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First published online 3 October 2007
doi: 10.1242/dev.009464

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Sulfated glycosaminoglycans are necessary for Nodal signal transmission from the node to the left lateral plate in the mouse embryo

Shinya Oki^1,*, Ryuju Hashimoto², Yuko Okui³, Michael M. Shen^4,, Eisuke Mekada⁵, Hiroki Otani², Yukio Saijoh^1,6 and Hiroshi Hamada^1,

¹ Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, and CREST, Japan Science and Technology Corporation (JST), 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan.
² Department of Developmental Biology, Center for Integrated Research in Science, Faculty of Medicine, Shimane University, 89-1 Enyacho, Izumo 693-8501, Japan.
³ Department of Biosignaling and Radioisotope Experiments, Center for Integrated Research in Science, Faculty of Medicine, Shimane University, 89-1 Enyacho, Izumo 693-8501, Japan.
⁴ Center for Advanced Biotechnology and Medicine and Department of Pediatrics, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
⁵ Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
⁶ Department of Neurobiology and Anatomy, Program in Human Molecular Biology and Genetics, and The Eccles Program in Human Molecular Biology and Genetics, University of Utah, Salt Lake City, UT 84112-5330, USA.

View larger version (26K): [in this window] [in a new window]   Fig. 1. Schematic representation of possible routes for Nodal signal transmission from the node to LPM in the mouse embryo. (A) Schematic transverse section of an E8.2 embryo showing endoderm (green), mesoderm (gray), ectoderm (orange), node (yellow) and Nodal-expressing (dark blue) cells. Internal (red arrow) or external (blue arrow) potential routes of Nodal signal transmission are indicated. (B,C) Two hypothetical mechanisms of Nodal signaling from the node (blue square) to LPM (blue hexagon). Nodal, red; type I and type II Activin receptors, green and purple; Cryptic, pink. In the direct transport model (B), Nodal produced at the node travels directly to LPM, where it is captured by Cryptic and induces Nodal expression. In the indirect signal-relay model (C), Nodal produced at the node binds to Cryptic and induces the expression of downstream gene products (X1, X2, Xn) that relay the Nodal signal to induce Nodal expression in LPM.

View larger version (50K): [in this window] [in a new window]   Fig. 2. Unresponsiveness of the endoderm to the Nodal signal. (A,E) Schematic transverse sections of an E8.0 mouse embryo showing the strategy for culture with (A), or injection of (E), recombinant Nodal or Activin. Recombinant proteins added to the culture medium or injected into the right LPM are colored pink. (B-D) In situ hybridization for Nodal mRNA in embryos that were recovered at the one-somite stage and cultured in the absence (B) or presence of recombinant Nodal (C) or Activin (D) until they developed to the six-somite stage. (F-H) In situ hybridization for Nodal mRNA in embryos recovered at the one-somite stage, injected with medium containing bovine serum albumin (F), recombinant Nodal (G) or Activin (H), and cultured to the six-somite stage. Arrowheads indicate ectopic expression of Nodal in the right LPM. l, left; r, right. Scale bar: 200 µm.

View larger version (76K): [in this window] [in a new window]   Fig. 3. The Nodal signal is not relayed indirectly between the node and LPM in mouse. (A) Schematic representation of a Cryptic transgene. An LPE isolated from a Cryptic genomic fragment was linked to the hsp68 promoter, Cryptic cDNA, an IRES, lacZ and a polyadenylation signal. Black and white boxes represent the open reading frame and untranslated regions, respectively, of Cryptic, with the arrow indicating the direction of transcription. (B,C) An E8.2 embryo harboring the transgene (Tg+) shows ß-galactosidase activity specifically in the LPM. (D,E) In situ hybridization for Cryptic mRNA in wild-type (D) or Cryptic-/-; Tg+ (E) embryos at E8.2. Cryptic is expressed only in LPM, not in the node (black arrowhead) or floor plate, of the Cryptic-/-; Tg+ embryo. (F-Q) In situ hybridization for Nodal (F-I), Lefty1 and Lefty2 (J-M) or Pitx2 (N-Q) transcripts in wild-type (F,J,N), Cryptic-/- (G,K,O), or Cryptic-/-; Tg+ (H,I,L,M,P,Q) embryos at E8.2. The expression of Nodal, Lefty2 and Pitx2 in LPM is lost in Cryptic-/- embryos (G,K,O), but is rescued by the transgene in Cryptic-/-; Tg+ embryos (H,L,P). Red arrowheads (I,M,Q) indicate ectopic gene expression in the right LPM of Cryptic-/-; Tg+ embryos, which probably results from a defect in the midline barrier (asterisks in L and M). H,I,L,M are all at the five-somite stage. a, anterior; fp, floor plate; lp, LPM; p, posterior; pA, polyadenylation signal. Scale bar: 200 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 4. Immunofluorescence detection of 3xMyc-tagged Nodal at the node. (A) Schematic representation of a Nodal transgene. Tandem node-specific enhancers (NDEs) were linked with the hsp68 promoter, Nodal cDNA (encoding the 3xMyc tag positioned four amino acids downstream from the Nodal cleavage site, arrowhead), IRES, lacZ and pA. (B) An E8.2 mouse embryo harboring the transgene (Tg+) exhibits ß-galactosidase activity only at the node. (C-E) In situ hybridization for Nodal (C,D) or Pitx2 (E) mRNA in Nodalneo/neo (C) or Nodalneo/neo; Tg+ (D,E) embryos at E8.2. The expression of the transgene (black arrowhead) rescues the loss of Nodal and Pitx2 expression in LPM (red arrowheads). The level of expression of the rescued Nodal and Pitx2 is lower than that in the wild-type embryo because the neo gene inserted into the endogenous Nodal gene prevents Nodal expression in the LPM from being amplified to the maximum level. (F-J) Transverse frozen sections of E8.0 Tg+ embryos were subjected to immunofluorescence analysis either with antibodies to Myc (F) and to ß-galactosidase (G), with the merged image shown in H, or with antibodies to Myc and to ZO-1 (I) or laminin (J), with the fluorescence signals being merged with the differential interference contrast and DAPI image (blue) in I,J. The basement membrane is indicated by white dots (I). c, crown cell of the node; ec, ectoderm; m, mesoderm. Scale bars: 200 µm in B-E; 20 µm in F-H; 5 µm in I-J.

