1. Male-to-Female Sex Reversal in Mice Lacking Fibroblast Growth Factor 9
Jennifer S. Colvin, Rebecca P. Green, Jennifer Schmahl, Blanche Capel, David M. Ornitz 
Cell 
Volume 104, Issue 6, Pages (March 2001)
DOI: /S (01)

2. Figure 1
Mammalian Reproductive Development and Targeted Disruption of Fgf9
(A) Mammalian reproductive development. At E11.0, males and females have indifferent gonads. Associated mesonephroi contain a Mullerian duct (MD, pink) and a Wolffian duct (WD, blue). Between E11.0 and E13.5, male-specific changes include: (1) proliferation (green) at the coelomic epithelium; (2) migration of mesonephric cells (arrows) into the testis; and (3) organization of testicular cords (hatched ovals). red ovals, basal lamina. In males, testosterone (T) from Leydig cells induces WD development into the epididymis, vas deferens, and seminal vesicles, and MIS from Sertoli cells induces MD regression. In females, the WD regresses, and the MD develops into the oviduct and uterus. (E18.5 panels of 1A adapted from Behringer et al. (1994).)
(B) Diagram of an E13.5 testicular cord. Sertoli cells (s) surround germ cells (blue nuclei) within the cord. A basal lamina (red line) and peritubular myoid cells (p) surround the cord. l, Leydig cells.
(C) Structure of the Fgf9 genomic locus, targeting vector, and mutant allele. Protein coding regions of exons (black boxes), untranslated regions (white boxes), Southern blot probes, and PCR primers are shown.
(D and E) Detection of homologous recombination in ES clones (D) and genotyping of embryos (E) by Southern blot. The 3′ probe detects a 7.0 kb EcoRI fragment in the genomic locus and a 4.8 kb fragment in the mutant allele. The 5′ probe detects a 10.0 kb SpeI fragment in the genomic locus and a 5.5 kb fragment in the mutant allele.
(F) PCR genotyping of embryos showing fragments from the genomic locus (310 bp; primers P1/P4) and the mutant allele (234 bp; P1/N1).
(G) RT-PCR analysis of expression from the mutant allele. Transcript joining exons 1 and 2 (primers P1/P2) and transcript joining exons 1 and 3 (P1/P3) were detected in wild-type and heterozygote brain RNA, but not in Fgf9−/− RNA. Fgf14 primers were used to control for RNA quality.
(H) Sex genotyping PCR generated a Zfy fragment in XY samples and a Rapsyn fragment in all samples
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3. Figure 2
Fgf9 Gonad Expression and E18.5 Reproductive System Morphology
(A–D) In situ hybridization for Fgf9 in E12.5 embryos. Gonadal germ cells exhibit alkaline phosphatase activity (A and C). Fgf9 is expressed in the XY gonad (arrow in [B]), but not immediately below the coelomic epithelium (ce). The XX gonad (arrow in [D]), and mesonephroi (m) in XX and XY embryos, lack Fgf9 expression.
(E–H) E18.5 internal reproductive organs. (E) XY control, showing testis (t), epididymis (e), and bladder (b). (F) Hypoplastic Fgf9−/− (F9−/−) testis. (G) Phenotypically female Fgf9−/− XY reproductive system, with ovaries (o), oviducts (arrow), and fused uteri (u). (H) XX control. ([E] and [F] are shown at approximately 2.5× the magnification of [G] and [H].)
(I–P) H & E sections of E18.5 gonads. (I) Control testis showing testicular cords (tc), germ cells (g), Sertoli cells (s), interstitial Leydig cells (arrow), and a capsule (asterisk). (J) Fgf9−/− testis with slightly disorganized cords (containing Sertoli and germ cells), interstitial Leydig cells (arrow), and a capsule (arrowheads). The cords in (J) were the most organized in the section and were in a region with extensive mesenchyme (arrow in [P]). (K) Mixed histology in a grossly ovarian Fgf9−/− XY gonad. Testicular cords contain germ and Sertoli cells. (L) Ovarian histology in an Fgf9−/− XY gonad. There are no cords, and a single-cell-layer epithelium surrounds the gonad (arrowhead). (M) Control testis with cords in extensive mesenchyme (m). (N) Fgf9−/− testis with somewhat disorganized cords (arrows) and lack of distinct regions of loose connective tissue. ([M] and [N] are shown at 0.4× the magnification of [I]–[L].) (O) Control XY testis (t), vas deferens (v), and epididymis (e). (P) Fgf9−/− testis with a hypoplastic epididymis. ([J] and [P] show the same testis.) (Panels labeled only XY or XX are from controls (+/+ or +/−). Panels labeled MXYF9−/− are from phenotypically male Fgf9−/− XY mice, and panels labeled FXYF9−/− are from phenotypically female Fgf9−/− XY mice.)
