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. Author manuscript; available in PMC: 2008 Apr 1.
Published in final edited form as: Dev Biol. 2006 Dec 19;304(1):182–193. doi: 10.1016/j.ydbio.2006.12.028

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

Alice Davy 1, Philippe Soriano 1,1
PMCID: PMC1892242  NIHMSID: NIHMS20505  PMID: 17223098

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.

Keywords: Neural crest cells, somites, ephrin, signaling

Introduction

Eph receptors and ephrins are cell surface signaling proteins that were first identified for their role in axon guidance in vertebrates, and which have been shown subsequently to regulate a number of developmental processes in vertebrates and invertebrates. Moreover, there is growing evidence that these proteins also function in the adult, both in pathological and physiological situations (Pasquale, 2005; Poliakov et al., 2004; Surawska et al., 2004). At the cellular level, Eph receptors and ephrins control cell migration and cell sorting as well as gap junction communication (Davy et al., 2006). Eph and ephrins have a unique bidirectional mode of action in which both receptors and ligands can activate downstream signaling cascades (Bruckner et al., 1997; Davy et al., 1999; Holland et al., 1996). The signaling cascade activated downstream of Eph receptors is referred to as forward signaling while the signaling cascade activated downstream of ephrins is known as reverse signaling. A number of studies using various model systems have suggested that both forward and reverse signaling cascades are required in vivo (Davy and Soriano, 2005).

There are two structurally distinct classes of ephrins: ephrins-A (A1 to A6) are tethered to the plasma membrane via a glycosylphosphoinositide (GPI) tail whereas ephrins-B (B1 to B3) are transmembrane proteins. At the protein level, ephrin-B1 and ephrin-B2 share a high degree of identity, while the third member of the ephrin-B family, ephrin-B3, is more distantly related. Although some of the proximal events contributing to the signal transduction cascade activated ephrins-B have been identified (Murai and Pasquale, 2003), the exact molecular mechanisms by which reverse signaling is achieved remain elusive. The cytoplasmic tail of transmembrane ephrins contains two distinct signaling domains: a stretch of tyrosines that are phosphorylated in response to Eph receptor binding and a PDZ-binding domain that allows for interaction with various PDZ-containing proteins. Eph/ephrin interaction induces a Src-dependent tyrosine phosphorylation of the cytoplasmic tail of ephrins-B leading to the recruitment of Grb-4 and the tyrosine phosphatase PTP-BL (Cowan and Henkemeyer, 2001; Palmer et al., 2002). It has been shown recently that Eph-induced activation of Src-family kinases involves processing of ephrins-B by PS1/γsecretase (Georgakopoulos et al., 2006). Unlike Grb-4, the interaction between ephrins-B and PDZ-containing proteins seems to be independent of Eph/ephrin binding. A number of PDZ-containing proteins have been shown to interact with ephrins-B (Lin et al., 1999), some of which contribute to reverse signaling (Lu et al., 2001). The importance of the PDZ-binding domain for the in vivo function of ephrins-B has been recently demonstrated in the mouse. Elimination of the PDZ binding domain of ephrin-B1 recapitulated some of the craniofacial phenotypes associated with loss of ephrin-B1 (Davy et al., 2004) and a similar mutation in ephrin-B2 led to lymphatic defects (Makinen et al., 2005). On the other hand, the in vivo relevance of tyrosine phosphorylation of ephrins-B has yet to be proven, since expression of a mutant form of ephrin-B2 that could not be phosphorylated yielded only minor phenotypes (Makinen et al., 2005).

The phosphorylatable tyrosines as well as the PDZ-binding domain are located in a carboxy-terminal 33-amino acid stretch that is 100% identical in all 3 ephrins-B indicating that these proteins might activate indistinguishable signaling cascades. In addition, ephrin-B1 and ephrin-B2 can bind a similar set of Eph receptors (although ephrin-B2 has a higher affinity for Eph-B4 and ephrin-B1 does not appear to bind Eph-A4) and Efnb1 and Efnb2 are co-expressed in a number of tissues and cells during early mouse embryonic development including endothelial cells, epithelial somites, neural tube and branchial arches. All of these observations raise the possibility that ephrin-B1 and ephrin-B2 might be functionally redundant and might compensate for each other during embryonic development. Redundancy has been described for Eph receptors and GPI-linked ephrins previously (for instance see (Feldheim et al., 2000; Orioli et al., 1996). On the other hand, the fact that Efnb1 and Efnb2 null mice exhibit profoundly distinct phenotypes (Adams et al., 1998; Compagni et al., 2003; Davy et al., 2004; Wang et al., 1998) argues that these proteins have specific, non-redundant functions.

