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

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.