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4. Figure 3 Gene Expression in E18.5 Reproductive Organs
(A–D) Immunohistochemistry for MIS on gonad sections. (A) Control testis showing MIS in Sertoli cells (photographed using 20× objective). (B) Fgf9−/− testis (20×, same gonad as in Figures 2J and 2P) with some irregular MIS staining. (C) Limited MIS in an Fgf9−/− XY gonad (10×). (D) No MIS in an Fgf9−/− XY gonad (10×).
(E–H) In situ hybridization for Scc on sections from the gonads in (A)–(D). (E) Control testis (5×) with Scc in interstitial Leydig cells. (F) Fgf9−/− testis (5×) showing Scc limited to regions of normal MIS staining. (G) Fgf9−/− XY gonad showing Scc only in the MIS-positive region of the gonad (10×). (H) No Scc in an Fgf9−/− XY gonad (10×). (Asterisk in [C] corresponds to the Scc-positive region in [G]. Dotted lines in [F]–[H] outline the gonad.)
(I–L) Coexistence of Wolffian and Mullerian duct derivatives in Fgf9−/− XY embryos. (I and J) H & E staining (I) and Wnt7a in situ hybridization (J) of uterine tissue (u) in an Fgf9−/− phenotypic male. This embryo showed reduced MIS expression (B), and had testicular and epididymal tissue (Figures 2J and 2P).
(K and L) H & E section from an Fgf9−/− XY phenotypic female with limited Scc expression (Figure 3G). (K) Note the epididymal-like tissue (arrowhead). The uterine horn shown (arrow) is shorter than XX controls. (L) Boxed area in (K) showing parallel ducts in the uterine horn
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5. Figure 4 Morphology and Gene Expression in E14.5 Gonads
(A) Control testis with testicular cords.
(B) A hypoplastic Fgf9−/− XY gonad without visible cords.
(C) Female morphology of an Fgf9−/− XY gonad.
(D) Control XX gonad.
(E–G) H & E stained gonad sections (40×). (E) Control testis with testicular cords (asterisk) in extensive mesenchyme (m). In cords, Sertoli cells (arrowheads) surround germ cells (arrow). (F) Rudimentary cords (asterices) and reduced mesenchyme in an Fgf9−/− XY gonad. Cords are less organized and less compact than XY controls (E). (G) Ovarian histology in an Fgf9−/− XY gonad.
(H–J) Immunohistochemistry for MIS on gonad sections (20×). (H) Control testis showing MIS-positive Sertoli cells in cords. (I) Irregular MIS staining in an Fgf9−/− XY gonad. (J) No MIS in an Fgf9−/− XY gonad.
(K–M) In situ hybridization for Scc on gonad sections (10×). (K) Control testis showing Scc in Leydig cells. (L and M) No Scc in an Fgf9−/− XY gonad (L) and in a control ovary (M).
(N–P) Immunohistochemistry for SSEA-1 on gonad sections (20×). (N) Control testis showing SSEA-1-positive germ cells clustered in cords. (O) An Fgf9−/− XY gonad that lacked testicular cords showing SSEA-1-positive germ cells throughout the gonad. (P) Control ovary. These sets of panels are from the same gonads: (E), (H), and (K); (B), (F), (I), and (L); and (G) and (J)
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6. Figure 5
Impaired Cord Formation and Sertoli Cell Differentiation in E13.5 Fgf9−/− XY Gonads
(A–H) Toluidine blue stained plastic sections of gonads (63×). (A) Control testis with Sertoli cells (s) surrounding germ cells (g) in a cord, peritubular myoid cells (p) lining the cord, and mesenchyme (m) around the cord and adjacent to the coelomic epithelium (left). (B) Control ovary with germ (g) and somatic cells (sc) dispersed throughout. (C) Fgf9−/− XY gonad with disorganized cord structures containing germ (g) and somatic cells (sc). A few mesenchymal cells (m) sit on cord surfaces. (D) Rudimentary cord in another Fgf9−/− XY embryo, showing lack of mesenchymal cells (m). (E–H) Lower power images (20×) of gonads in (A)–(D). (E) Control testis showing extensive mesenchyme around well-defined cords. (F) Control ovary lacks distinct mesenchymal zones. (G) Fgf9−/− XY gonad showing less compact cords and smaller mesenchymal regions (arrows) than control testis. (H) An Fgf9−/− XY gonad lacking distinct mesenchymal regions.