Studies in zebrafish, Xenopus and chick embryos have implicated Eph/ephrins in neural crest cell (NCC) migration. In the chick, Efnb1 is expressed in the posterior half of the somites and disruption of Eph/ephrin interaction led to trunk NCC migration defects (Krull et al., 1997). In Xenopus, EphA4/EphB1 receptors and Efnb2 are expressed in adjacent domains in the hindbrain region and inhibition of EphA4/EphB1 function led to abnormal NCC migration (Smith et al., 1997). In the mouse, Efnb2 expression is restricted to the posterior half of the somites and in vitro, Eph-expressing NCC avoided ephrin-containing stripes (Wang and Anderson, 1997) suggesting that ephrin-B2 acts as a repulsive cue for trunk NCC. Surprisingly, no trunk NCC migration defects have been reported to date in Efnb2 or Efnb1 deficient mice (Adams et al., 1998; Compagni et al., 2003; Davy et al., 2004; Wang et al., 1998).

To better assess the role of ephrin-B2 signaling in NCC in the mouse, we have generated Efnb2 null or tyrosine phosphorylation mutant mouse lines as well as Efnb1/Efnb2 double heterozygous mutant mice. We found that Efnb2 null embryos exhibited trunk NCC migration defects concomitant with somite defects. In addition, we show that Efnb1/Efnb2 double heterozygous mutant embryos exhibited phenotypes in a number of NCC-derivatives that were not previously known targets of Eph/ephrin signaling. Expression of one copy of the phosphorylation mutant was sufficient to rescue the defects associated with the loss of Efnb2. Together these results uncover novel roles for ephrin-B2 in NCC, show that ephrin-B2 exerts most of its embryonic functions independently of tyrosine phosphorylation and suggest that ephrin-B1 and ephrin-B2 are functionally redundant in certain developmental contexts.

Materials and methods

Mice

The Efnb2GFP allele was generated by inserting the H2B-GFP cDNA cassette at the MluI site in the first exon of the Efnb2 gene. The MluI-XbaI fragment encompassing the start codon of the Efnb2 gene was replaced with the H2BGFP cDNA. The Efnb2F5 allele was generated by fusing a partial cDNA (harboring Y to F mutations) at the NcoI site in the first exon of Efnb2. The mutations were introduced in ES cells by homologous recombination. Efnb2GFP and Efnb2F5 mice were maintained in 129S4/C57Bl6J and 129S4 congenic backgrounds, respectively. Mice were housed in microisolator racks in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, and experimentation was reviewed by the Hutchinson Center Institutional Review Committee. Genotyping was done by PCR using the following sets of primers for the Efnb2GFP allele: GFP-F: 5′-GCAAGAAGGCGGTGACTAAGGCGC-3′; GFP-R: 5′-GGCCGCCGCCAGTGCTTGAGGTCG-3′. The Efnb2F5 mouse line was genotyped with the following set of primers that amplifies both the mutant and wild type alleles: F5-F: 5′-CGGCTTGGGCATGGCCATGGC-3′; F5-R1: 5′-AAGAGAAGGCTCGTGGACACC-3′; F5-R2: 5′-CTGAAATTCTAGACCCCAGAGG-3′.

Whole mount in situ hybridization

Embryos were fixed in 4% PFA overnight and subsequently dehydrated in PBT (PBS/1%Tween)/methanol. Embryos were stored in 100% methanol at 4°C until processed for in situ hybridization as described previously (Riddle et al., 1993). Hybridization with DIG-labeled riboprobes was performed at 70°C. DIG-labeled riboprobes were detected with an AP-conjugated anti-DIG antibody (Roche). BM purple (Roche) or NBT/BCIP (Roche) were used as a substrate for the alkaline phosphatase. At least 3 embryos of each genotype were used for each probe. We obtained the probe for Sox10 from Michael Wegner (Kuhlbrodt et al., 1998), for Meox1 from Chris Wright, for Myogenin from Norbert Kraut, for Sema3F from David Ginty and for Uncx4.1 from Peter Gruss.

Whole mount immunohistochemistry

Neurofilament staining was performed on PFA-fixed embryos (n=3). Briefly, embryos were dehydrated and rehydrated in methanol/PBT (PBS containing 1%Tween 20) and incubated in blocking solution (PBS containing 1% BSA and 5% sheep serum) for 1 hour at room temperature. Embryos were then incubated overnight in primary antibody diluted 1/150 in blocking solution. The 2H3 anti-Neurofilament antibody developed by Thomas M. Jessell and Jane Dodd was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Embryos were washed extensively, blocked again and incubated overnight in secondary antibody (1/75 dilution of HRP-conjugated anti-mouse from SIGMA). Next day, the embryos were stained using the DAB kit (Vector Laboratories Inc.).

X-gal staining

X-gal staining was performed as previously described (Friedrich and Soriano, 1993).

