Wnt4 coordinates directional cell migration and extension of the Müllerian duct essential for ontogenesis of the female reproductive tract

Human Molecular Genetics, 2016, Vol. 25, No. 6 1059–1073 doi: 10.1093/hmg/ddv621 Advance Access Publication Date: 31 December 2015 Original Article ORIGINAL ARTICLE Wnt4 coordinates directional cell migration and extension of the Müllerian duct essential for ontogenesis of the female reproductive tract 1 Oulu Centre for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, Laboratory of Developmental Biology, InfoTech Oulu, University of Oulu, PO Box 5000, FIN-90014 Oulu, Finland and 2Biocenter Oulu, University of Oulu, 90220 Oulu, Finland *To whom correspondence should be addressed at: Biocenter Oulu, Laboratory of Developmental Biology, Aapistie 5, University of Oulu, PO Box 5000, FIN-90220 Oulu, Finland. Tel: +358 407470939; Fax: +358 85315037; Email: seppo.vainio@oulu.fi Abstract The Müllerian duct (MD) is the anlage of the oviduct, uterus and upper part of the vagina, the main parts of the female reproductive tract. Several wingless-type mouse mammary tumor virus (MMTV) integration site family member (Wnt) genes, including Wnt4, Wnt5a and Wnt7a, are involved in the development of MD and its derivatives, with Wnt4 particularly critical, since the MD fails to develop in its absence. We use, here, Wnt4EGFPCre-based fate mapping to demonstrate that the MD tip cells and the subsequent MD cells are derived from Wnt4+ lineage cells. Moreover, Wnt4 is required for the initiation of MD-forming cell migration. Application of anti-Wnt4 function-blocking antibodies after the initiation of MD elongation indicated that Wnt4 is necessary for the elongation as well, and consistent with this, cell culture wound-healing assays with NIH3T3 cells overexpressing Wnt4 promoted cell migration by comparison with controls. In contrast to the Wnt4 null embryos, some Wnt4monomeric cherry/monomeric cherry (Wnt4mCh/mCh) hypomorphic mice survived to adulthood and formed MD in ∼45% of cases. Nevertheless, the MD of the Wnt4mCh/mCh females had altered cell polarization and basement membrane deposition relative to the controls. Examination of the reproductive tract of the Wnt4mCh/mCh females indicated a poorly coiled oviduct, absence of the endometrial glands and an undifferentiated myometrium, and these mice were prone to develop a hydro-uterus. In conclusion, the results suggest that the Wnt4 gene encodes signals that are important for various aspects of female reproductive tract development. Introduction The mammalian sex ducts are formed from the female Müllerian duct (MD) and the male Wolffian duct (WD) during embryogenesis. The paired MD, also called the paramesonephros, represents the primordium of the oviduct, the uterus and the upper part of the vagina, and is an integral part of the embryonic urogenital system. The MD initially forms in both the male and the female, but degenerates later in the male under the influence of the antiMüllerian hormone (1,2). The MD was described more than 200 years ago, but there are still lively discussions going on even today about its origin. It is now well-established that the MD cells do not originate from the WD cell population (2–4), even though the WD does provide † Present address: Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Received: July 25, 2015. Revised and Accepted: December 21, 2015 © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1059 Renata Prunskaite-Hyyryläinen1,†, Ilya Skovorodkin1, Qi Xu1, Ilkka Miinalainen2, Jingdong Shan1 and Seppo J. Vainio1, * 1060 | Human Molecular Genetics, 2016, Vol. 25, No. 6 Figure 1. The MD is constructed of cells of Wnt4EGFPCre lineage via cell migration, as depicted in time-lapse imaging. (A–D″) Selected snapshots from the time-lapse movie presented in Supplementary Material, Movie S3 depict an organ culture of a Wnt4EGFPCre; mT/mG embryonic urogenital ridge. (A) The MD primordium forms in the anterior part of the urogenital ridge in close proximity to the WD. Note the GFP+ Wnt4 lineage cells (A′ and A″ arrows). The CoE serves as a landmark for the migrating MD-forming cells (A′–D″, arrowhead). (B–B″) Micrographs depict a cell located in the MD primordium that has generated extensions (arrow) between the WD and the CoE (arrowhead). (C–C″) After differentiation of the tip cells (arrow) the MD-forming cells start to migrate in a posterior direction. (D–D″) The cellular processes protrude though the extracellular matrix between the WD and the CoE, which is positive for GFP+ (arrowheads). (E–E′″) Cryo-sections of the genital ridges of Wnt4EGFPCre; mT/mG embryos. (E) Merged images of the GFP+ (green) and mTomato (red) fluorescent cells marking Wnt4 lineage. (E′) The GFP+ cells of Wnt4+ lineage are seen (in green). The yellow arrowheads point to the Wnt4+ lineage cells contributing to MD assembly. Note that, there are also GFP− cells among the MD cells. Cells of Wnt4+ lineage are also present in the CoE, while the WD is negative for GFP. (E′″) Hoechst staining depicts the nuclei of the cells. The dotted line underlines the WD and the solid line the MD. Scale bars (A–D) 250 µm, (A′–D′) 50 µm, (A″–D″) 10 µm and (E–E′″) 50 µm. Human Molecular Genetics, 2016, Vol. 25, No. 6 Results Initiation of MD growth requires Wnt4 signalling The MD fails to form in Wnt4 knock-out embryos and only the anterior MD precursor cells differentiate, as depicted by the Wnt7a MD marker in in situ hybridization (Supplementary Material, Fig. S1, compare B with A, arrow) (13). To understand better how Wnt4 coordinates MD development, we examined the location of lineages derived from cells that have expressed Wnt4 by making the use of Wnt4EGFPCre mice crossed with floxed RosaR26R LacZ (R26R LacZ) so that the LacZ gene was flanked by LoxP sites and became activated by the Cre reaction. Although no LacZ staining was seen in the controls at any of the stages analysed (Supplementary Material, Fig. S1C, E, G and I), the Wnt4EGFPCre; R26RLacZ embryos had positive cells scattered within the urogenital ridge at E12.5-E15.5 (Supplementary Material, Fig. S1D, F, H and J). These were more numerous in the anterior side of the urogenital ridge at E12.5 (Supplementary Material, Fig. S1D, arrow), whereas later, at E14.5, they tended to accumulate in the posterior portion (Supplementary Material, Fig. S1H, arrow). Such observations led us to hypothesize that Wnt4 may be a factor that controls MD development by promoting cell migration. To study this, we performed lineage tracing of the cells expressing Wnt4 by time-lapse imaging of ex vivo urogenital ridge explant cultures from Wnt4EGFPCre; RosaR26R YFP (R26R YFP) embryos (Supplementary Material, Movies S1 and S2). The movies (...truncated)