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- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
Background: The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail-bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear. Results: We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β, and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co-expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell-matrix interactions regulating BMP signalling at this site of caudal morphogenesis. Conclusions: Laminin532 could interact with α6-containing integrin to direct differentiation of the specialised VER cells from surface ectoderm. Developmental Dynamics 241:1808–1815, 2012. © 2012 Wiley Periodicals Inc.
- Top of page
- RESULTS AND DISCUSSION
- EXPERIMENTAL PROCEDURES
After completion of gastrulation, the tail begins to form in the caudal-most region of the mouse embryo, beginning with the generation of the tail-bud. From embryonic day (E) 9.5 onwards, the tail-bud contains a morphologically distinct group of ectodermal cells known as the ventral ectodermal ridge (VER). The VER is a source of signals that regulate tail development, and contains progenitor cells that contribute to the ventral midline ectoderm of the tail (Gruneberg,1956; Goldman et al.,2000). Ablation of the VER leads to defects in somitogenesis and tail elongation (Goldman et al.,2000). However, the mechanisms through which the VER exerts its effect on these processes have not been fully elucidated.
One candidate player in mediating the effects of the VER is the bone morphogenetic protein (BMP), Bmp2. Bmp2 is expressed in the cells of the VER, while the BMP antagonist, noggin, is expressed in the immediately adjacent mesoderm of the ventral tail-bud. Noggin expression is no longer detected in the ventral mesoderm after ablation of the VER (Goldman et al.,2000). Although the signals from the VER that regulate noggin expression are unclear, it is suggested that Bmp2 could induce noggin production, as part of a negative feedback loop (Goldman et al.,2000). The signals that control the restricted expression of Bmp2 in the VER have yet to be identified.
The BMPs form a large subclass of the transforming growth factor β (TGFβ) superfamily of signalling molecules, with demonstrated involvement in embryonic events including neurulation and dorso-ventral patterning (Mehler et al.,1997). BMPs have been specifically implicated in regulating outgrowth and patterning of the Xenopus tail-bud (Beck et al.,2001) and, together with nodal, in function of the zebrafish tail organizer (Agathon et al.,2003; Fauny et al.,2009). The BMP signalling pathway is well characterised (Attisano and Wrana,2002), and its activity can be monitored by analyzing the expression of downstream genes such as Cadherin6, Rhob (Sela-Donenfeld and Kalcheim,1999), and Msx1 and Msx2 (Marazzi et al.,1997; Suzuki et al.,1997; Kettunen and Thesleff,1998).
BMP signalling is regulated by extracellular antagonists including chordin, chordin-like 1 (Chrdl1; also called neuralin1), follistatin, and noggin, and by the intracellular antagonists Smad6 and Smad7 (Attisano and Wrana,2002; Rider and Mulloy,2010). We previously described how Bmp2 signalling is modulated by its antagonists, and by sonic hedgehog (Shh), during the process of spinal neural tube closure (Ybot-Gonzalez et al.,2007). In addition to these well-described BMP regulators, other factors, such as the extracellular matrix components collagen IV, heparan sulphate proteoglycans, and laminins, have also been found to play a role in modulating BMP signalling (Belenkaya et al.,2004; Wang et al.,2008; Dolez et al.,2011). It is unclear whether any of these extracellular modulators are involved in the regulation of BMP signalling in the VER.
One group of potential extracellular modulators of BMP signalling are the laminins, which are major glycoprotein components of basement membranes. Laminins have been implicated in many biological processes, including cell adhesion, migration, and differentiation (Colognato and Yurchenco,2000; Miner and Yurchenco,2004). At least 16 different laminin variants exist, and their expression in basement membranes is spatially and developmentally regulated (Tunggal et al.,2000; Yurchenco et al.,2004; Aumailley et al.,2005; Tzu and Marinkovich,2008). Laminins are heterotrimers containing an α, β, and γ chain in a cross-like three-dimensional structure (Colognato and Yurchenco,2000). To date, five distinct α chains, three β chains, and three γ chains have been described, and their various combinations define the different laminin isoforms (Miner et al.,1997; Patton et al.,1997; Miner and Yurchenco,2004). Basement membranes can contain more than one laminin isoform (Yurchenco et al.,2004; Miner,2008) but, owing to the intracellular assembly of the laminin heterotrimer prior to its secretion, co-expression of α, β, and γ chain mRNAs in a particular cell type is obligatory for production of each specific laminin isoform.
Cellular responses to laminin are determined in part by a group of transmembrane receptors known as integrins (Miranti and Brugge,2002). The integrin family is composed of 24 α, β heterodimeric members that mediate the attachment of cells to the extracellular matrix (Barczyk et al.,2010). Integrins containing the α3 and α6 subunits have been described as receptors for laminin, regulating activities such as organization of the basement membrane and differentiation of several epithelial cell types (Sorokin et al.,1990; Kadoya et al.,1995; Walker and Menko,1999). Interestingly, during osteoblast differentiation, Bmp2 has been reported to stimulate the expression of αV and β integrins, which, in turn, are essential for Bmp2 activity (Lai and Cheng,2005).
In an effort to gain insight into the factors controlling Bmp2 signalling in the VER, we have studied the mRNA expression of Bmp2 signalling components, together with the protein and mRNA expression patterns of all known laminin chains, in the tail-bud of the mouse embryo. We also examined expression of the α3 and α6 integrin subunits. Taken together, our results suggest the existence of a previously undescribed laminin variant that may be implicated in the regulation of Bmp2 responsiveness in the VER via interaction with α6-containing integrin.