QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 24 October 2007
doi: 10.1242/dev.010249

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guioli, S. Articles by Lovell-Badge, R. Search for Related Content PubMed PubMed Citation Articles by Guioli, S. Articles by Lovell-Badge, R.

PITX2 controls asymmetric gonadal development in both sexes of the chick and can rescue the degeneration of the right ovary

Division of Stem Cell Biology and Developmental Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

View larger version (82K): [in this window] [in a new window]   Fig. 1. Expression profile of L-R asymmetry markers within the chick genital ridges before and after sex determination. (Top three rows) Transverse sections stained for fibronectin (Fn, red), N-cadherin (N-Cad, red), and DMRT1 plus Fn (in green and red, respectively) at E5 (HH26-27), E7 (HH30-31) and E12 (HH38). At E5 and E7, ZZ and ZW embryos show similar L-R differences, and only sections from ZW embryos are shown. Arrows in the ZZ E12 panel highlight areas of the left epithelium that are still DMRT1-positive. (Bottom two rows) Expression of ER (green) or ER and LHX9 (red) at HH30-31 (left) and HH38 (right). L, left; R, right. Scale bars: 50 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Fluorescence images of transverse sections from gonads of chick embryos treated with lindane. (A) E8.5 (HH35) embryo with complete situs inversus. The gonads display inversion of the L-R differences within the epithelium as shown by fibronectin (Fn) and DMRT1; DAPI staining of DNA (blue). (B) E7 (HH31) ZZ embryo with gut heterotaxia double stained for DMRT1 and fibronectin, or for N-cadherin (N-Cad). (C) E7 (HH31) ZW embryo with gut heterotaxia double stained for ER and LHX9. In B and C, both left and right gonads display a left pattern consistent with development as left isomers. L, left; R, right. Scale bar: 50 µm.

View larger version (103K): [in this window] [in a new window]   Fig. 3. Pitx2 expression is asymmetric during gonadal development. (A) Pitx2 expression between E2 and E12. (i) E2 (HH13) transverse section across the posterior part of the somitic region showing asymmetric expression of Pitx2 in the left splanchnic and somatic lateral plate mesoderm. (ii) At E3 (HH20), Pitx2 is asymmetrically expressed in the left gonadal region and weakly in the mesonephros. (iii) At E4.5 (HH25), Pitx2 asymmetric expression is maintained in the left side of the dorsal mesentery and in the adjacent gonad, but is lost in the mesonephros. (iv-vi) Pitx2 expression is maintained in the left gonad of both sexes at HH31(E7), HH36(E10), HH38(E12). (B) Pitx2c expression in left gonad and mesentery (E5; HH26). (C) Pitx2 expression in E7 (HH31) gonads from samples injected with lindane. An embryo with complete situs inversus expresses Pitx2 in the right gonad (i), whereas three out of four embryos with gut heterotaxia express Pitx2 bilaterally (ii) and one only in the left gonad (iii). IM, intermediate mesoderm; LPM, lateral plate mesoderm; L, left; R, right; LG, Left gonad; RG, right gonad; Me, mesentery; Ms, mesonephros; So, somite. Scale bars: 100 µm.

View larger version (46K): [in this window] [in a new window]   Fig. 4. Sections from three samples infected with RCAS-Pitx2a virus at HH8-10, screened at E7 (HH31) for expression of Pitx2 in the right gonad and analysed with the battery of asymmetry markers. (A,B) ZW and ZZ chick embryos with bilateral expression of Pitx2. The gonads are left isomers as shown by staining with DMRT1, fibronectin (Fn), N-cadherin (N-Cad) (white line demarks the epithelium-medulla border), and ER plus LHX9. (C) ZZ embryo with very patchy expression of Pitx2 in the right gonad: its phenotype is ambiguous, with some epithelial DMRT1 and localised deposits of Fn, but some areas of the epithelium have flattened (arrows). (D) E7.5 (HH31-32) embryo with gonadal left isomerism following RCAS-Pitx2a infection. Pitx2 expression is detected using either a probe for all Pitx2 isoforms or one specific for Pitx2c. L, left gonad; R, right gonad. Scale bars: white, 50 µm; black, 100 µm.

View larger version (116K): [in this window] [in a new window]   Fig. 5. E12-13 (HH38-39) gonads infected with RCAS-Pitx2a virus in ovo at HH8-10 analysed for the expression of H2AX, ER plus LHX9, and Pitx2. (A) E12 uninfected wild-type and (B,C,D) three infected samples showing some degree of right gonad `puffiness'. Of the panels showing antibody staining for H2AX and for ER plus LHX9, in B the right gonad is indistinguishable from the left, whereas in C and D localised superficial areas of the right gonad contain clusters of meiotic germ cells and these areas are positive for epithelial ER. In the panels showing in situ hybridisation for Pitx2, expression is in the entire epithelium (B) or in localised areas (C,D) of right gonads. Co-localisation of Pitx2 and the left epithelial markers is evident. Arrowheads in C,D indicate left-like areas. L, left gonad; R, right gonad. Scale bars: white, 100 µm; black, 1 mm.

View larger version (121K): [in this window] [in a new window]   Fig. 6. Expression of L-R identity markers in gonads from chick embryos electroporated with RCAS-Pitx2c and GFP DNAs at HH15-16 and screened at E7-8 (HH31-34) for GFP expression. (A-C) Sections of the gonads from three infected embryos (A,B,C are ZZ, ZW and ZW, respectively) showing the epithelial expression of DMRT1 (DM) and accumulation of fibronectin (Fn) found along the A-P axis (Ai,Bi,Ci). These samples also express epithelial ER (ER) to different degrees (Aii,Bii,Cii). The induction of epithelial ER in the right gonad is independent of the sex as it is also present in the male embryo (Aii). HA-tag antibody staining shows a partial colocalisation between PITX2 and ER (Bii). Double-staining for ER and LHX9 (LH) shows colocalisation within the cortical layer of the infected right gonad (Cii). (D) High levels of ectopic PITX2 induce the formation of `humps' at the surface epithelium (white arrows) and lack of a proper border between gonad and mesentery (i). The epithelial cells within the humps still express ER plus LHX9 (ii), DMRT1 (i,iii) and accumulate high levels of fibronectin (iii). Scale bars: 50 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 7. Analysis of cortical differentiation in the right gonad of chick embryos electroporated with RCAS-Pitx2c at HH15-16 and screened at E12 (HH38). (Top row) Transverse sections along the A-P axis of one right gonad containing germ cells clusters (DMRT1-positive) surrounded by fibronectin (Fn). (Bottom row) Left-like cortex areas are positive for ER plus LHX9 and contain germ cells positive for DMRT1 plus H2AX. The HA antibody staining (right) shows widespread expression of ectopic PITX2 found in extensively infected right gonads. The line marks left-like cortex areas. Scale bars: 50 µm.