Histology and immunofluorescence

H&E staining and sectioning was performed on paraffin embedded samples. The size of the salivary gland was evaluated on photomicrographs of H&E stained section using the Photoshop software. Immunofluorescence was performed on frozen sections using a monoclonal anti-ephrin-B1 antibody (25H11, a kind gift from Wieland Huttner; Stuckmann et al., 2001). Briefly, sections were incubated in blocking solution (PBS containing 5% Horse serum) for 1 hour at room temperature and incubated with the 25H11 antibody (1/25) overnight. Sections were then incubated with an anti-rat antibody conjugated to Cy3.

Results

Neural crest migration defects in Efnb2 null mice

We have generated a mouse line harboring a null mutation in the Efnb2 gene by inserting a cDNA coding for a fusion protein between histone 2B (H2B) and the green fluorescent protein (GFP) in the first exon of the Efnb2 gene (Fig. 1). This mutation places the expression of H2B-GFP under the control of the Efnb2 promoter and creates a null allele for Efnb2 (referred to herein as Efnb2GFP). Loss of Efnb2 expression in Efnb2GFP/GFP homozygous embryos was confirmed by RT-PCR on RNA isolated from embryonic day (E) 10.5 embryos (Fig. 1B). Intercrosses between heterozygous parents failed to produce any live homozygous offspring. Consistent with previous observations (Wang et al., 1998; Adams et al., 1998), homozygous embryos died at about E10.5 from cardiovascular defects (Fig. 1C and data not shown). Analysis of whole embryos at various stages of development revealed that nuclear H2B-GFP was readily visible by epifluorescence and could be used as a marker of ephrin-B2 expression in living embryos (Fig. 1D). To confirm that the expression pattern of H2B-GFP was similar to the documented expression pattern of Efnb2, we examined tissues in which Efnb2 expression has been reported previously such as blood vessels (Fig. 1Dd), branchial arches (Fig. 1De), somites (Fig. 3), the nervous system and the retina (data not shown). In all tissues examined the expression of H2B-GFP was identical to the reported endogenous expression of Efnb2. These results indicate that H2B-GFP can be used as a bona fide marker for Efnb2 expression in mice carrying the Efnb2GFP allele.

Figure 1. Generation of an Efnb2 null mouse line.

Figure 1

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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 Efnb2GFP/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 Efnb2GFP/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 3. Somite defects in Efnb2 null embryos.

Figure 3

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A. Epifluorescence images of Efnb2+/GFP embryos (a) and Efnb2GFP/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 Efnb2GFP/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 Efnb2GFP/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.

Using Sox10 as a marker of migrating NCC and the combination of Wnt1Cre/R26R alleles for Cre recombinase-based lineage tracking of NCC derivatives, we observed a general reduction in the population of cranial NCC in Efnb2 null embryos (Fig. 2Aa–d). The stream of NCC migrating toward the second branchial arch (BA) was reduced in size, consistent with a previous report (Adams et al., 2001). In addition we observed a depletion of NCC migrating to the first BA (Fig. 2Ab, d). Consistent with the depletion of cranial NCC, the first BA was often smaller and hypoplastic in Efnb2 null embryos (Supplementary Fig. 1). In fact, 12/21 Efnb2GFP/GFP embryos exhibited a severe reduction in the size of the first BA (Fig. 2Ad). Depletion of NCC was accompanied by an increase cell death (assessed by Nile Blue staining) in the Efnb2GFP/GFP embryos (data not shown). To test whether this phenotype was specific to NCC, we used an antibody specific for neurofilament to label differentiating neurons. In Efnb2GFP/GFP embryos, neurofilament staining showed a reduction in the fifth (trigeminal) cranial ganglion (a NCC derivative) but not in mid-brain neurons (Fig. 2Ae, f). These results indicate that loss of Efnb2 leads to a selective reduction in cranial NCC.

Figure 2. Neural crest cell defects in Efnb2 null embryos.

Figure 2

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A. Efnb2+/GFP embryos (a, c, e) and Efnb2GFP/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 Efnb2GFP/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.

We also observed a disruption of trunk NCC migration in our Efnb2 null embryos (Fig. 2B). In the Efnb2GFP/GFP embryos, Sox10-positive trunk NCC detached from the main migrating streams and migrated in a scattered fashion across somites (Fig. 2Bb). In addition to this scattered migration, loss of segmented migration of trunk NCC could be observed in the most posterior part of Efnb2GFP/GFP embryos (Fig. 2Bd). Histological sections showed that NCC invade the posterior part of somites in Efnb2 mutant embryos (Fig. 2Bf). Trunk NCC migration defects were observed in all of the Efnb2 null embryos analyzed (n=11), however, the severity of the defects was variable from embryo to embryo (data not shown).

