnb2 null embryos. B. Efnb2^+/GFP embryos (a, c) and Efnb2^GFP/GFP embryos (b, d) were stained with the following markers: Sox10 (a, b) and Wnt1Cre/R26R (c, d). Scattered migration of trunk NCC can be seen in Efnb2 homozygous null embryos, as well as loss of segmented migration (brackets). Histological sections of X-gal stained control (e) and Efnb2 mutant (f) embryos show that NCC invade the posterior (P) half of somites in mutant embryos. In control embryos, NCC migration is restricted to the anterior (A) half of somites. The black lines mark somite boundaries based on the position of the dermomyotome.

Figure 3. Somite defects in Efnb2 null embryos

A. Epifluorescence images of Efnb2^+/GFP embryos (a) and Efnb2^GFP/GFP embryos (b) showing that the somitic distribution of GFP-positive cells of Efnb2 null embryos is disrupted. B. Dorsal view of sections of Efnb2^+/GFP (a–c) and Efnb2^GFP/GFP embryos (d–f). Actin staining (a, d) and Efnb2 expression (b, e) show that cells expressing higher levels of GFP are abnormally clustered at the medial edge of somites in mutant embryos. The mesoderm (m) visible laterally in the pictures of Efnb2 null embryo (d–f) is due to an incomplete closure of the body wall. m: mesoderm; nt: neural tube; s: somite. C. Expression of Uncx4.1, Meox1 and Sema3F was detected by in situ hybridization on control embryos (a, c, e) and Efnb2^GFP/GFP embryos (b, d, f). Expression of uncx4.1 is decreased and diffuse, while polarized expression of Meox1 and Sema3F is lost in mutant embryos. So: Somite numbers indicated for both control (highest number) and mutant (lowest number) embryos. One number indicates that both control and mutant embryos had the same number of somites. Anterior (A) is to the right and posterior (P) is to the left in all pictures shown.

Figure 4. Delayed somite differentiation in Efnb2 mutants

A. Expression of Twist (a, b) and Myogenin (c–f) in wild type (a, c, e) and Efnb2^GFP/GFP embryos (b, d, f) show that somite differentiation is impaired in Efnb2 null embryos. So: Somite numbers.

Figure 5. Generation of Efnb2^F5 mice

A. Efnb2 wild type locus (a), targeting vector (b) and targeted locus (c). B. Epifluorescence images of an Efnb2^F5/GFP (a) and Efnb2^GFP/GFP (b) embryos.

Figure 6. Phenotypes in Efnb1/Efnb2 double mutant embryos

A. H&E staining of salivary glands from E15.5 embryos shows that salivary gland from Efnb1^+/−/Efnb2^+/GFP double mutant (c) is smaller than either wild type (a) or Efnb1^+/− single mutant (b) salivary glands. B. Efnb1^+/−/Efnb2^+/GFP double heterozygous E18.5 embryos (b, d) exhibit an “open eye” phenotype. Eyelids are fused in E18.5 control embryos (a, c). C. Immunofluorescent staining of ephrin-B1 (a) and epifluorescent image of H2B-GFP (b, c) show that ephrin-B1 is expressed in the dermis (D) while ephrin-B2 is most highly expressed in the epidermis (E) in the developing embryo. Ephrin-B2 expression is upregulated at the tip of the eyelids (asterisk in c). Paraffin section of a X-gal stained-E14.5 embryo carrying the Wnt1Cre/R26R alleles showing that the dermis of the growing eyelids (arrows) is derived from NCC (d).