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First published online 24 December 2003
doi: 10.1242/dev.00951

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Requirement of Lim1 for female reproductive tract development

¹ Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA
² Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA
³ Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA
⁴ Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, Center for Neurobiology and Behavior, Columbia University, New York, NY 10032, USA

View larger version (83K): [in a new window]   Fig. 1. Dynamic expression of Lim1 during Müllerian duct formation. (A-F) Lim1tlz expression in the developing urogenital system. Cranial mesonephric region at E11.5 (A) and E12.0 (B). Ventral view at E12.5 (C) and at E13.5 in females (E) and males (F). Cross-section of the anterior mesonephric region at E12.5 counterstained with Eosin (D). (G,H) Lim1 expression detected using whole-mount in situ hybridization at E12.5 (G) and E13.5 (H) in the female genital system. Asterisks point to the posterior end of the Müllerian duct and broken lines indicate the gonad. g, gonad; k, kidney (metanephros); m, mesonephros; md, Müllerian duct; wd, Wolffian duct. Scale bars: 250 µm in A-C,G,H; 100 µm in D; 500 µm in E,F.

View larger version (67K): [in a new window]   Fig. 2. Sexual dimorphic expression pattern of Lim1 in the developing reproductive tract. Lim1tlz expression in the developing urogenital system in females (A,C,E,G,I) and males (B,D,F,H,J). (A,B) Ventral view at E14.5. Note that Lim1tlz expression in the Müllerian duct is thinner in males compared to its expression in females. (C,D) Ventral view at E15.25. Lim1tlz expression in the Müllerian duct is discontinuous in the anterior region of males (open arrow in D). (E,F) Ventral view at E15.5. Lim1tlz expression in the Müllerian duct is fragmented in males (arrow in F). (G,H) Ventral view at E16.5. Lim1tlz expression in the Müllerian duct is restricted to a few vesicles in males (arrow in H). (I,J) Lateral view at E17.5. Lim1tlz is expressed in the presumptive oviduct, but not in the uterus in females (I). The open arrowhead indicates the boundary of the presumptive oviduct and uterus. Lim1tlz expression is upregulated in the male reproductive tract (J). a, adrenal; e, epididymis; k, kidney (metanephros); md, Müllerian duct; od, oviduct; ut, uterus; v, vas deferens; wd, Wolffian duct. Scale bar: 500 µm.

View larger version (63K): [in a new window]   Fig. 3. Lim1 expression in MIS signaling mutant mice. (A-D) Lateral view of the testicular region in wild-type (A), Misr2–/– (B), Mis–/– (C) and Wnt7a–/– (D) male embryos at E15.75. Fragmentation of Lim1tlz expression in the Müllerian duct is observed in wild-type embryos (arrows in A), which is suppressed in MIS signaling-deficient mutants (B-D). (E-J) Ventral view of the urogenital system in wild-type (E,G,I) and MT-hMIStg/+ transgenic (F,H,J) female embryos. (K,L) Ventral view of the testicular region in wild-type (K) and MT-hMIStg/+ transgenic (L) male embryos. Note that fragmentation of Lim1tlz expression in the Müllerian duct is enhanced in MIS transgenic males. k, kidney (metanephros); md, Müllerian duct; od, oviduct; ut, uterus; wd, Wolffian duct. Scale bars: 250 µm in A-D; 500 µm in E-L.

View larger version (48K): [in a new window]   Fig. 4. Absence of the female reproductive tract in Lim1–/– female neonates. (A,B) Newborns of wild-type (A) and Lim1–/– (B). (C,D) Gross view of the female reproductive system from wild-type (C) and Lim1–/– (D) newborns. (E,F) High magnification of the anterior gonadal region in C and D, respectively. Note that Lim1–/– XX newborns have normal ovaries (ov). (G,H) Longitudinal sections of the urogenital system from wild-type (G) and Lim1–/– (H) female newborns. Note that neither epithelium nor mesenchyme of the female reproductive tract is found in Lim1–/– XX mice (asterisk in H). b, bladder; cv, cervix; ge, genitalia; mg, midgut; od, oviduct; ov, ovary; pb, pubic bone; r, rectum; ut, uterus; vg, vagina. Scale bars: 5 mm in A,B; 500 µm in C-H.

View larger version (23K): [in a new window]   Fig. 5. Strategy to generate female mouse chimeras. (A) Schematic diagram of female chimera analysis. Lim1–/–;Rosa26tg/+ XO ES cells were generated from Lim1–/–;Rosa26tg/+ XY ES cells (Shawlot et al., 1999) by spontaneous loss of the Y chromosome by screening with an Y-chromosome-specific repeat probe, Y353/B. These XO ES cells were injected into XX blastocysts to generate chimeric female mice. XX blastocysts can be distinguished from XY blastocysts by using X-linked ubiquitous GFP male mice for breeding. Chimeric mice were recovered at different stages of embryogenesis and processed for ß-gal staining to visualize Rosa26-marked ES cell-derived XO cells. The yolk sac was collected for reconfirming sexes of injected blastocysts by PCR genotyping for the Sry gene. (B) Southern blot analysis of XO ES cells. Genomic DNA of XO ES cells was blotted after EcoRI digestion and hybridized with a Lim1 probe. The same blotted membrane was hybridized again with Y-chromosome-specific Y353/B probe. Genomic DNA from male and female tails was used for comparison. Note that these XO cells show the same pattern as female tail.

View larger version (70K): [in a new window]   Fig. 6. Cell-autonomous requirement of Lim1 for epithelium development of the female reproductive tract. (A-B) Craniofacial development of female chimeras at E18.5. XO cells derived from ES cells are stained blue by ß-gal staining. High contribution of Lim1–/– cells in chimeras cased head truncations. (C-F) The female reproductive tract of female chimeras. XO cells derived from ES cells are stained blue by ß-gal staining and wild-type XX cells derived from blastocysts are pink by eosin staining. Lim1–/– cells are not present in the epithelium in the uterus (arrow in D) and the oviduct (arrow in F) but present in the surrounding mesenchyme of the both tissues. In control experiments, wild-type XO cells can contribute to the epithelium of the uterus (arrow in C) and the oviduct (arrow in E). (G,H) The reproductive tract of female chimeras at E12.5. Lim1–/– XO cells are not present in the epithelium of the developing Müllerian duct (arrow in H). Lim1–/– XO cells are also not found in the epithelium of the Wolffian duct (arrowhead in H). Scale bars: 5 mm in A,B; 50 µm in C-F; 25 µm in G,H.

View larger version (74K): [in a new window]   Fig. 7. Lim1 expression in mouse mutants with Müllerian duct abnormalities. (A-D) Ventral view of the right urogenital system in wild-type (A), Wnt4–/–(B), Pax2–/–(C) and Wnt7a–/–(D) embryos at E12.5. (E,F) Ventral view of the anterior urogenital system in Wnt4+/– (E) and Wnt4–/–(F) embryos at E11.5. Arrows in F indicate Lim1tlz expressing presumptive Müllerian duct precursor cells. (G,H) Lateral view with high magnification. Note that Lim1tlz-positive Müllerian duct precursor cells do not show funnel-shaped invagination (arrow in H). Broken lines indicate the gonad. k, kidney (metanephros); m, mesonephros; md, Müllerian duct; wd, Wolffian duct. Scale bars: 250 µm in A-F; 100 µm in G,H.