Somite defects in Efnb2 null embryos

Ephrin-B2 is expressed in the posterior half of somites (Fig. 3) and therefore may control trunk NCC migration non-autonomously, by acting as a repulsive cue for Eph-expressing NCC. Alternatively, loss of ephrin-B2 could impair somite development, which might in turn impinge on trunk NCC migration. To better understand the underlying cause of the trunk NCC migration defects observed in Efnb2 null embryos, we analyzed somite development in these mutants. In the absence of Efnb2, cells that normally express Efnb2 (GFP-positive) were abnormally distributed across the somites (Fig. 3Aa, b) and clumping of GFP-positive cells could be observed at the caudal boundary of somites (Fig. 3Ab). To analyze this phenotype in more detail, we analyzed the distribution of GFP-positive cells on sections of control and mutant embryos. In the control embryo, epithelial cells expressing high levels of GFP were distributed throughout the caudal half of somites (Fig. 3Ba–c). In Efnb2 null embryos the difference in the level of GFP expression between the caudal and rostral half of somites was attenuated (Fig. 3Bd–f). In addition, despite the fact that epithelial cells expressing higher levels of GFP were found close to the caudal boundary, these cells were clustered at the medial edge of the somites and did not extend laterally (Fig. 3Bd–f). These observations indicate that loss of Efnb2 might affect the patterning of somites. To test for this we monitored the expression of a number of markers of somite polarity. At 20–22 somite stage, the expression of Uncx4.1, a marker of the caudal compartment appeared polarized in Efnb2GFP/GFP embryos, however, the level of expression was decreased as compared to control embryos and the boundary between the expressing and non-expressing domain was not as sharp as in the control embryos (Fig. 3Ca, b). The reduction in Uncx4.1 expression levels was already evident at the 10 somite stage (Supplementary Fig. 2). The expression level of Meox1, another marker of the caudal compartment, was also diminished in Efnb2 null embryos (Fig. 3Cc,d), consistent with a loss of caudal properties in somites. However, the decrease in the expression level of markers of the caudal compartment was not accompanied by an expansion of the rostral compartment since Tbx18, a gene expressed anteriorly in somites, appeared identical in control and Efnb2 null embryos (data not shown). These results indicate that loss of Efnb2 perturbs the patterning of somites.

Sema3F, which is expressed in somites, has recently been implicated in regulating the migration of trunk NCC (Gammill et al., 2006). At the 19–20 somite stage, Sema3F was expressed in the posterior part of somites in control embryos (Fig. 3Ce), however, the polarized expression of Sema3F was completely lost in Efnb2 null embryos (Fig. 3Cf). To rule out the possibility that the phenotypic differences in the mutant embryos were secondary to developmental delays associated with angiogenic defects, we examined the expression of this marker at an earlier stage of development. The loss of polarized Sema3F expression was already evident at the 14 somite stage, even though mutant embryos are otherwise indistinguishable from control embryos (Fig. 3Cg, h). This data shows that loss of Efnb2 perturbs the distribution of Sema3F in somites.

To test whether the changes in polarity markers observed in Efnb2GFP/GFP embryos were concomitant with impaired somite differentiation, we analyzed the expression of Myogenin (a marker of myogenic differentiation) and Twist (a marker of epithelial somites that later becomes restricted to the sclerotome and dermatome). At the 26-somite stage, the expression pattern of Twist indicated that the somites were less mature in the mutant embryos (Fig. 4a, b). Consistent with this, the expression of Myogenin was barely detectable in Efnb2 null embryos at the 26-somite stage (Fig. 4c, d). Myogenin expression was already delayed in Efnb2GFP/GFP at the 18-somite stage, even though control and mutant embryos had similar number of somites (Fig. 4e, f), indicating that these phenotypes are not secondary to the developmental delay caused by the defective angiogenesis. These results indicate that loss of Efnb2 perturbs somite patterning and impairs somite differentiation.

Figure 4. Delayed somite differentiation in Efnb2 mutants.

Figure 4

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A. Expression of Twist (a, b) and Myogenin (c–f) in wild type (a, c, e) and Efnb2GFP/GFP embryos (b, d, f) show that somite differentiation is impaired in Efnb2 null embryos. So: Somite numbers.

Tyrosine phosphorylation of ephrin-B2 is not required for NCC migration

To ask whether tyrosine phosphorylation was required for the function of ephrin-B2 in somite patterning and NCC development, we generated a mouse line expressing a mutant form of ephrin-B2 in which all of the tyrosine residues were mutated to phenylalanine, therefore preventing tyrosine phosphorylation of ephrin-B2 (Fig. 5A). As a control for the targeting strategy, we also created a mouse line carrying a wild type ephrin-B2 cDNA that showed no developmental phenotypes (data not shown). Genotyping of 10 day old pups showed that Efnb2F5/F5 homozygous animals were recovered at the expected Mendelian ratio (Table 1). In addition, one copy of the Efnb2F5 allele was sufficient to rescue the developmental defects associated with the loss of Efnb2, since Efnb2F5/GFP were completely viable and fertile (Fig. 5B and Table 1). These mutants did not present craniofacial defects and exhibited normal development of the axial skeleton, suggesting that tyrosine phosphorylation of ephrin-B2 is dispensable for somite patterning and NCC development. However, these results could also indicate that ephrin-B1 might compensate for the loss of ephrin-B2 reverse signaling in tissues where both ephrins are expressed, such as endothelial cells, somites and NCCs. To test for this possibility, we mated Efnb2F5/F5 mice with Efnb1Y/− mice (Davy et al., 2004) to generate double mutants. As a control, we also crossed Efnb2+/GFP mice with Efnb1 mutants. Removing one copy of Efnb2 in an Efnb1 mutant background led to phenotypes that were not observed in Efnb1 single mutants (Table 2 and Fig. 6), indicating that Efnb1 and Efnb2 interact genetically. In contrast, Efnb2F5/F5 mutant mice lacking Efnb1 only exhibited the phenotypes associated with the loss of Efnb1 (Table 2 and data not shown). These results show that the lack of embryonic phenotype in Efnb2F5/F5 mice is not due to functional compensation by ephrin-B1.

