Open Access Highly Accessed Research article

Foxl2 functions in sex determination and histogenesis throughout mouse ovary development

José Elias Garcia-Ortiz12, Emanuele Pelosi1, Shakib Omari1, Timur Nedorezov1, Yulan Piao1, Jesse Karmazin1, Manuela Uda3, Antonio Cao3, Steve W Cole4, Antonino Forabosco5, David Schlessinger1* and Chris Ottolenghi16*

Author Affiliations

1 Laboratory of Genetics, NIA/NIH-IRP, Baltimore, USA

2 División de Genética, Centro de Investigación Biomédica de Occidente, CMNO-IMSS, Guadalajara, México

3 Istituto di Neurogenetica e Neurofarmacologia, Consiglio Nazionale delle Ricerche, Cittadella Universitaria di Monserrato, Monserrato, Cagliari, Italy

4 Department of Medicine, Division of Hematology-Oncology, UCLA School of Medicine, Los Angeles, CA 90095-1678, USA

5 Unità di Genetica Medica, Università di Modena, Modena, Italy

6 Current address: UMR-S747 Inserm-Université Paris Descartes, Paris, France

For all author emails, please log on.

BMC Developmental Biology 2009, 9:36  doi:10.1186/1471-213X-9-36

Published: 18 June 2009

Additional files

Additional file 1:

The PCA plot from main text Figure 2 (x-axis: PC1; y-axis: PC2) in which the gonadal samples are coded to highlight additional features. A) distinct colored symbols and shapes indicate distinct experimental source (detailed next), sex, genotype, and developmental stage (detailed in the margins). "E" indicates embryonic day; "P" indicates postnatal day. B) color coding now is for the datasets of origin; distinct colors thus represent distinct experiments performed by different laboratories (with the number of samples per dataset that is given in brackets; see Methods). Shape-coding, as in A.

Format: PDF Size: 30KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 2:

Developmental markers for the PCA, thus including all the markers identified as significant by Focus analysis. The list in A (top 6455 probes) was used for the main figures; the lists of the genes that were most significantly associated with the first and second principal components (PC1 and PC2) are given in B and C, resp.

Format: XLS Size: 956KB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data

Additional file 3:

Genes that were down- (A, C) or up-regulated (B, D) in Foxl2-null ovaries aged 13.5 dpc through birth compared to age-matched controls. Scores (second column from left) are standard deviations of four parameters for differential expression obtained with the Focus program (see text). A-B: overall ("multiwise") comparison of three developmental stages using linear contrasts with Focus (i.e., 6 genotype × stage conditions). C-D: overlap of the gene lists obtained by three separate pairwise analyses each confined to age-matched conditions (i.e., each developmental stage was analyzed separately; Focus scores were averaged over the three analyses).

Format: XLS Size: 1MB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data

Additional file 4:

Genes that are consistently down- (C) or up- (D) regulated in all three pathological conditions involving Foxl2 loss that we have tested. A-B. Genes that are down- or up- regulated (A-B, resp.) in double knockout ovaries lacking Foxl2 and either Wnt4 or Kit relative to controls. C-D: genes shared between such two models of double knockout ovaries and the ovaries lacking Foxl2 alone (from additional files 3).

Format: XLS Size: 146KB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data

Additional file 5:

Microarray gene expression profiles of the genes that were assayed by real-time PCR in Main Figure 2. For each gene, normalized expression intensities (y-axis) are represented as a fraction of the maximum mean value observed (the latter being set to 100).

Format: PDF Size: 24KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 6:

Genes that were differentially expressed in single knockout ovaries lacking genes different from Foxl2. A-B, genes that were down- or up-regulated in Wnt4-null ovaries aged 15.5 dpc relative to controls aged 13.5 dpc through birth. C-J, genes that were down- (C, E, G, I) or up- (D, F, H, J) regulated in Lhx8- (C, D), Nobox- (E, F), Foxo3-null (G, H) newborn ovaries or Emx2-/- (I, J) 10.5 dpc bipotential gonads relative to age-matched controls.

Format: XLS Size: 3.3MB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data

Additional file 7:

Overlap between the lists of Foxl2- or Wnt4-dependent genes. A, downregulated, B, upregulated in both single knockout ovaries relative to controls, as determined from additional files 3 and 6.

