Developmental and sexual dimorphic atlas of the prenatal mouse external genitalia at the single-cell level

Abstract

Birth defects of the external genitalia are among the most common in the world. Proper formation of the external genitalia requires a highly orchestrated process that involves special cell populations and sexually dimorphic hormone signaling. It is clear what the end result of the sexually dimorphic development is (a penis in the male versus clitoris in the female); however, the cell populations involved in the process remain poorly defined. Here, we used single-cell messenger RNA sequencing in mouse embryos to uncover the dynamic changes in cell populations in the external genitalia during the critical morphogenetic window. We found that overall, male and female external genitalia are largely composed of the same core cellular components. At the bipotential stage of development (embryonic day or E14.5), few differences in cell populational composition exist between male and female. Although similar in cell population composition, genetic differences in key sexual differentiation developmental pathways arise between males and females by the early (E16.5) and late (E18.5) differentiation stages. These differences include discrete cell populations with distinct responsiveness to androgen and estrogen. By late sexual differentiation (E18.5), unique cell populations in both male and female genitalia become apparent and are enriched with androgen- and estrogen-responsive genes, respectively. These data provide insights into the morphogenesis of the external genitalia that could be used to understand diseases associated with defects in the external genitalia.

One Tool for Many Jobs: Divergent and Conserved Actions of Androgen Signaling in Male Internal Reproductive Tract and External Genitalia

“…The androgen-independent phase involves the transformation of the primitive cloaca into the genital tubercle, the precursor of the penis. At the end of embryonic development, the genital tubercle morphs into the penis that consists of the distal dorsal glans, distal ventral glans, proximal glans, prepuce, corporal bodies, and urethra epithelium ( 67 ). The distal dorsal glans, distal ventral glans, and urethral epithelium have distinct cellular origins.…”

“…To accomplish this event, extensive androgen-dependent communication occurs between the urethral epithelium and proximal glanular mesenchyme. Although the urethral epithelium expresses Ar mRNA and protein throughout development ( 67 , 73 ), AR in the urethral epithelium is not required for urethral closure and proper penis formation ( 70 , 74 ) ( Figure 3 ). Conversely, inactivation of Ar in the surrounding mesenchyme causes severe cases of hypospadias, indicating the necessity of mesenchymal androgen signaling for urethra closure ( 70 , 74 – 76 ).…”

“…The contractility capability of the proximal glanular mesenchyme is not only induced directly by androgens, but also by androgen-dependent morphogen pathways from the urethra and proximal mesenchyme. The WNT signaling pathway, identified through the Gene Ontology analyses on the proximal mesenchyme, is specifically enriched in the proximal glanular mesenchyme of the penis ( 67 ). The main WNT ligands, Wnt5a, Wnt6, Wnt7a, Wnt10a and Wnt2 , are expressed throughout the penis, with Wnt5a being the main effector in the proximal glanular mesenchyme ( 82 , 83 ).…”

“…The distal ventral glanular mesenchyme subsequently interacts with the urethra to form a tube and complete the closure of the urethra. Similar to the proximal glans, the distal ventral glans express myosin light chain 12a ( Myl12a ) and Myl6 and is enriched with actin-cytoskeleton signaling pathway components, suggesting that it may exert similar mechanical forces on the distal urethra ( 67 ). The expression of these genes are also elevated in the male distal ventral glans compared to the female, an indication that they could be controlled by androgens.…”

“…Other than the androgen signaling, the distal ventral glans has the highest expression of estrogen receptor (ER) compared to the rest of the penis ( 67 ). Estrogen receptor and AR can be both antagonistic and agnostic to one another depending on hormone levels and tissue types ( Figure 3 ) ( 88 ).…”