Figure 5. Generation of Efnb2F5 mice.

Figure 5

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A. Efnb2 wild type locus (a), targeting vector (b) and targeted locus (c). B. Epifluorescence images of an Efnb2F5/GFP (a) and Efnb2GFP/GFP (b) embryos.

Table 1.

Efnb2F5 homozygous and hemizygous embryos are viable.

Efnb2+/F5x Efnb2+/F5 Efnb2+/F5x Efnb2+/GFP
Genotypes Efnb2+/+ Efnb2+/F5 Efnb2F5/F5 Efnb2+/+ Efnb2+/F5 Efnb2+/GFP Efnb2F5/GFP
Ratios (number) 22% (28) 50% (62) 28% (36) 18.0% (11) 37.9% (22) 22.5% (13) 20.7% (12)
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The ratio for each genotype is indicated as a percentage. The total number of pups of each genotypes is also presented.

Table 2.

Genetic interaction between Efnb1 and Efnb2.

Efnb2F5/F5x Efnb2+/F5; Efnb1+/−
Genotypes B2+/F5/B1+/+ B2+/F5/B1Y/− B2F5/F5/B1+/+ B2F5/F5/B1Y/−
Ratios (number) 38% (23) 19.5% (12) 23% (14) 19.5% (12)
Efnb2+/GFPx Efnb1+/−
Genotypes B2+/+/B1+/+ B2+/+/B1Y/− B2+/GFP/B1+/+ B2+/GFP/B1Y/−
Ratios (number) 35% (19) 22% (12) 43% (23) - (0)
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The ratio for each genotype is indicated as a percentage. The total number of pups of each genotypes is also presented. For clarity, Efnb1Y/− males and Efnb1+/− females have been grouped under the genotype Efnb1Y/−, since they have similar phenotypes. Efnb2F5/F5/Efnb1Y/− mice are viable while Efnb2+/GFP/Efnb1Y/− die perinatally.

Figure 6. Phenotypes in Efnb1/Efnb2 double mutant embryos.

Figure 6

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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).

Redundancy between ephrin-B1 and ephrin-B2 in NCC derivatives

To better characterize the genetic interaction between Efnb1 and Efnb2, we further analyzed the Efnb1/Efnb2 double heterozygous embryos. Although ephrin-B1 heterozygous females display phenotypes not observed in homozygous females or hemizygous males due to X-inactivation and subsequent sorting of ephrin-B1 expressing and non-expressing cells (Compagni et al., 2003; Davy et al., 2004), all the phenotypes we discuss below were the same for Efnb1Y/−/Efnb2+/GFP males and Efnb1+/−/Efnb2+/GFP females (double mutants are henceforth referred to as Efnb1/Efnb2 mutants). While Efnb1/Efnb2 mutant embryos exhibited normal trunk NCC migration and somite development (Supplementary Fig. 3A), they presented defects in a number of tissues that have not previously been known to require EphB/ephrinB signaling including the salivary gland and the dermis (Fig. 6), and the cartilage of the trachea (Supplementary Fig. 2C, D). In 3/3 Efnb1/Efnb2 mutant embryos the salivary gland was ~ 40% smaller and less mature than in control embryos (Fig. 6A and Supplementary Fig. 3B). Cultures of wild type and Efnb1/Efnb2 mutant salivary glands indicated that the branching morphogenesis defect is rescued in vitro (data not shown), indicating that the phenotype observed in vivo could be due to defective growth factor signaling. Failure to close the eyelids during late embryogenesis was observed in 11/13 Efnb1/Efnb2 embryos (Fig. 6B). While ephrin-B1 is expressed in the dermis (Fig. 6Ca), ephrin-B2 is normally expressed in the epidermis (Fig. 6Cb). Interestingly, expression of ephrin-B2 is up-regulated in the dermis of the growing eyelids (Fig. 6Cc). Lineage tracing using Wnt1Cre/R26R alleles showed that the dermis of the eyelids is derived from NCC (Fig. 6Cd). Similarly, Efnb1 and Efnb2 were found to be co-expressed in the NCC-derived mesenchyme of the salivary gland (data not shown). These results demonstrate that ephrin-B1 and ephrin-B2 are co-expressed in mesenchymal cells of neural crest origin and suggest that they exert redundant functions in these cells.

