Cessation of gastrulation is mediated by suppression of epithelial-mesenchymal transition at the ventral ectodermal ridge

Sho Ohta Kentaro Suzuki Katsuro Tachibana Hideaki Tanaka Gen Yamada ) In the gastrula stage embryo, the epiblast migrates toward the primitive streak and ingresses through the primitive groove. Subsequently, the ingressing epiblast cells undergo epithelial-mesenchymal transition (EMT) and differentiate into the definitive endoderm and mesoderm during gastrulation. However, the developmental mechanisms at the end of gastrulation have not yet been elucidated. Histological and genetic analyses of the ventral ectodermal ridge (VER), a derivative of the primitive streak, were performed using chick and mouse embryos. The analyses showed a continued cell movement resembling gastrulation associated with EMT during the early tailbud stage of both embryos. Such gastrulation-like cell movement was gradually attenuated by the absence of EMT during tail development. The kinetics of the expression pattern of noggin (Nog) and basal membrane degradation adjacent to the chick and the mouse VER indicated a correlation between the temporal and/or spatial expression of Nog and the presence of EMT in the VER. Furthermore, Nog overexpression suppressed EMT and arrested ingressive cell movement in the chick VER. Mice mutant in noggin displayed dysregulation of EMT with continued ingressive cell movement. These indicate that the inhibition of Bmp signaling by temporal and/or spatial Nog expression suppresses EMT and leads to the cessation of the ingressive cell movement from the VER at the end of gastrulation. INTRODUCTION During early embryogenesis, the primitive streak first appears as a thickening of the epiblast at the posterior marginal zone, the future caudal end of the embryo (Bellairs, 1986; Eyal-Giladi et al., 1991; Vakaet, 1984; Stern, 1990). The mediolateral intercalating cell movement and convergent extension is responsible for the progression of the primitive streak (Chuai et al., 2006; Lawson and Schoenwolf, 2001). The epiblast migrates toward the primitive streak and ingresses through the primitive groove to become the definitive endoderm and mesoderm. Such ingressing cells undergo epithelial-mesenchymal transition (EMT) and migrate into the blastocoel to form the mesodermal layer including the presomatic mesoderm (PSM), intermediate mesoderm and lateral plate mesoderm corresponding to the head-trunk region along the anterior-posterior (AP) axis (Shook and Keller, 2003). This developmental process is referred as gastrulation. Several bone morphogenetic protein (Bmp) genes (e.g. Bmp2, Bmp4, Bmp7) are expressed adjacent to the primitive streak, and BMP signaling has been shown to be essential for mesoderm formation from the gastrula to the somitogenesis stage (Komatsu et al., 2007; Mishina et al., 1995; Ohta et al., 2004). In addition, BMP signaling can induce EMT during the formation of neural crest (Liem, Jr et al., 1995; Liu and Jessell, 1998). The regulatory mechanism of EMT has been shown to involve the direct suppression of E-cadherin expression by Snail (Slug), one of the zinc-finger transcription factors (Batlle et al., 2000; Bolos et al., 2003; Cano et al., 2000). Consequently, epithelial cells acquire mobility through the reduction of cell-cell adhesion and differentiate into mesenchymal cells. The primitive streak and Hensens node are replaced by a bulblike structure, the tailbud, consisting of a morphologically uniform mass of mesenchyme, during the late gastrula stage (Schoenwolf, 1979a; Schoenwolf, 1981). The late primitive streak contributes to the ventral ectodermal ridge (VER) which is the thickened ectodermal tissue located at the tailbud ventrodistally, following the caudal elongation of the tailbud (Catala et al., 1995; Schoenwolf, 1981; Tam and Beddington, 1987; Wilson and Beddington, 1996). The histological similarity between the VER and the apical ectodermal ridge (AER) suggests that the VER is the signaling center for tail development and it positively regulates tail elongation through modulating proliferation of mesodermal cells during tail development (Cohn and Tickle, 1996; Globus and Vethamany Globus, 1976; Gruneberg, 1956; Reiter and Solursh, 1982). However, there is no direct evidence that the VER directly regulates the proliferation of mesodermal cells (Goldman et al., 2000). Gastrulation is a fundamental process of embryogenesis and many studies have investigated the developmental mechanism of the initiation, induction and/or patterning of mesoderm during gastrulation. However, the developmental processes at the end of gastrulation have not been elucidated so far. Since the VER is derived from the primitive streak of late gastrula stage, histological and genetic analyses of the VER are expected to reveal the developmental mechanisms at the end of gastrulation. This study identified one of the regulatory mechanisms controlling the cessation of ingressive cell movement from the VER at the end of gastrulation. MATERIALS AND METHODS Animals Fertilized White Leghorn chicken eggs were purchased from Marui Farm (Izumi, Japan). Eggs were incubated at 38.5C and staged according to the Hamburger and Hamilton classification (Hamburger and Hamilton, 1951). noggin (Nog) mutant mice have been previously described (Brunet et al., 1998; McMahon et al., 1998; Bachiller et al., 2000; Suzuki et al., 2003). In situ hybridization for gene expression analysis Whole-mount in situ hybridization was performed by standard procedures using probes for mouse (m) Bmp2, chicken (c) Bmp2, cBmp4, cBmp7, mBmp7 and cNog (Haraguchi et al., 2000) (kindly provided by B. L. Hogan, Duke University Medical Center, Durham, NC; T. Nohno, Kawasaki Medical School, Kurashiki, Japan; C. Tickle, University of Dundee, UK; B. Houston, University of Dundee, UK; M. Yoshida, IMEG, Kumamoto, Japan; and Y. Takahashi, Nara Institute of Science and Technology, Nara, Japan, respectively), mBmp4 (Jones et al., 1991), mNog (McMahon et al., 1998). Section in situ hybridization was performed by standard procedures using probes of mSnail (Snai1) and cSlug (kindly provided by A. Nieto, Instituto Cajal, Madrid, Spain, and H. Tanaka, Kumamoto University, Kumamoto, Japan). Developmental cell fate analysis DiI (1,1 -dioctadecyl-3,3,3 ,3-tetramethylindocarbocyanine perchlorate; Molecular Probes) was used as a fluorescent lineage labeling reagent at a concentration of 0.05% in 0.3 M sucrose to label both mouse and chick ectoderm including the VER. The culture of mouse tail grafts was performed as described previously (Goldman et al., 2000). Immunohistochemistry Paraffin sections of embryos fixed in 4% paraformaldehyde (PFA) were prepared with a microtome (MICROM, Germany). Immunostaining was performed by standard procedures. The specimens were incubated at 4C overnight with the following primary antibodies: anti-GFP antibody (mouse monoclonal, Roche); anti-E-cadherin (mouse monoclonal, BD Bioscience); anti-laminin (rabbit monoclonal, Sigma); anti-pSmad1.5.8 antibody (rabbit polyclonal, Cell Signaling Tec (...truncated)