Format: XLS Size: 329KB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data

Additional file 8:

KEGG classification of the gene lists relevant to Table 3. The gene lists are derived from additional files 3 (down and up in Foxl2-null fetal ovaries or in Wnt4-null fetal ovaries relative to wild-type controls (from additional files 3 and 6). A-B, Foxl2-null ovaries vs wild-type; C-D, Wnt4-null ovaries vs wild-type. All KEGG categories with p-value less than 0.1 by Fisher exact test (comparison of proportions) are included. Columns from left to right: Pathway name, numbers of genes for the knockout and for the wild-type ovaries, p-value, proportions for the knockout and for the wild-type ovaries, and finally two standard gene identifications. The 10% FDR threshold p-value is 0.0077 for A-B, and 0.0054 for C-D.

Format: XLS Size: 357KB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data

Additional file 9:

Lists of anti-testis genes that were inferred to have an action independent of Foxl2 (A), Wnt4 (B) or both (C). comparison of knockout ovaries to both ovary and testis (see text for details on the approach).

Format: XLS Size: 1.6MB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data

Additional file 10:

Real-time PCR showing Foxl2 transcript levels in Foxl2 transgenic mouse embryonic gonads. XX or XY trangenic embryos were compared to wildtype littermates.

Format: PDF Size: 13KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 11:

Real-time PCR validation in Foxl2 transgenic mouse embryos and wildtype littermates. This panel includes Foxl2-dependent genes (based on knockout models, see text) that are induced at higher levels in Foxl2 transgenic XX gonads and are either weakly induced (Plxnc1, Tcf4, Odz4) or not induced (Gpc4, Foxp1, Mllt3, Akr1c14) in Foxl2 transgenic XY gonads.

Format: PDF Size: 98KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 12:

Oocyte and somatic cell-enriched gene lists represented by the corresponding probes in the Affymetrix MOE430 v.2 (A) and Agilent mouse developmental (B) platforms. These lists were used for GSEA analysis presented in additional file 13. They correspond to the top-scoring hits from additional microarray analyses (see text) with an arbitrary cut-off at the 50th or 100th rank (labeled as "Top50" and "Top100"). Additional labels (see Methods for details): OvFetSoma, enriched in fetal ovary somatic cells relative to bipotential gonad and to testis (Sf1-positive cells); FetalOo, enriched in newborn oocytes relative to postnatal growing oocytes; OoGrow, enriched in postnatal growing oocytes relative to newborn oocytes; SomaGrow, enriched in postnatal growing somatic cells by an indirect analysis, i.e., enriched in postnatal versus newborn whole-organ ovary preparation and excluded from the "OoGrow" markers; "testis determination markers", genes known to be involved in gonadal XY sex reversal when inactivated.

Format: XLS Size: 59KB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data

Additional file 13:

GSEA plots for the study of Foxl2-gene dosage effects. Any deviation from random of the distribution of 9 lists of marker genes given in additional file 12 is tested between the following conditions. Labels at the top of each panel correspond to the acronyms that denote the lists of additional file 12. A blue-red gradient along the x-axis indicates increasing differential expression toward either condition under comparison. Values for the relevant statistical parameter are shown on the y-axis by a green line. Thus, the highest vs lowest peak of this curve relative to a horizontal black line (baseline) provide estimates for the degree of enrichment toward either the "red" or the "blue" condition, respectively. A, Foxl2-null (E16ko, red) vs Foxl2+/- heterozygous ovaries at E16.5 dpc (E16het, blue); B, E16het (red) vs Foxl2+/+ wild-type ovaries at E16.5 dpc (E16wt, blue); C, E16ko (blue) vs E16wt (red); D, Foxl2-null (P7ko, red) vs Foxl2+/- ovaries at 7 dpn (P7het, blue); E, P7het (red) vs Foxl2+/+ wild-type ovaries at 7 dpn (P7wt, blue); F, P7ko (red) vs P7wt (blue).

Format: PDF Size: 1.5MB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 14:

Representative gene expression levels (microarray) that illustrate Foxl2-gene dosage effects in newborn (P0) or 7 dpn ovaries (P7). Top panel: somatic cell genes; bottom panel: oocyte genes. Note that P7 heterozygous ovaries (2nd group of bars from right) express high levels of oocyte genes and low levels of somatic genes compared to age-matched wild-type ovaries (right-most group of bars).

Format: PDF Size: 108KB Download file

This file can be viewed with: Adobe Acrobat Reader

Open Data

Additional file 15:

Comparison of our in vivo gene list from Foxl2-null ovaries with a published in vitro gene list. Our list is from additional files 3. The in vitro gene list is from an ovarian cell line transiently transfected with Foxl2 (see text).

Format: XLS Size: 22KB Download file

This file can be viewed with: Microsoft Excel Viewer

Open Data