Discussion

Ephrin-B2 in cranial NCC

In this manuscript we document that Efnb2 is required for NCC development. We observed a number of defects in cranial NCC in Efnb2 null embryos. Streams of migrating NCC to the first and second BA were reduced, possibly due to cell death. As a consequence of this depletion of cranial NCC, the first BA was small and hypoplastic while formation of the fifth ganglion was impaired. Interestingly, expression of Sema3F appeared unchanged in the cranial region of Efnb2 null embryos (data not shown). In addition to this early function of Efnb2 in cranial NCC, we have also uncovered a role for Efnb2 in cranial NCC-derivatives by generating and analyzing Efnb1/Efnb2 double mutants. Although we have shown previously that loss of Efnb1 leads to craniofacial defects (Davy et al., 2004), the Efnb1/Efnb2 double mutants exhibited additional phenotypes that were not observed in Efnb1 single mutants. The Efnb1/Efnb2 mutants were born with an open eye phenotype which is usually linked to an epidermal migration defect (Xia and Kao, 2004). In the developing embryo, ephrin-B1 and ephrin-B2 were co-expressed in the dermis at the leading edge of the eyelids, suggesting that ephrin-B1 and ephrin-B2 might perform redundant function to regulate epidermal migration non-autonomously. We have also noted a delay in growth and maturation of the salivary gland in Efnb1/Efnb2 embryos. In the double mutants, the gland was smaller, less branched and EGF production was decreased. Since ephrin-B1 and ephrin-B2 are expressed in the mesenchyme in the gland while EphB2 is expressed in the epithelium (J. Bush and P.S., unpublished observation), an attractive possibility is that ephrin-B1 and ephrin-B2 regulate epithelial branching in the salivary gland non-autonomously by activating forward signaling. Interestingly, involvement of EphA/ephrinA signaling in regulating growth factor-induced branching morphogenesis has been reported previously (Miao et al., 2003).

Ephrin-B2 regulates trunk NCC migration

In the trunk, we observed the disruption of segmented migration of trunk NCC. However, segmented migration was not lost entirely, possibly reflecting the fact that other molecules are involved in controlling this process, including other Eph/ephrins (McLennan and Krull, 2002), as well as other guidance molecules such as semaphorins/neuropilins (Gammill et al., 2006; Kawasaki et al., 2002) and Slit/ROBO (Jia et al., 2005). The fact that this phenotype was not reported previously (Adams et al., 1998; Wang et al., 1998) might be due to the use of more sensitive markers than available previously, including the Wnt1Cre/ROSA26R lineage tracing system (Chai et al., 2000; Jiang et al., 2002). The defect in trunk NCC migration observed in Efnb2 null embryos is consistent with previous studies in the chick and the mouse that had proposed that transmembrane ephrins act as repulsive cues for migrating NCC expressing Eph receptors (Krull et al., 1997; Wang and Anderson, 1997). Our observations are also consistent with previous work showing that disruption of segmented NCC migration in Delta-1 null mice correlated with loss of expression of Efnb2 in the posterior part of somites (De Bellard et al., 2002). Here we show that loss of Efnb2 perturbs the distribution of Sema3F. Interestingly, Sema3F has been shown recently to act as a repulsive cue for migrating trunk NCC in the mouse (Gammill et al., 2006) and it has been shown in zebrafish embryos that aberrant expression of class-3 Semaphorins leads to abnormal cranial NCC migration (Yu and Moens, 2005). Thus, one possibility is that mislocalization of Sema3F leads to the trunk NCC migration defects in Efnb2 null embryos. Consistent with this hypothesis, the trunk NCC migration defects reported in the Sema3F null embryos were more severe than the defects we observed in Efnb2 null embryos (Gammill et al., 2006). Alternatively, it is also possible that multiple pathways cooperate in guiding trunk NCCs, perhaps by converging on the regulation of common cellular targets. Recent work has indeed shown that semaphorin signaling can regulate integrins, which are also a target of ephrins (Kruger et al., 2005; Nakamoto et al., 2004). Based on these results, we propose that in addition to directly regulating the migration of trunk NCCs via repulsion (Krull et al., 1997; Wang and Anderson, 1997), ephrin-B2 also regulates the migration of trunk NCCs by controlling the expression of somitic genes.

