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First published online 14 November 2007
doi: 10.1242/dev.008151

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

¹ Center for Animal Resources and Development (CARD), Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan.
² JSPS research fellow, Chiyoda-ku 1-8, Tokyo 102-8472, Japan.
³ Department of Anatomy, Fukuoka University School of Medicine, Fukuoka, Japan.
⁴ Department of Developmental Neurobiology Graduate School of Medical Sciences, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan.

View larger version (85K): [in this window] [in a new window]   Fig. 1. Histological and fate map analysis of chick tail development. (A) A schematic illustration of chick caudal embryogenesis. cl, cloaca; cm, cloacal membrane; hg, hind gut; ps, primitive streak, tb, tailbud; ver, ventral ectodermal ridge. (B) The tailbud of chick embryos at HH stage 18. The white line in B indicates the approximate level of the section shown in C. Scale bar: 400 µm. (C) Transverse section of the chick tailbud. no, notochord; nt, neural tube; psm, presomatic mesoderm; tvm, tail ventral mesoderm. Bar: 100 µm. (D) High magnification of the box in C, showing the chick VER and the adjacent region. Scale bar: 50 µm. (E) Immunostaining with laminin and E-cadherin of the chick VER. Bar: 50 µm. (F) Fate mapping of the tailbud ectoderm including the chick VER. The ectoderm was labeled with DiI at HH stage 16 and 20 (left panels). Middle and right panels show distribution of labeling after 24 hours. The white line in the middle panels indicates the position of the sections shown on the right. hl, hind limb. Scale bars: whole mounts, 800 µm, 400 µm; sections, 100 µm.

View larger version (118K): [in this window] [in a new window]   Fig. 2. Bmp(s) and Nog expression pattern and degradation of the basal membrane. Gene expression analysis for the tailbud at HH stage 17. (A,D) cBmp4, (B,E) cBmp7, (C,F) cNog. Arrows indicate expression in the ventrolateral region. Scale bars: 400 µm (A-C); 100 µm (D-F). (G) The kinetics of the cNog expression pattern and basal membrane degradation. The arrow indicates the primitive groove. Note that cNog is expressed in the ventrolateral region of the TVM at HH stage 17-18 (black arrowheads) and that basal membrane degradation occurs where there is a low level of cNog expression (white arrowheads). Until HH stage 24, cNog expression extended to the entire TVM and the basal membrane was formed along the entire ventral ectoderm. Scale bars: 100 µm.

View larger version (108K): [in this window] [in a new window]   Fig. 3. Nog overexpression in the tailbud using sonoporation. (A) A schematic drawing of the sonoporation procedure. (B-E) GFP expression after Sonoporation. The white lines in B and D indicate the position of the sections in C and E. Scale bars: 400 µm (B,D); 50 µm (C,E). (F) Morphology of the tailbud of control and Nog-overexpressing chick embryos. Scale bar: 200 µm. (G) pCAβ-mNog-IRES-GFP or pCAβ-IRES-GFP were transduced into the caudal region of the chick tailbud. The surface ectoderm of the tailbud was labeled with DiI after sonoporation. The distribution of DiI-labeled cells in control and Nog-overexpressing embryos was observed after 24 hours. Scale bars: 200 µm (left); 100 µm (middle and right). (H,I) Hematoxylin and Eosin staining of control and Nog-overexpressing embryos. (J,K) GFP expression in control and Nog-overexpressing embryos. (L,M) Immunostaining of pSmad (red) and E-cadherin (green) in control and Nog-overexpressing embryos. (L',M') High-magnification view of boxed area in L and M. (N,O) Immunostaining of E-cadherin (green) and laminin (red) in control and Nog-overexpressing embryos. (P,Q) cSlug expression in control and Nog-overexpressing embryos. Scale bar: 50 µm.

View larger version (107K): [in this window] [in a new window]   Fig. 4. Bmp2-overexpression in chick embryos. (A,B) Immunostaining of E-cadherin (green) and laminin (red) in GFP and human BMP2 (hBmp2)-overexpressing embryos. (C,D) High-magnification view of boxed area in A and B. (E,F) Double-staining for cSlug (blue) and E-cadherin (brown) of GFP or hBmp2-overexpressing embryos. Note that the cells expressing both cSlug and E-cadherin are in the ventral ectodermal layer in the BMP2-overexpressing embryos (arrows).

