Development of the human penis and clitoris.

First and second-trimester human fetal specimens were collected free of patient identifiers after elective termination of pregnancy (Committee on Human Research approval at the University of California at San Francisco, IRB# 12–08813). Gender was determined by inspection with a dissecting microscope for Wolffian (mesonephric) and Müllerian (paramesonephric) ductal morphology. For the youngest specimens at the indifferent stage (8 to 10 weeks), PCR of the SRY- chromosomal sequences was used to determine sex ( Cui et al., 1994 ) ( Li et al., 2015 ). Heel-toe length was used as a determinant of gestational age ( Drey et al., 2005 ; Mercer et al., 1987 ; Mhaskar et al., 1989 ). Human fetal specimens were then examined by optical projection tomography ( Li et al., 2015 ), lightsheet fluorescence microscopy ( Isaacson et al., 2018a ), and scanning electron microscopy ( Shen et al., 2016 ) as previously described. Transverse and sagittal serial paraffin sections of human fetal specimens were prepared and stained with hematoxylin and eosin or immunostained using antibodies for cytokeratins (KRT) 6, 7, 10 and 14, androgen receptor, Foxa1, Mafb, Runx1/2/3, Notch, the proliferation marker Ki67, and the apoptotic marker caspase 3 ( Li et al., 2015 ; Overland et al., 2016 ). Immunostaining was detected using horseradish-peroxidase-based Vectastain kits (Vector Laboratories, Burlingame, CA) or by immunofluorescence. Negative controls deleted the primary antibody ( Robboy et al., 2017b ).