Somite defects in Efnb2 null embryos

In absence of Efnb2, both somitic polarity and somitic differentiation markers were perturbed. To date, the importance of Eph/ephrins signaling in somitogenesis has been most strongly established in zebrafish. Perturbation of Eph/ephrin signaling by overexpression of dominant negative forms of Eph and ephrins in zebrafish leads to severe segmentation defects and reduced or disturbed MyoD expression (Durbin et al., 1998). Abnormal expression of several ephrins and Eph receptors has also been shown to correlate with somite segmentation defects in the fss (fused somites) zebrafish mutant. In this mutant, restoration of Eph/ephrin signaling was sufficient to rescue most aspects of boundary formation (Barrios et al., 2003; Durbin et al., 2000). Finally, it has been shown recently that integrin/fibronectin and Eph/ephrin systems function cooperatively in maintaining somite boundaries in zebrafish embryos (Koshida et al., 2005). Our results indicate that in the mouse, somite segmentation often proceeds normally in absence of Efnb2, although we have obtained a few embryos exhibiting abnormal somite morphology. These results are consistent with the fact that no defects in somite development have been reported in EphA4 null mice (EphA4 binds to ephrin-B2 and is expressed in the rostral compartment of somites). Interestingly, it has been reported recently that forced expression of the transcription factor Mesp2 in somitic cells in mouse embryos leads to the activation of EphA4 and the repression of Uncx4.1 (Nakajima et al., 2006), consistent with what we have observed in Efnb2 null embryos. The fact that somite segmentation proceeds normally in absence of Efnb2 or EphA4 raises the possibility that other members of the Eph receptor or ephrin family might compensate for the loss of either gene. No somite defects were observed in either Efnb1 null embryos (data not shown) or Efnb1/Efnb2 double heterozygous mutant embryos indicating that ephrin-B1 is not required for somite formation and that one copy of Efnb2 is sufficient for normal somite development, even in absence of Efnb1. However, it would be interesting to analyze somite development in the mouse, in the conditional absence of both Efnb1 and Efnb2 in somitic cells. Similarly, the analysis of conditional EphA4/Efnb2 double null embryos might further our understanding of the role of these proteins in somite development.

Ephrin-B2 reverse signaling in mouse development

A number of genetic studies in the mouse have addressed the question of ephrin-B2 reverse signaling in vivo. Adams et al. showed that a mutant version of ephrin-B2 lacking the cytoplasmic tail was able to rescue a cranial NCC migration defect but not the cardiovascular defect that are observed in Efnb2 null embryos. From these results the authors concluded that ephrin-B2 reverse signaling was essential for angiogenesis but not for cranial NCC migration (Adams et al., 2001). This conclusion was challenged later on with the generation of a mouse line in which the cytoplasmic tail of ephrin-B2 was removed and replaced by β–galactosidase. Embryos carrying this allele were born alive but died shortly after birth presumably because of cardiac malformations (Cowan et al., 2004). These data indicated that the cytoplasmic tail of ephrin-B2 and therefore reverse signaling was not required for angiogenesis, but was important for cardiac valve formation. More recently, point mutations have been introduced in the cytoplasmic tail of ephrin-B2 that either disrupt interactions with PDZ-containing proteins or abolish tyrosine phosphorylation. These studies have revealed a post-natal role for the PDZ-binding domain of ephrin-B2 since mice homozygote for the ephrinB2ΔV mutation were born alive but died prematurely because of defects in remodeling of the lymph vasculature (Makinen et al., 2005). On the other hand, mice expressing a mutant version of ephrin-B2 that can not be tyrosine phosphorylated (ephrinB25Y) survived until adulthood and presented mild defects in the lymph vasculature that could be due to the fact that the PDZ binding domain might have been disrupted in this allele (Makinen et al., 2006). Taken together, these results are consistent with the notion that ephrin-B2 reverse signaling is not required for early vascular development.

We found that all of the phenotypes associated with loss of Efnb2, including the loss of cranial NCCs and the defective patterning of somites could be rescued by expression of a mutant form of ephrin-B2 that can not be tyrosine phosphorylated, indicating that tyrosine phosphorylation of ephrin-B2 is not essential for embryonic development. Because ephrin-B1 and ephrin-B2 are co-expressed in many cell types and tissues, one possible explanation for the lack of embryonic phenotype in the Efnb2F5/F5 mice was functional compensation. The results presented in this manuscript indicate that this is not the case since eliminating Efnb1 in Efnb2F5/F5 background did not uncover embryonic phenotypes. On the other hand, Efnb1/Efnb2 double heterozygotes exhibited phenotypes that had not been observed in Efnb1 single mutants, indicating that Efnb1 and Efnb2 interact genetically.

In conclusion, our results show that in addition to its role in angiogenesis, ephrin-B2 also plays an important role in NCC and somite development during mammalian embryogenesis. Remarkably, we provide evidence that these functions are tyrosine phosphorylation–independent. These results, combined with other studies in the mouse (Makinen et al., 2005), suggest that ephrin-B2 acts primarily as a ligand to activate Eph-induced forward signaling during early embryogenesis.

