Figure 5. Developmental dynamics of the ovarian ligaments correlate with ovary morphogenesis.

(A) Maximum intensity projections from confocal Z-stacks of whole urogenital complexes at E15.5, E16.5, and E18.5 immunostained for TUJ1 (red) and FOXL2 (cyan—left and middle panel only), and counterstained with Hoechst nuclear dye (grayscale). Images on the bottom row are close-ups of the regions outlined in the top row images. (B) Maximum intensity projections from confocal Z-stacks of the whole urogenital complex of XX SF1-eGFP mice at E14.5 (left) and P0 (right), immunostained for GFP (green) and TNC (E14.4, red) or aSMA (P0, red), and counterstained with Hoechst nuclear dye (grayscale). The E14.5 sample was imaged from the ventral side and the P0 from the dorsal. The left bottom panel is a high magnification view of the area outlined in the left top panel. White arrows point to the continuity of the CSL from the ovary to the adrenal. (C) Maximum intensity projections from confocal Z-stacks of whole ovary/mesonephros complexes at E14.5 and E18.5 immunostained for FOXL2 (cyan) and TNC (red), and imaged from the medial side. White arrows point to the continuity of the CSL between the medial ridge of the ovary and the infundibulum. (D) Maximum intensity projections from confocal Z-stacks of whole ovary/mesonephros complexes at E15.5, E16.5, and E17.5 immunostained for RUNX1 (cyan) and aSMA (red), and imaged from the ventral side. White arrows point to the population of RUNX1+ cells linking the medial edge of the mesovarium to the infundibulum, and to the CSL. (E) 3D models generated by isosurface segmentation of lightsheet images taken of whole ovary/mesonephros complexes at E14.5 (left) and E17.5 (right) stained with Hoechst nuclear dye (grayscale), and false-colored to illustrate the three regions of the BL (purple, mesometrium; yellow, mesosalpinx; green, mesovarium), the CSL (red), and the ovary (cyan). Each row represents a different view of the same ovary for each stage (from top to bottom: medial; ventral; lateral; dorsal). Yellow asterisks indicate the location of the infundibulum of the presumptive oviduct for reference. Ad, adrenal; CSL, cranial suspensory ligament; K, kidney; Mm, mesometrium; Mn, mesonephros; Ms, mesosalpinx; Mv, mesovarium; OC, ovarian capsule; Ov, ovary; Ovi, oviduct; Ut, uterus. Scale bars, 100 μm.

Figure 5—figure supplement 1. The mesovarium and cranial suspensory ligament tether the ovary to the rest of the urogenital complex.

(A) Confocal images of whole ovary/mesonephros complexes at E15.5, E16.5, and E18.5 immunostained for aSMA (green), and counterstained with Hoechst nuclear dye (grayscale). Samples in the top two rows were imaged from the dorsal side and samples in the bottom row from the medial side. (B) 3D models generated by isosurface segmentation of lightsheet images taken of whole ovaries at E14.5, E17.5 and P0 immunostained for FOXL2 (cyan) and TNC (E14.5, red) or labeled with AF647-Hydrazine to reveal Elastin expression (E17.5, P0, red). All samples were counterstained with Hoechst nuclear dye (grayscale). Top panels represent the dorsal view, middle panels represent the medial view, and small bottom panels illustrate ventral and lateral views of the same ovary. White dashed arrows show the direction to the body wall. Yellow asterisks indicate the location of the infundibulum of the presumptive oviduct for reference. Compasses on the bottom left of each panel indicate the orientation of the ovary for the entire row: CSL, cranial suspensory ligament; D, dorsal; H, hilum; L, lateral; M, medial; MD, Müllerian duct; Mm, mesometrium; Mn, mesonephros; Ms, mesosalpinx; Mv, mesovarium; OA, ovarian artery; OC, ovarian capsule; Ov, ovary; Ovi, oviduct; Ut, uterus; V, ventral. Scale bars, 100 μm.