Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development

Abstract

Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely ephrin-B2 and the ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and ephrin molecules play an important role in these clinically significant midline cell-cell adhesion and fusion events.

Mutational analyses of UPIIIA, SHH, EFNB2, and HNF1β in persistent cloaca and associated kidney malformations

“…In contrast, the Efnb2 lacZ/lacZ homozygous mice manifest severe anorectal malformations, with high imperforate anus in the males and persistent cloaca in females. These data, combined with the noted hypospadias in the male EphB2 and EphB3 compound mutant mice, suggested that B-subclass signaling plays a major role in early murine development of the urogenital and anorectal structures (24), perhaps due to altered adhesion/ repulsion signals between the embryonic tissues that express these genes. Thus far, no human disease has been associated with EFNB2 mutations, but, based on the mouse data, the gene appeared to be an excellent candidate gene to screen in humans with persistent cloaca.…”

“…In mutant Bsubclass Eph or Efn mice was identified an additional role during urogenital and anorectal development. Efnb2 is expressed in the normal urogenital sinus and cloacal epithelium of mice, while the EphB2 receptor is predominantly expressed in the adjacent mesoderm of the urorectal septum that migrated towards the caudal midline during septation (24,40). Male mice with Efnb2 lacZ/+ exhibited hypospadias, a midline birth defect of the penile urethra, and delayed closure of the perineum.…”

“…Evidence for this contention derives from genetic studies in mice because animals with genotypes Gli2 +/− /Gli3 −/− , Gli2 −/− /Gli3 +/− (23), Efnb2 lacZ/lacZ (24) and Hoxa13 −/− /Hoxd13 −/− (45) have persistent cloaca, whereas mice with other combinations of mutations in these pathways have recto-vaginal/rectourethral fistula (Gli2 −/− ) (23), anal stenosis/ectopic anus (Gli3 −/− ) (23), hypospadias (Efnb2 +/lacZ , Ephb2 −/− /Ephb3 −/− ) (24) and urethral-vaginal/recto-vaginal fistula with uterine malformations (Hoxa13 +/− /Hoxd13 −/− ) (45), showing that, in genetic terms, persistent cloaca lies within the same spectrum of malformations as these more mild urethral/anorectal malformations. If this can be taken as a paradigm for human disease, then it would be logical to seek mutations of UPIIIA, SHH, EFNB2 and HNF1β in a similar, wider spectrum of malformations.…”

“…In addition, we screened three other genes, Sonic Hedgehog (SHH: 7q36), Ephrin B2 (EFNB2: 13q33) and Hepatocyte Nuclear Factor 1 β(HNF1β: 17cen-q21.3). As detailed in the Discussion, SHH (8,17,(21)(22)(23), EFNB2 (24) and HNF1β (25)(26)(27)(28)(29) are expressed in the developing renal, genital and alimentary tracts, and have been functionally implicated in the normal development of these structures.…”

Role for ephrinB2 in postnatal lung alveolar development and elastic matrix integrity

“…Moreover, although in principle ephrinB2-PDZ interactions could affect ephrinB2 trafficking and membrane targeting (Urena et al, 1999;Altschuler et al, 2003) and thereby indirectly influence EphB receptor activation, the overall health of ephrinB2 ⌬V/⌬V animals and the successful apical epithelial targeting of unbound ephrinB2 ⌬V protein in pseudoglandular stage lung (G.W. and R.K., unpublished observations) suggests that substantial signaling downstream of EphB receptors is preserved in this allele (see also discussion in Cowan et al, 2004;Dravis et al, 2004;Makinen et al, 2005). Finally, it should be pointed out that there is mounting evidence that ephrinBs can modulate other signaling pathways independently of Eph receptors (Foo et al, 2006;Lee et al, 2006) and that the repertoire of PDZ domain containing proteins bound to ephrinB ligands could change in the presence versus the absence of stimulating receptor (Torres et al, 1998;Bruckner et al, 1999;Palmer et al, 2002).…”