Lessons learned from developmental biology show a remarkable degree of conservation of pathways/signaling molecules to program developmental processes across species. Alongside these roles, eutherian mammals have further utilized distinct developmental strategies to home developing embryos in the womb, and to nurture their growth through the processes of implantation, decidualization and placentation. This study presents two major unexpected findings. First, we show that Msx genes, known for their critical roles in development, particularly in craniofacial and limb development, have profound effects on implantation. Second, we present evidence suggesting that this role for Msx genes may be mediated by altering epithelial cell integrity through Wnt5a and E-cadherin/β-catenin complex formation.
Delayed implantation (diapause), a condition of suspended animation, is widespread in mammals. Nearly 100 mammals in seven orders exhibit this condition under various physiological and environmental conditions (Lopes et al., 2004
; Renfree and Shaw, 2000
), but the underlying mechanism by which the uterus and the embryo temporarily achieve quiescence and then resume implantation under favorable conditions remains largely unknown. The failure to initiate implantation in delayed implanting uteri lacking Msx1/2 strongly suggests that Msx
genes are crucial for conferring readiness (neutral phase) to respond to an implantation cue. A comparative study on Msx
genes in mammals which undergo delayed implantation may provide valuable information.
The relationship between Msx1 and LIF is also intriguing. Our present results suggest a positive-negative feedback loop between Msx1 and LIF in regulating each other’s activity. We propose that increasing Msx1 expression with rising P4 levels superimposed by preimplantation estrogen on day 4 facilitates Lif expression, and increased levels of LIF then downregulate Msx1 prior to implantation. The fact that Msx1 expression persists in Lif−/− mice and that Lif is downregulated with loss of Msx genes suggests that the regulation of one is influenced by the other, but their effects on implantation are mediated by different mechanisms, since implantation fails to occur in females deleted of Msx genes even after LIF administration.
The absence of Bmp2
and aberrant Cox2
expression at the site of blastocyst apposition in mice deleted for Msx
genes suggests that the epithelial-mesenchymal interactions prerequisite for implantation are disturbed. Defective implantation in these mice suggests that Msx
genes are required for optimal uterine receptivity with appropriate epithelial cell integrity for normal blastocyst attachment and invasion. Upregulation of Wnt5a in uteri deleted of Msx
genes and binding of Msx1
suggest that this morphogen is a potential downstream target of Msx
genes and is important in regulating cell polarity. While Wnt5a is involved in uterine gland formation (Mericskay et al., 2004
), the present results suggest that Wnt5a may also orchestrate epithelial apical-basal polarity by regulating E-cadherin/β-catenin complex formation. Our present in vivo
and in vitro
findings are consistent with previous work in cell culture systems that demonstrate roles for Wnt5a in directing cell polarity and cytoskeleton reorganization by influencing E-cadherin/β-catenin complex formation and adhesion receptors (Medrek et al., 2009
; Mericskay et al., 2004
; Witze et al., 2008
Increased association of E-cadherin and β-catenin in Msx
-deleted uteri with heightened Wnt5a levels suggests a non-canonical Wnt pathway without apparent changes in Wnt/β-catenin signaling. However, a role for Wnt/β-catenin cannot completely be ruled out, since there is evidence that Wnt5a and Wnt11 can form complexes and activate both non-canonical and canonical pathways in Xenopus
axis formation (Cha et al., 2008
). This suggests that nuclear β-catenin dependent and independent pathways are not always mutually exclusive.
Wnt5a can stabilize E-cadherin/β-catenin complex formation by increasing β-catenin phosphorylation by casein kinase 1 in cultured mammary epithelial cells (Medrek et al., 2009
), and ROR2 can mediate non-canonical Wnt signaling in the presence or absence of Fzd (Green et al., 2008
). Our results showing the presence of putative Wnt5a receptors Fzd2 and ROR2 and the downstream kinase CK1ε in the LE suggests that this signaling pathway is a potential mediator of Wnt5a’s effects in the uterus in the context of Msx
genes. It is interesting that ROR2 mediates the transcriptional activity of Msx2 and Dlx5 (Matsuda et al., 2003
) and that expression patterns of ROR2, Wnt5a
genes show many similarities during development (Green et al., 2008
; Satokata et al., 2000
; Satokata and Maas, 1994
; Yamaguchi et al., 1999
). This suggests that Msx-Wnt5a-ROR2 pathway is also involved in uterine biology. Whether other effector pathways of Wnt5a are active in implantation will require further investigation.
The aberrant uterine luminal configuration, in the absence of Msx genes, notably its failure to assume a slit-like architecture and form a crypt, lend support to the importance of LE integrity in uterine biology and implantation. Since the expression of Msx genes is undetectable in stromal cells before, during and after implantation, the compromised decidualization in mice deleted of uterine Msx genes suggests that the defective LE function leads to poor stromal cell proliferation and differentiation to decidualization, ultimately compromising pregnancy outcome. The role of Msx genes in epithelial-mesenchymal interactions during development recapitulates their similar functions in epithelial-stromal interaction during implantation. Upregulation of Wnt5a in both the epithelium and stroma in uteri missing Msx genes is consistent with a role of these homeotic proteins in epithelial-stromal interaction.
Our findings have led us to believe that Msx
genes play crucial roles in transitioning the uterus from the prereceptive to the receptive to the nonreceptive state. The importance of Msx1/Msx2 in human implantation is underscored by microarray gene expression analyses showing that both genes are downregulated during the window of implantation in women (Kao et al., 2002
; Mirkin et al., 2005
; Riesewijk et al., 2003
), similar to which occurs preceding implantation in mice. The findings that Msx
genes are critical to uterine receptivity and maintaining uterine readiness to implantation without altering the ovarian hormone levels or uterine sensitivity to these hormones are of high relevance to female fertility. These findings raise the possibility that clinicians may be able to develop new strategies to improve implantation rates in IVF programs by temporarily increasing uterine levels of Msx to extend the uterine responsiveness to implantation prior to embryo transfer. In the same vein, further uncovering the role of Msx may aid in developing non-hormonal contraceptives.