The Rete Ovarii undergoes dynamic changes during development alongside the ovary.

(a,b,c,d top row) Diagrams depicting the development of the three regions (EOR-magenta; CR-cyan; IOR-yellow) of the RO from E16.5-P7 in the whole ovarian complex (ovary-light pink; oviduct-grey). (a,b,c,d bottom rows) Maximum intensity projection from confocal Z-stacks of whole ovary/mesonephros complexes at E16.5 (a), E18.5 (b), P0 (c) and P7 (d) immunostained for PAX8 (yellow), GFRa1 (cyan) and KRT8 (magenta). a,b,c,d second row are composite images, while the third row show separate panels for KRT8 and and the bottom row show panels for GFRa1. *Note that GFRa1 and KRT8 do not co-localize and are specific to the CR and EOR, respectively. All figures are dorsal views of the ovary. Yellow asterisk indicates opening of the infundibulum for reference. Scale bar – 100um

The EOR is highly integrated with its extra-ovarian environment suggesting multifaceted communication.

(a) Top panel is a whole ovarian complex maximum intensity projection of the confocal Z-stack at E18.5 immunostained for ENDOMUCIN (magenta) and PAX8 (cyan). Bottom panel is an optical section showing vasculature tightly surrounding the DDT of the EOR (magenta). (b) Top panel is a whole ovarian complex maximum intensity projection of the confocal Z-stack at E18.5 immunostained for aSMA (magenta) and KRT8 (cyan). Bottom panel is an optical section showing the EOR tightly ensheathed by smooth muscle (magenta). (c) Top panel is a whole ovarian complex maximum intensity projection of the confocal Z-stack at E18.5 immunostained for TUJ1 (magenta) and PAX8 (cyan). Bottom panel is an optical section showing direct contacts between the EOR and neuronal projections (magenta). (d) EOR at P0 immunostained for PAX8 (yellow), F4/80 (magenta) and LYVE-1 (cyan). Top and bottom panel are maximum intensity projection of the confocal Z-stack. Bottom panel shows the absence of LYVE1 macrophages (cyan) proximate to the EOR, in contrast to the closely associated F4/80 macrophages (magenta in top image). Arrowheads show regions devoid of LYVE1 macrophages where F4/80 macrophages are present. Scale bar – 100um

Fluid moves from the EOR to the ovary.

(a,b) Schematic of dextran injections (syringe) for each group (a-EOR injections, b-ovary injections) (Oviduct-grey, ovary-light pink, IOR-yellow, CR-cyan, EOR-magenta). (Second row) Maximum intensity projection of a confocal Z-stack of whole ovarian complexes at P7, where Dextran was injected into the EOR. Presence of Dextran in the ovary shows that when dextran is injected into the EOR, it diffuses throughout the ovary (dextran-cyan, E-CADHERIN-magenta). (bottom row) Maximum intensity projection from confocal Z-stacks of whole ovary/mesonephros complexes at P7 where Dextran was injected into the ovary. Absence of Dextran signal in the EOR shows that dextran did not travel into the EOR when injected into the ovary near the IOR. Scale bar – 100um

The EOR and CR acquire the potential for fluid transfer

(a,b,c,d) Cells of the CR acquire E-CADHERIN soon after birth. Maximum intensity projection from confocal Z-stacks of whole ovary/mesonephros complexes at E16.5-P7 immunostained for PAX8 (magenta) and E-CADHERIN (cyan). Arrowhead shows the region of the CR where E-CADHERIN expression is negligible at E16.5 (a), low at E18.5 (b) and fully expressed at P0 (c) and P7 (d). (e) Optical section from confocal Z-stacks of RO complexes at P0 immunostained for E-CADHERIN (yellow), CNN1 (magenta), and aSMA (cyan). Boxed regions show the absence of CNN1 in the aSMA+ sheath around the EOR at P0, and (f) its acquisition by P7, suggesting gain of contractility. (g) The RO expresses cilia marker ARL13b by P0. Maximum intensity projection from confocal Z-stacks of RO complexes at E18.5 immunostained for E-CADHERIN (cyan) and ARL13b (magenta). Outlined boxes show all regions of the EOR have ciliated cells. Scale bar – 100um (h). Airyscan optical section from confocal Z-stacks of RO complexes at E18.5 immunostained for KRT8 (cyan) and ARL13b (magenta). Scale bar – 25um

Cells of the EOR secrete proteins essential for vesicle transport.

Candidate list of 15 proteins identified by Mass Spectrometry. Bolded proteins were selected for validation due to role in vesicle and protein transport.

Presence of SNARE-complex members suggests a role for secretion.

(a) Optical section from confocal Z-stacks of PAX8-rtTa; Tre-H2B-GFP (cyan) EOR at E18.5 using HCR for detection of Clu expression (magenta). (b) Optical section from confocal Z-stacks of PAX8-rtTa; Tre-H2B-GFP (cyan) EOR at E18.5 using HCR for Stxbp2 (magenta). (c) Optical section from confocal Z-stacks of PAX8-rtTa; Tre-H2B-GFP (cyan) EOR at P7/8 using HCR for Clu (magenta). (d) Optical section from confocal Z-stacks of PAX8-rtTa; Tre-H2B-GFP (cyan) EOR at P7/8 using HCR for Stxbp2 (magenta). (e) Optical section from confocal Z-stacks of PAX8-rtTa; Tre-Cre; Rosa-mTmG (cyan) EOR at E18.5 immunostained for STX3 (magenta). Outlined higher resolution image acquired with Airyscan (f) Optical section from confocal Z-stacks of PAX8-rtTa; Tre-Cre; Rosa-mTmG (cyan) EOR at E18.5 immunostained for RAB11 (magenta) Outlined higher resolution image acquired with Airyscan Scale bar – 100um

Validation of Igfbp2 expression in the RO.

(a) Maximum intensity projection of confocal Z-stacks of PAX8-rtTa; Tre-H2B-GFP (cyan) EOR at E18.5 using HCR for detection of IGFBP2 expression (magenta). (b) Maximum intensity projection of confocal Z-stacks of PAX8-rtTa; Tre-H2B-GFP (cyan) EOR at P7/8 using HCR for detection of IGFBP2 expression (magenta). Bottom panels are outlined higher resolution image acquired with Airyscan. Scale bar top panel – 100um. Scale bar top panel – 25um