Supplementary Material

Supplementary Figure 1. Histology of the first branchial arch

H&E staining of paraffin sections of a wild type and Efnb2GFP/GFP E9.5 embryo, at the level of the first branchial arch. The branchial arch is smaller and hypoplastic in the mutant embryo.

Supplementary Figure 2. Polarity marker

Uncx4.1 expression in wild type (a) and Efnb2GFP/GFP embryos (b). Expression of Uncx4.1 is decreased unilaterally in the Efnb2 null embryo (arrow).

Supplementary Figure 3. Novel phenotypes in Efnb1/Efnb2 double mutants

A. Expression of Myogenin is normal in Efnb1 mutant embryos (a) and in Efnb1/Efnb2 double mutant embryos (b). Expression of Sox10 shows that migration of trunk NCC is normal in Efnb1/Efnb2 double mutant embryos (c). B. Immunofluorescent detection of EGF (red) on frozen sections from wild type (a) or B1Y/−/B2+/GFP E17.5 (b) embryos. Nuclei were labeled blue with DAPI. Western-blot analysis to detect EGF in protein lysates of salivary glands isolated from E16.5 embryos, either Efnb2+/GFP (B2), Efnb1+/− (B1) or ephrinB1Y/−/Efnb2+/GFP (B1B2) (c). Insoluble fractions of the protein lysates were analyzed by western-blot using an anti-EGF antibody (Sigma). EGF levels are decreased in ephrinB1Y/−/Efnb2+/GFP salivary glands. C. The trachea was isolated from E18.5 embryos, either Efnb2+/GFP (a), Efnb1+/− (b) or Efnb1+/−/Efnb2+/GFP double mutants (c) and cartilaginous tracheal rings were stained with Alcian Blue. D. H&E staining of paraffin sections of lungs isolated from E18.5 Efnb2 heterozygous control embryos (a) and Efnb1Y/−/Efnb2+/GFP double heterozygous embryos (b). Perinatal lethality of the Efnb1/Efnb2 was probably due to the severe malformation of the tracheal rings since this phenotype was concomitant with poorly inflated lungs. Malformation of the tracheal rings was observed in 4/4 Efnb1/Efnb2 embryos.

Acknowledgments

The Wnt1-Cre mice were kindly provided by Andy McMahon. We thank Philip Corrin and Marc Grenley for excellent technical assistance; and our laboratory colleagues and for critical reading of the manuscript. A.D. is a Canadian Institute of Health Research postdoctoral fellow. This work was supported by grants HD24875 and HD25326 from the National Institute of Child Health and Human Development to P.S.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure 1. Histology of the first branchial arch

H&E staining of paraffin sections of a wild type and Efnb2GFP/GFP E9.5 embryo, at the level of the first branchial arch. The branchial arch is smaller and hypoplastic in the mutant embryo.

NIHMS20505-supplement-a.pdf (98.8KB, pdf)
Supplementary Figure 2. Polarity marker

Uncx4.1 expression in wild type (a) and Efnb2GFP/GFP embryos (b). Expression of Uncx4.1 is decreased unilaterally in the Efnb2 null embryo (arrow).

NIHMS20505-supplement-b.pdf (36.3KB, pdf)
Supplementary Figure 3. Novel phenotypes in Efnb1/Efnb2 double mutants

A. Expression of Myogenin is normal in Efnb1 mutant embryos (a) and in Efnb1/Efnb2 double mutant embryos (b). Expression of Sox10 shows that migration of trunk NCC is normal in Efnb1/Efnb2 double mutant embryos (c). B. Immunofluorescent detection of EGF (red) on frozen sections from wild type (a) or B1Y/−/B2+/GFP E17.5 (b) embryos. Nuclei were labeled blue with DAPI. Western-blot analysis to detect EGF in protein lysates of salivary glands isolated from E16.5 embryos, either Efnb2+/GFP (B2), Efnb1+/− (B1) or ephrinB1Y/−/Efnb2+/GFP (B1B2) (c). Insoluble fractions of the protein lysates were analyzed by western-blot using an anti-EGF antibody (Sigma). EGF levels are decreased in ephrinB1Y/−/Efnb2+/GFP salivary glands. C. The trachea was isolated from E18.5 embryos, either Efnb2+/GFP (a), Efnb1+/− (b) or Efnb1+/−/Efnb2+/GFP double mutants (c) and cartilaginous tracheal rings were stained with Alcian Blue. D. H&E staining of paraffin sections of lungs isolated from E18.5 Efnb2 heterozygous control embryos (a) and Efnb1Y/−/Efnb2+/GFP double heterozygous embryos (b). Perinatal lethality of the Efnb1/Efnb2 was probably due to the severe malformation of the tracheal rings since this phenotype was concomitant with poorly inflated lungs. Malformation of the tracheal rings was observed in 4/4 Efnb1/Efnb2 embryos.

NIHMS20505-supplement-c.pdf (194.2KB, pdf)

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