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First published online 16 January 2008
doi: 10.1242/dev.012856

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Mechanism of asymmetric ovarian development in chick embryos

Yoshiyasu Ishimaru^1,2, Tomoko Komatsu², Megumi Kasahara¹, Yuko Katoh-Fukui², Hidesato Ogawa², Yoshiro Toyama³, Mamiko Maekawa³, Kiyotaka Toshimori³, Roshantha A. S. Chandraratna⁴, Ken-ichirou Morohashi^2,*,, and Hidefumi Yoshioka^1,*

¹ Department of Natural Sciences, Hyogo University of Teacher Education, 942-1, Shimokume, Kato, Hyogo 673-1494, Japan.
² Division for Sex Differentiation, National Institute for Basic Biology, National Institutes of Natural Sciences, Myodaijicho, Okazaki, Aichi 444-8787, Japan.
³ Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan.
⁴ Retinoid Research, Departments of Chemistry and Biology, Allergan, Irvine, CA 92623, USA.

View larger version (55K): [in this window] [in a new window]   Fig. 1. L-R asymmetric development and gene expression in developing gonads of chick. (A,B) The number of cortical and medullary cells was determined as described (see Materials and methods and Fig. S2E,F in the supplementary material). The relative fold changes in the number of cortical (A) and medullary (B) cells are plotted, with the numbers in the right cortex and right medulla of the female gonad at stage 27 set at 1. (C-F) Cell proliferation was examined at stage 27 (C) and stage 29 (D). The developing gonads in BrdU-labeled embryos were sectioned and subjected to immunostaining for BrdU (green) and cytokeratin (red) (C,D). Relative fold changes in the number of BrdU-positive cells in the gonadal cortex and medulla are plotted, with the numbers in the right cortex (E) and right medulla (F) of the female gonad at stage 27 set at 1. (G-L) The expression of RALDH2 (G), CYP26A1 (H), RAR (I), PITX2c (J), Ad4BP/SF-1 (K) and LHX9 (L) in the embryonic gonad at stage 27 was examined in both sexes by whole-mount in situ hybridization. Sections of female (ZW) gonads are shown. R and L indicate right and left, respectively. ZW and ZZ indicate female and male, respectively. Scale bars: 100 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 2. Effects of RA signaling on the expression of PITX2c, Ad4BP/SF-1 and cell proliferation in chick. (A-F) RA and RAA beads were implanted into the left (A,B) and right (C,D) presumptive gonad regions of both sexes, respectively, at stage 18-19. As controls, DMSO beads were implanted (E,F). 48 hours after implantation (stage 27), the embryos were subjected to whole-mount in situ hybridization with Ad4BP/SF-1 (A,C,E) and PITX2c (B,D,F) probes, then the embryos were sectioned. Sections of female (ZW) samples are shown. Arrowheads in A and C indicate affected expression of Ad4BP/SF-1. (G-N) The effects of implantation of RA (G) and RAA (I) beads on cell proliferation were examined at stage 27 in female embryos. DMSO beads were used as a control (H,J). BrdU was injected into the operated embryos 1 hour before fixation, then gonads were stained with antibodies for BrdU (green) and cytokeratin (red). Effects of RA (K) and RAA (M) beads on cortical thickness were examined by staining with anti-cytokeratin antibody 72 hours after the implantation (stage 29). DMSO beads were used as controls (L,N). (O-R) Cell proliferation was analyzed quantitatively in gonads of embryos implanted with RA (O,P), RAA (Q,R) and DMSO beads at stage 27. Total DAPI-stained cells (data not shown) and BrdU-positive cells in the gonadal cortex (cytokeratin-positive) (O,Q) and medulla (cytokeratin-negative) (P,R) were counted. Relative fold changes in the number of BrdU-positive cells in the cortex and medulla are plotted, with the number of BrdU-positive cells in the right cortex of DMSO-bead-implanted embryos set at 1. Scale bars: 100 µm in A-J; 50 µm in K-N.

