Requirement for basement membrane laminin α5 during urethral and external genital development

Abstract

Hypospadias, a congenital malformation of the penis characteristic of an abnormal urethral orifice, affects 1 in every 125 boys, and its incidence is rising. Herein we test the hypothesis that the basement membrane protein laminin α5 (LAMA5) plays a key role in the development of the mouse genital tubercle, the embryonic anlage of the external genitalia. Using standard histological analyses and electron microscopy, we characterized the morphology of the external genitalia in Lama5 knockout (LAMA5-KO) mouse embryos during both androgen-independent genital tubercle development and androgen-mediated sexual differentiation. We compared regulatory gene expression between control and LAMA5-KO by in situ hybridization. We also examined the epithelial structure of the mutant genital tubercle using immunofluorescence staining and histological analyses of semi-thin sections. We found that Lama5 was expressed in both ectodermal and endodermal epithelia of the cloaca. The LAMA5-KO displayed a profound external genital malformation in which the genital tubercle was underdeveloped with a large ectopic orifice at the proximal end. In older embryos, the urethra failed to form a tubular structure and was left completely exposed. These defects were not associated with a significant alteration in regulatory gene expression, but rather with a defective ectodermal epithelium and an abnormal disintegration of the cloacal membrane. We conclude that LAMA5 is required in the basement membrane to maintain normal architecture of the ventral ectoderm during genital tubercle development, which is essential for the formation of a tubular urethra. Perturbation of LAMA5, and possibly other basement membrane components, may cause hypospadias in humans.

Keywords: Basement membrane; External genitalia; Hypospadias; Laminin α5; Urethra.

Figure 1. Cloacal expression of laminin α5 in e10.5 embryos

(A–C) Double immunofluorescence staining using E-Cadherin and LAMA5 antibodies. A) E-Cadherin was detected in both ectodermal and endodermal epithelia. Note that the two types of epithelia made direct contact in the cloacal membrane (boxed region). B) LAMA5 was detected in the basement membrane of both ectodermal (arrows) and endodermal (arrowheads) epithelia. However, it was not present between the two epithelia in the cloacal membrane (boxed region). C) Merged image. cm, cloacal membrane; ecto, ectodermal epithelium; endo, endodermal cloacal epithelium. Bars in all panels represent 50 μm.

Figure 2. Morphological comparison between control and LAMA5-KO GTs

(A–H) Scanning electron microscopy (SEM) images of control and LAMA5-KO GTs at e12.5 (A, B), e13.5 (C, D), e15.5 (E, F), and e17.5 (G, H). For all experiments, only males were used. Note the presence of the large proximal opening in e13.5 (arrow in D) and e15.5 (arrow in F) mutant GTs, and the presence of a small proximal orifice in e15.5 control GT (arrowhead in E). Also note the presence of a distal urethral orifice in the e17.5 control male GT (arrowhead in G), and a completely exposed urethral epithelium (arrows in H) in the mutant male GT. Bars in A through D represent 400 μm, in E and F represent 600 μm, and in G and H represent 800 μm.

Figure 3. Histological analyses of e17.5 male and female WT and LAMA5-KO GTs

(A–D) H&E staining of penis (A, C) and clitoris (B, D) of e17.5 embryos. Note the tubular urethra in the control penis (A) and clitoris (B), and the exposed urethral epithelium in the mutant penis and clitoris (arrows in C and D). ure, urethra, v, ventral side, d, dorsal side. Bars in all panels represent 100 μm.

Figure 4. Gene expression analyses of e12.5 WT and LAMA5-KO GTs

(A–L) Whole mount in situ hybridization analyses of control (A, C, E, G, and I) and LAMA5-KO GTs (B, D, F, H, and J) using the indicated probes. Note the similar expression patterns and levels in both groups.

Figure 5. Disruption of the double-layered cloacal membrane in the LAMA5-KO GTs

(A–C and E–G) Double immunofluorescence staining on transverse GT sections from e12.5 control (A–C) and LAMA5-KO (E–G) embryos. E-Cadherin was detected in both the ectodermal and urethral epithelia (A and E), whereas KRT14 was expressed in the ventral ectodermal epithelium (B and F). Note that in the LAMA5-KO, the KRT14-expressing ectodermal surface epithelium at the ventral midline (cloacal membrane) was missing (white arrows in F and G). (D, H) Shh in situ hybridization on e12.5 control (D), and LAMA5-KO (H) embryos. Note that the Shh-expressing urethral cells were covered by Shh-negative ectodermal epithelium in the control (D), while in the KO GT, the outermost layer of the midline epithelium was clearly Shh-positive (arrows in H). Bars in all panels represent 100 μm.

Figure 6. Defective ectodermal epithelium in the cloacal membrane of e10.5 LAMA5-KO mutants

(A, B) Toluidine blue-stained semi-thin sections of e10.5 control (A) and LAMA5-KO (B) cloaca. Note the difference in thickness (double headed arrows) and the disorganized contact between ectoderm and endoderm in the mutant cloacal membrane (boxed region). Bars in all panels represent 50 μm.