View larger version (60K): [in this window] [in a new window]   Fig. 5. The caudal dysmorphogenesis induced by Nog overexpression. (A,B) Dorsal view of the tail of control and Nog-overexpressing chick embryos at HH stage 35-36. (C,D) Skeletal pattern of the tail in control and Nog-overexpressing chick embryos. py, pygostyle. Scale bar: 800 µm. (E,F) Frontal view of the external genitalia of control and Nog-overexpressing chick embryos. gt, genital tubercle; sl, sulcus phalli; t, tail. Scale bar: 200 µm. (G,H) Histological analysis of the hind gut region of control and Nog-overexpressing chick embryos. Cl, cloaca; cm, cloaca membrane; re; rectum; tg, tail gut. Scale bar: 100 µm. (I,J) Hind limb fusion (arrowhead) in Nog-overexpressing chick embryos. hl; hind limb, t; tail. Scale bar: 200 µm. (J') High magnification of the boxed area in J.

View larger version (106K): [in this window] [in a new window]   Fig. 6. A histological analysis of the mouse VER and kinetics of mNog expression pattern and basal membrane degradation during mouse tail development. (A) The tailbud of a mouse embryo at E10.5. The white line indicates the approximate level of the section shown in B. Scale bar: 400 µm. (B) Transverse section of the mouse tailbud. No, notochord; Nt, neural tube; PSM, presomatic mesoderm; Tg, tail gut; TVM, tail ventral mesoderm; VER, ventral ectodermal ridge. Scale bar: 100 µm. (C) High magnification of the boxed region in B. Scale bar: 50 µm. (D) Immunostaining with laminin (red) and E-cadherin (green) of the mouse VER. Scale bar: 50 µm. (E) The kinetics of mNog expression pattern and basal membrane degradation. An arrow indicates the primitive groove. Scale bar: 50 µm. Note the low level mNog expression in the TVM at E9.0 (red arrowhead) and the associated basal membrane degradation underlying the ectoderm derived from the late primitive streak (white arrowheads). Prominent mNog expression was localized in the TVM and basal membrane was formed underlying the mouse VER until E10.0-10.5. Scale bars: black, 50 µm; white, 10 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 7. Phenotypic analyses of Nog mutant mice. (A,B) Lateral view of the tailbud in wild-type and Nog mutant mice at E10.0. Scale bar: 800 µm. (C,D) Hematoxylin and Eosin staining of tailbud sections of wild-type and Nog mutant mice. Scale bar: 100 µm. (E,F) High magnification of the boxed region in C,D. (G,H) Immunostaining of pSmad (red) and E-cadherin (green). Note that pSmad signals were detected in the prospective VER region of Nog mutant mice, and that E-cadherin expression was negative in the same region (arrowheads). Scale bar: 50 µm. (G',H') High-magnification view of boxed area in G and H. Note that the pSmad signal is localized in the nuclei. (I,J) Immunostaining of laminin. (K,L) Double staining of mSnail (blue) and E-cadherin (brown). Note that mSnail was detected in the region without E-cadherin expression in Nog mutant mice (arrowheads). Scale bar: 50 µm. (M,N) Distribution of DiI-labeled cells after 24 hours in culture. (M',N') DiI-labeling at the ventral surface ectoderm of the tailbud. Scale bar: 100 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 8. Schematic diagram showing the cessation of gastrulation. During the early tailbud stage of chick embryos, cNog expression is initiated at the ventrolateral region of the TVM (purple indicated the Nog-expressing cells). BMP signaling could induce EMT with basal membrane (green) degradation. The chick VER shows gastrulation-like ingressive cell movement (pink indicates the ingressing cells). In the late tailbud stage of the chick embryos, cNog expression extends to the entire TVM (purple; the Nog-expressing cells) and inhibits Bmp signaling, which results in the suppression of EMT and basal membrane formation. In the early tailbud stage of mouse embryos, mNog expression in the TVM is low (light purple). The ectoderm derived from the late primitive streak shows ingressive cell movement with basal membrane degradation (pink indicates the ingressing cells and green indicates the basal membrane). In the late tailbud stage of mouse embryos, mNog is prominently expressed in the entire TVM (purple indicates the Nog-expressing cells) and inhibits BMP signaling, which results in the suppression of EMT and basal membrane formation.

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