View larger version (84K): [in this window] [in a new window]   Fig. 3. Function of PITX2 in the gonadal cortex before sexual differentiation. (A-I) Chick embryonic fibroblasts producing Pitx2- (A-C) and Pitx2-en- (D-F) encoding viruses were implanted into the right and left presumptive gonad regions of both sexes, respectively, at stage 11-12. As a control, cells producing AP-encoding virus were implanted into the right presumptive gonad regions (G-I). Distribution of the virus was examined by in situ hybridization for mouse Pitx2 (A,D) and by AP staining (Morgan and Fekete, 1996) (G). Arrowheads in A,D,G indicate viral distribution in the developing gonads. 72 hours after implantation (stage 27), embryos were subjected to whole-mount in situ hybridization for RALDH2 (B,E,H) and Ad4BP/SF-1 (C,F,I). Arrowheads in B,C,E,F indicate altered expression of RALDH2 and Ad4BP/SF-1. Sections of female (ZW) samples are shown. (J-U) The effect of exogenous Pitx2 (J), Pitx2-en (L) and AP (K,M) on cell proliferation was examined in female embryos at stage 27. BrdU was injected into the operated embryos 1 hour before fixation, and their gonads were stained with antibodies for BrdU (green) and cytokeratin (red) (overlays in J-M). The effect of misexpressed Pitx2 (N), Pitx2-en (P) and AP (O,Q) on cortical thickness was examined by staining with anti-cytokeratin antibody at stage 29. Cell proliferation was analyzed quantitatively in gonads misexpressing Pitx2 (R,S) or Pitx2-en (T,U) at stage 27. AP was used as a control. The total number of DAPI-stained cells (data not shown) and BrdU-positive cells in the gonadal cortex (cytokeratin-positive) (R,T) and medulla (cytokeratin-negative) (S,U) was counted. Relative fold changes in BrdU-positive cell numbers in the cortex and medulla are plotted with the numbers in the right cortex and medulla misexpressing AP set at 1. Scale bars: 100 µm in A-M; 50 µm in N-Q.

View larger version (70K): [in this window] [in a new window]   Fig. 4. Function of Ad4BP/SF-1 in the gonadal cortex before sexual differentiation. (A-E) Chick embryonic fibroblasts producing Ad4BP/SF-1 (A,B,D) and AP (C,E)-encoding viruses were implanted into the right presumptive gonad of both sexes at stage 11-12. The distribution of Ad4BP/SF-1-encoding viruses was examined by in situ hybridization for chick Ad4BP/SF-1 (A). As chick Ad4BP/SF-1 was used for the implantation study, in situ hybridization detected both endogenous and exogenous expression. The arrowhead in A indicates viral distribution in the developing right gonadal cortex, where Ad4BP/SF-1 gene is not endogenously activated (see Fig. 1K). The effects on cell proliferation (B,C) and cortical thickness (D,E) were examined as described in Fig. 3. (F,G) Relative numbers of BrdU-positive cells in the cortex (F) and medulla (G). Scale bars: 100 µm in A-C; 50 µm in D,E.

View larger version (51K): [in this window] [in a new window]   Fig. 5. Effect of RA, PITX2 and Ad4BP/SF-1 on cyclin D1 expression.(A-H) Beads soaked in RA (B), RAA (C) and DMSO (D), and chick embryonic fibroblasts expressing Pitx2 (E), Pitx2-en (F), Ad4BP/SF-1 (G) and AP (H) were implanted into embryos of both sexes as described in the legends to Figs 2 and 3. An untreated embryo is shown in A. Embryos were fixed at stage 27, and cyclin D1 expression was examined by whole-mount in situ hybridization. Sections of female (ZW) samples are shown. Arrowheads (B,C,E-G) indicate altered expression of cyclin D1. Scale bars: 100 µm.

View larger version (88K): [in this window] [in a new window]   Fig. 6. Effects of RA signaling and Ad4BP/SF-1 on the expression ER and CYP26A1. (A-K) Beads soaked in RA (B,G), RAA (C,H) and DMSO (D,I), and chick embryonic fibroblasts expressing Ad4BP/SF-1 (E,J) and AP (F,K) were implanted into embryos of both sexes as described in Figs 2 and 3. An untreated embryo is shown in A. The embryos were fixed at stage 27, and the expression of ER (A-F) and CYP26A1 (G-K) was examined by whole-mount in situ hybridization. Sections of female (ZW) samples are shown. Arrowheads in B and C, and in G and H, indicate affected expression of ER and CYP26A1, respectively. Scale bars: 100 µm.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Asymmetric gonad development in sexually differentiating chick embryos. Expression domains of PITX2c, Ad4BP/SF-1, RALDH2, ER and cyclin D1 before sexual differentiation (stages 18 and 27) in the chick embryonic gonad, along with expression patterns of aromatase and ER at sex-determined stages (stages 29 and 30) in the female chick embryonic gonad are illustrated. The events shown in the sex-independent process occur in both sexes. L-R differential genetic and functional cascades are indicated at stage 27, in accordance with our findings. The genes in black and gray are expressed and suppressed genes, respectively.

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