Our research group is investigating a novel genetically engineered murine model manifesting the unanticipated phenotype of hypospadias and genitourinary/anorectal malformations. The Eph and ephrin gene families () are known for their roles in cell–cell signaling, cell sorting32
, axonal guidance during neuronal development33,34
, delineation of embryonic cellular boundaries35
and epithelial–mesenchymal transitions38
. The Eph receptors, the largest subclass of receptor tyrosine kinases, and their membrane-bound ephrin ligands are two large highly conserved gene families expressed throughout invertebrates and vertebrates. Both the Ephs and ephrins are subdivided into A and B classes. For the most part, A-class Ephs bind to A-class ephrins and B-class Ephs bind to B-class ephrins, although there are a few exceptions to this rule. To understand their interactions, two cells are depicted in , one expressing a B-class Eph molecule and the other expressing a B-class ephrin molecule. Upon activation of the EphB molecule by the ephrin-B molecule, a set of intracellular signals are activated within the EphB-expressing cell, which has been called the “forward” signal. Similarly, upon activation of the ephrin-B molecule by the EphB molecule, the “reverse” signal is activated within the ephrin-B-expressing cell. For ephrin-B2, it is clear that two ligands include EphB2 and EphB3. Thus, these molecules establish a system for cell–cell interactions and bidirectional signaling. Activation of these molecules alters cytoskeletal elements leading to alterations in cell morphology and in the nervous system, and cell–cell adhesive or repulsive events.
Figure 4 The Eph and ephrin gene families are divided into A and B classes. Protein functional motifs are depicted by shapes; A-class ephrins are bound to the cell membrane by glycosylphosphotidy linositol (GPI) linkage while the B-class ephrins are transmembrane (more ...)
In order to better characterize the independent nature of “reverse” signaling, our group created a mutant mouse wherein the intracellular domain of the ephrin-B2 molecule was substituted by a lac Z cassette (ephrin-B2lacZ). The adult male heterozygous mice () manifest hypospadias at approximately 30–40% penetrance, independent of genetic background. demonstrates a normal adult wild-type mouse with a normal penis and tubularized urethra (). In contrast, demonstrates the ephrin-B2lacZ/+ heterozygous hyspospadic adult mouse wherein the anogenital distance is decreased and the penis is hypospadic, with an open urethral plate as indicated in . Given that ephrin-B2 binds to EphB2 and EphB3, adult mice functionally null for both EphB2 and EphB3 (EphB2ki/ki;EphB3Δ/Δ compound homozygotes) also manifested perineal hypospadias and a reduced perineal distance (), confirming the key role of this signaling pathway. In these hypospadic mice, there is no sign of insufficient virilization, as judged by prostate and seminal vesicle size.
Figure 5 (A) Adult male wild-type (+/+) mouse depicting normal anus (arrowhead) and penis (arrow). (B) Adult male ephrin-B2lacZ/+ heterozygous(+/−) mouse with normal anus (arrowhead), reduced anogenital distance and perineal hypospadias (arrow). (C) Cross (more ...)
Figure 6 (A) Adult male wild-type (+/+) mouse depicting normal anus (arrowhead) and penis (arrow). (B) Adult male EphB2ki/ki;EphB3Δ/Δ compound homozygote mouse with normal anus (arrowhead), reduced anogenital distance and perineal hypospadias (arrow). (more ...)
To understand the maldevelopment of the hypospadias in these adult mutant mice, the mutant mouse embryos were studied. In , β-gal staining reveals that the ephrin-B2lacZ
protein is localized within the endodermal cells of the urethral plate. In these heterozygous mutant mice, urethral tubularization is incomplete, yielding perineal hypospadias. When compared to the wild-type animals as seen in the top panel, the bottom panel also clearly reveals that there is a defect of a persistent cloaca in these males. In the series of images in , whole-mount visualization of these same embryos gives another view of the perineal defect at low and high power magnification. The tail has been amputated and is in the upper area of the images. The genital tubercle is then seen coming out towards the viewer in the lower portion of the images. shows a wild-type animal at embryonic day 16, demonstrating complete septation of both the urogenital sinus and the anorectal canal with closure of the perineum in the midline. In comparison, two EphB2χ/χ
mutant littermates are seen in panels 8B and C. These two littermates with identical genotypes show phenotypic variability, with the mouse seen in panel B being less severely affected than the mouse in C, but it is clear that when they are both compared to the wild-type littermate, the perineum has not closed and there is an open cloaca. These data therefore suggest that ephrin-B2 and EphB2/EphB3 are key molecules involved in septation events in the perineum. This concept was further confirmed when we examined the ephrin-B2lacZ/lacZ
homozygous mice. As seen in , the newborn wild-type male (left panel) demonstrates a normal anorectal canal, normal bladder and completely tubularized urethra. In contrast, on the right, the newborn male ephrin-B2lacZ/lacZ
homozygous littermate has a high imperforate anus. The arrow indicates the location of the rectourethral fistula entering at the base of the bladder neck. Female ephrin-B2lacZ/lacZ
homozygous mice demonstrate persistent cloaca (). This work has recently been published39
. Taken together, these murine data reveal the key role of B-subclass Eph/ephrin signaling in normal midline fusion events of the perineum, cloaca and external genitalia, and reveal gene dosage sensitivity, yielding hypospadias, anorectal malformations and cloaca.
Figure 7 Upper panel: consecutive cross sections of embryonic day 17 (E17) wild-type mouse penis demonstrating tubularized urethra (arrow) and normal septated anus (asterisk). From left to right, the sections progress from the distal penis inward into the perineum. (more ...)
Figure 8 Low power (upper panel) and high power (lower panel) view of the perineum of three E16 littermates with the amputated tail (t) and genital tubercle (g) viewed at top and bottom of photos, respectively. (A) EphB2χi/+;EphB3Δ/Δ mouse (more ...)
Figure 9 Left: sagittal section of E18 male wild-type mouse demonstrating normal spine, anorectum and male urethra. Right: sagittal section of E18 ephrin-B2lacZ/+ male with normal spine and high imperforate anus with rectourethral fistula at the bladder neck. (more ...)
Figure 10 Left: sagittal section of E18 female wild-type mouse demonstrating normal spine, bladder (B), anorectum–hindgut (HG), vagina (V) and female urethra (asterisk). Right: sagittal section of E18 ephrin-B2lacZ/+ female with normal spine and high persistent (more ...)
To discriminate between EphB2-mediated forward (cell autonomous) and reverse (non-cell autonomous) signaling, a EphB2χ
mutation producing a kinase-inactive C-terminally truncated EphB2–β-gal fusion protein was bred to generate EphB2χ/χ
compound heterozygotes. The EphB2χ
- encoded EphB2–β-gal fusion protein exhibits normal protein trafficking and ability to stimulate reverse signaling in adjacent ephrin-expressing cells.