Embryo implantation is a gateway to pregnancy success. A crosstalk between the blastocyst and uterus is vital to implantation. Formation of a healthy implantation chamber (crypt) is critical for normal embryo implantation; an abnormal crypt results in subfertility. Planar cell polarity (PCP) is a classic downstream target of the non-canonical Wnt5a signaling pathway. Uterine deletion of Vang- like protein 2 (Vangl2), a major PCP component, severely derails crypt formation, embryo spacing, and decidualization, ultimately compromising pregnancy outcomes. We have found that crypts invariably originate with preexisting glands conferring direct communication between the implanting blastocyst and glands. Heparin-binding EGF-like growth factor (HB-EGF) is exclusively expressed in the crypt epithelium surrounding the blastocyst, and beads preloaded with HB-EGF, if transferred to day 4 pseudopregnant uteri, evoke implantation-like changes similar to normal pregnancy. However, these responses fail to occur in uteri missing Vangl2, suggesting an intersection of Vangl2 with HB-EGF signaling. HB-EGF executes its function via the EGF family of receptors (Erbbs 1-4) as homodimers or heterodimers and induces tyrosine phosphorylation. This proposal will test a novel hypothesis that HB-EGF signaling intersects with PCP signaling by engaging the ErbBs in implantation. HB-EGF working via ErbBs 1-4 induces tyrosine phosphorylation. Our preliminary results show a physical association of Vangl2 with ErbB2 and ErbB3, suggesting that these receptors, if activated by HB-EGF, transphosphorylate Vangl2. The following two specific aims will study interactions between PCP and HB-EGF signaling in implantation using mouse models: Specific aim 1 will test the hypothesis that HB-EGF signaling intersects with Vangl2 signaling to direct the formation of an appropriate glands-crypt assembly for direct cross-talk between the implanting blastocyst and the glands. This interlocking of two different signaling pathways involves phosphorylation of members of the ErbB family of receptors followed by transphosphorylation of Vangl2. Specific aim 2 will test the hypothesis that the functional coupling of HB-EGF and Vangl2 signaling in implantation requires the presence of ErbB1, ErbB2 and/or ErbB3 in the uterine epithelium and/or stroma. We will use conditional deletion of ErbB1, ErbB2 and/or ErbB3 using Pgr-Cre and Ltf- iCre drivers to explore the Vangl2 phosphorylation status in the uterus, and correlate these results with pregnancy phenotypes. Successful completion of this project will reveal a conceptual attribute to the field of implantation and pregnancy biology that a growth factor signaling pathway intersects with PCP signaling in forming implantation chambers (crypts) for pregnancy success.

Heparin-binding EGF-like growth factor (HB-EGF) via EGF family of receptors intersects with planar cell polarity (PCP) signaling to confer successful embryo implantation; but the mechanism by which this intermingling of two signaling pathways is functionally and molecularly executed remains unknown. The proposed research will test the hypothesis that Vangl2, a major PCP signaling component, undergoes transphosphorylation that is achieved by autophosphorylation of specific EGF family of receptors as homo- or heterodimers by HB-EGF in the uterus. Upon completion, the results will bring new perspectives in the field of uterine biology with respect to embryo implantation and pregnancy events that will be applicable to reproductive health of women.