(I–P) TEM images of XY gonads. (I) Control testicular cord with a central germ cell (g) and peripheral Sertoli cells (s). Peritubular myoid cells (p) line the cord. (J) Boxed region of (I) showing cisternae of RER (arrow) in a Sertoli cell. (K) Fgf9−/− XY testicular cord. A peritubular myoid cell (p) is in close contact with presumptive Sertoli cells (s). (L) Boxed region of (K). Cisternae of RER (arrows) are smaller and less abundant than in (J). (M) Fgf9−/− XY gonad with a disorganized, cord-like cell cluster lined by mesenchymal cells (asterisk). Arrow indicates gap between cord and mesenchymal cells. (N) Cytoplasmic blebs (arrows) between a presumptive Sertoli cell (s) and a mesenchymal cell (asterisk). (O) Control testicular cord showing cytoplasmic interdigitation (arrow) and close contact (arrowheads) between Sertoli cells. (P) Boxed region of (M) showing gaps (arrowheads) between cells within a rudimentary cord. Scale bars: (I), (K), and (M) 4.5 μm; (J) and (L) 0.54 μm; (N) 1.3 μm; (O) 0.35 μm; (P) 0.67 μm
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7. Figure 6
Proliferation in E12.5 and Sox9 Expression in E12.5 and E18.5 Gonads
(A–C) In situ hybridization for Sox9 on E12.5 gonad sections (10×). (A) Control testis showing Sox9 in Sertoli cells. (B) Sox9 in an Fgf9−/− XY gonad. (C) No Sox9 in an Fgf9−/− XY gonad. (Dotted line outlines the gonad.)
(D–F) MIS immunohistochemistry on sections (10×) from the gonads in (A)–(C). (D) Control testis. Sertoli cell MIS staining delineates organizing testicular cords. Note unstained mesenchyme between MIS-positive cords and the coelomic epithelium (ce). (E) Fgf9−/− XY gonad with diffuse MIS staining and reduced mesenchyme beneath the coelomic epithelium. (F) No MIS in an Fgf9−/− XY gonad.
(G–I) BrdU immunohistochemistry on gonad sections. (Dotted red lines show height of area counted in proliferation analysis.) (G) Control testis with BrdU-labeled Sertoli, germ (large round nuclei, arrowhead), and mesenchymal cells (spindle-shaped nuclei, arrows). Germ cells are excluded from a zone of mesenchyme (m) below the coelomic epithelium (top). (H) The XY Fgf9−/− gonad shown in (C) and (F) showing fewer BrdU-labeled mesenchymal cells (arrow) than in (G). Note germ cells (arrowhead) just below the coelomic epithelium. (I) Control ovary resembles (H).
(J) Cell proliferation in E12.5 gonads (see Experimental Procedures). Gonad sections from BrdU-exposed embryos were stained using an anti-BrdU antibody. Counts of BrdU-labeled nuclei within a set area were determined for total cells (solid bars), somatic cells (open bars), and germ cells (shaded bars). For each parameter, Fgf9−/− XY gonads were significantly different (asterices) from control testes (P < ), but not significantly different from control ovaries (P > 0.1). Error bars show standard deviation.
(K–M) Sox9 in situ hybridization on E18.5 XY gonad sections (5×). (K) Control testis showing Sox9 in testicular cords. (L) Sox9 in an Fgf9−/− testis with extensive MIS-positive cords (see Figure 3B). (M) Limited Sox9 in an Fgf9−/− XY gonad with a few MIS-positive cords (see Figure 3C). Scale bar in (g): 40 μm
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8. Figure 7 FGF9 Induces Mesonephric Cell Migration in Culture
(A) XY gonads induce migration from the neighboring mesonephros. When an XY gonad from a “white” CD1 mouse is cultured next to a mesonephros from a “blue” ROSA26 mouse, migration results in extensive blue staining within the XY gonad.
(B) XX gonads do not induce extensive migration from the mesonephros.
(C) When XX gonads are cultured with FGF9 in the media, migration into the XX gonad is induced
Cell  , DOI: ( /S (01) )