View larger version (86K): [in this window] [in a new window]   Fig. 5. Immuno-TEM detection of 3xMyc-Nodal at the node. (A-D) Immunohistochemical staining for 3xMyc-Nodal in transverse frozen sections of E8.0 mouse embryos positive (A-C) or negative (D) for the transgene shown in Fig. 4A. (E-H) Ultrathin sections corresponding to the boxed regions shown in A to D, respectively, were examined by TEM at x1000. (I-M) The boxed regions in E-G are shown at higher magnification (x4000) in I-K, respectively. Black dots, cell boundaries; green dots delineate the nucleus. The boxed regions in J and K are shown at even higher magnification (x6000) in L and M, respectively, revealing apparent secretion of 3xMyc-Nodal at the basolateral membranes. Scale bars: 10 µm in A-H; 2.5 µm in I-K; 1 µm in L,M.

View larger version (73K): [in this window] [in a new window]   Fig. 6. Distribution of sulfated GAGs in the E8.0 mouse embryo. Transverse frozen sections of E8.0 embryos were treated with buffer only (A-C), heparitinase (Hase; D-F), chondroitinase (Case; G-I) or both enzymes (J-L) and were then subjected either to immunofluorescence analysis with antibodies to HS (10E4; A,D,G,J) or to CS (CS-56; B,E,H,K) or to staining with Alcian Blue (C,F,I,L). Red and blue indicate immunoreactivity and nuclear staining with DAPI, respectively, in the immunofluorescence images. Insets in A-C show the boxed regions at higher magnification. Scale bar: 100 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Interaction of Nodal with sulfated GAGs. Recombinant mouse Nodal was incubated with heparin-sepharose beads, which were then isolated and subjected to stepwise elution with NaCl (A) or with heparin, CS or HS (B). Input (IP), flow through (FT), washed (1-3) and eluted fractions were subjected to immunoblot analysis with antibodies to Nodal. The amount of Nodal in the various fractions obtained by elution with NaCl (normalized relative to that in the fraction containing the most Nodal) was quantitated by densitometry of the immunoblot shown.

View larger version (84K): [in this window] [in a new window]   Fig. 8. Sulfated GAGs are necessary for transmission of the Nodal signal from the node to the LPM. (A,J) Schematic representation of proteoglycans from normal (A) or chlorate-treated (J) mouse embryos. A serine residue (yellow) of the core protein (orange) is attached to the GAG chain (blue curved line), which is sulfated (blue circles) under normal conditions but not in cells treated with chlorate. (B-D,K-M) Transverse frozen sections of embryos cultured to the six-somite stage in the absence (B-D) or presence (K-M) of 15 mM sodium chlorate were subjected either to immunofluorescence analysis with antibodies to HS (B,K) or to CS (C,L) or to staining with Alcian Blue (D,M). (E-G,N-P) In situ hybridization for Nodal (E,N), GDF1 (F,O) or Cryptic (G,P) mRNAs in embryos cultured in the absence (E-G) or presence (N-P) of chlorate. (H,I,Q,R) Expression vectors for Nodal and GFP were co-injected into the right LPM of embryos at the headfold stage, which were then cultured to the six-somite stage in the absence (H,I) or presence (Q,R) of chlorate. The cultured embryos were examined for GFP fluorescence (H,Q) and then subjected to in situ hybridization for Nodal mRNA (I,R). It should be noted that the region of Nodal expression was much broader than the area expressing GFP (I,R), which is due to a competence of LPM for Nodal auto-activation. (S) The number and percentage of embryos with (blue) or without (white) Nodal expression in LPM after culture in the absence or presence of chlorate. The difference between the two culture conditions was statistically significant (P<0.001) by the two-tailed Fisher's exact probability test. Scale bars: 100 µm in B-D,K-M; 200 µm in E-I,N-R.

View larger version (80K): [in this window] [in a new window]   Fig. 9. CS is required for Nodal signal transmission from the node to LPM. (A,H) Schematic representation of proteoglycans from normal (A) or xyloside-treated (H) mouse embryos. Xyloside (purple) acts as primer for GAG elongation, resulting in the syntheses of unlinked GAGs and naked proteoglycans. (B-D,I-K) Transverse frozen sections of embryos cultured with 0.1% dimethyl sulfoxide vehicle (B-D) or 1 mM xyloside (I-K) to the six-somite stage were subjected either to immunofluorescence analysis with antibodies to HS (B,I) or to CS (C,J) or to staining with Alcian Blue (D,K). (E-G,L-N) In situ hybridization for Nodal (E,L), GDF1 (F,M) or Cryptic (G,N) mRNAs in embryos cultured in the absence (E-G) or presence (L-N) of xyloside. (O,P) Expression vectors for Nodal and GFP were co-injected into the right LPM of embryos before culture with xyloside. The resulting embryos were examined for GFP fluorescence (O) and then subjected to in situ hybridization for Nodal mRNA (P). (Q) The number and percentage of embryos with (blue) or without (white) Nodal expression in LPM after culture in the absence or presence of xyloside. Scale bars: 100 µm in B-D,I-K; 200 µm in E-G,L-P.