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doi: 10.1242/10.1242/dev.00408

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Nodal signaling induces the midline barrier by activating Nodal expression in the lateral plate

Masamichi Yamamoto, Naoki Mine, Kyoko Mochida, Yasuo Sakai^*, Yukio Saijoh, Chikara Meno and Hiroshi Hamada

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
^* Present address: E. Kennedy Shriver Center, Division of Developmental Neuroscience, 200 Trapelo Rd, Waltham, MA 02254, USA

View larger version (83K): [in a new window]   Fig. 1. Cre-mediated deletion of Foxh1 in LPM. (A-C) Mice harboring the Lefty2-3.0 Cre transgene were crossed with transgenic mice that harbor the reporter construct CAG-CAT-Z (Sakai and Miyazaki, 1997), which expresses lacZ in the presence of the Cre recombinase. Embryos containing both Lefty2-3.0 Cre and CAG-CAT-Z were stained with X-gal at E7.5 (A) and E8.2 (B). A transverse section at the plane indicated in B is shown in C. At E7.5, most of the mesoderm exhibited X-gal staining whereas the ectoderm and endoderm did not. At E8.2, the anterior region of LPM, paraxial mesoderm and the heart were positive for staining, whereas the posterior portion of LPM and midline structures (arrowhead in B), including the PFP, were negative. a, anterior; p, posterior; l, left; r, right. (D-G) Whole-mount in situ hybridization analysis of Foxh1 transcripts. Lateral views are shown for wild-type (WT) (D) and Foxh1c/- (F) embryos at E7.5 and anterior views for wild-type (E) and Foxh1c/- (G) embryos at E8.2. In the Foxh1c/- embryos, Foxh1 mRNA was not detected in the regions that were positive for X-gal staining in A-C.

View larger version (92K): [in a new window]   Fig. 2. Aberrant expression of Nodal, Lefty1, Lefty2 and Pitx2 in Foxh1c/- embryos. The expression of Nodal (A-E), Lefty1 plus Lefty2 (F,G) and Pitx2 (H-V) in wild-type (WT) and Foxh1c/- embryos was examined by whole-mount in situ hybridization. Embryos shown in A-H,J,L are at E8.2, whereas the others are at E9.5. Transverse sections at the planes indicated in I, K and M are shown in N-P, Q-S and T-V, respectively. All E8.2 embryos are anterior views, with the exception that left lateral views are shown for B and E. The insets in A, C, D and G show the node in posterior view of the embryos. The white line in the inset of G indicates the location of the node. Most Foxh1c/- embryos fail to exhibit left-sided gene expression, although some retain Nodal expression in a small region of left LPM adjacent to the node (arrowhead in E) and a low level of left-sided Pitx2 expression in various organs (arrowheads in T-V). a, anterior; p, posterior; l, left; r, right.

View larger version (125K): [in a new window]   Fig. 3. LR defects in the visceral organs of Foxh1c/- mice. Visceral organs and heart sections of wildtype (WT) and Foxh1c/- neonates are shown. Genotype is indicated at the top of each column. (A,B) Lobation of the lung. In the wild-type mouse (A), the left and right lungs have one and four lobes, respectively. In most Foxh1c/- mice (B), both left and right lungs have four lobes. AL, accessory lobe; CaL, caudal lobe; CrL, cranial lobe; ML, medial lobe; H, heart; LL, left lobe. (C,D) The azygos vein (az) of the wild-type mouse is located on the left side (C). In most Foxh1c/- mice, the azygos vein is located on the right side (D) or both sides. ao, aorta. (E,F) Hypoplasia of the spleen (sp) in the Foxh1c/- mouse (F). st, stomach. (G,H) Visceral organs including the stomach are reversed in many Foxh1c/- mice (H). du, duodenum; li, liver. (I-K) The heart apex is located on the left side of the wild-type mouse (I), but is either in the middle (J) or on the right side (K) of Foxh1c/- mice. la, left atrium; lv, left ventricle; ra, right atrium; rv, right ventricle. (L,M) The right renal vein is located anteriorly to the left renal vein in the wild-type mouse (L). In Foxh1c/- mice, the relative positions of left and right renal veins are reversed or the two veins are located at the same level (M). cvc, caudal vena cava; ki, kidney; rv, renal vein; aa, abdominal aorta. (N) Aberrant positioning of the portal vein (po) in the Foxh1c/- mouse. (O-T) Frontal sections of the heart. In most Foxh1c/- mice, the heart manifests severe malformations, including transposition of the great arteries (P,S) and double outlet of the right ventricle (Q,T). pa, pulmonary artery.

View larger version (93K): [in a new window]   Fig. 4. Induction of Nodal and Lefty1 by transplanted left LPM. (A) Schematic representation of the tissue transplantation system. (B-K) Expression of Nodal (N in B) and Nodal plus Lefty1 (N+L1 in C-K) was examined by whole-mount in situ hybridization 3 hours after the indicated type of transplantation. Genotypes of the recipients and donors are shown in red and blue, respectively, at the top of each panel. Donor tissue was derived from left LPM of the indicated embryos. The transplant sites in the host embryos are indicated by square brackets (B,C,E-K). Donor tissue was transplanted to the anterior region of LPM in host embryos, with the exception of the embryo shown in H,J, which received the transplant in the paraxial mesoderm (IPAM). Two representative Foxh1c/- embryos that received a transplant derived from the left LPM of wild-type (WT) embryo are shown in F,G. Endogenous Nodal expression in the transplant is detectable in G but undetectable in F. Transverse sections at the planes indicated in C are shown in D and E; black and white arrowheads indicate left and right PFP, respectively. (J,K) Magnified views of the regions boxed in H,I, respectively. Arrowheads in J,K indicate Lefty1 expression induced in left PFP.

View larger version (74K): [in a new window]   Fig. 5. Induction of Nodal and Lefty1 by a Nodal expression vector. Expression vectors for Nodal plus EGFP (enhanced green fluorescent protein) (A-H) or those for caALK4 plus EGFP (I,J) were introduced by electroporation into the anterior region of right LPM of a wild-type (WT) embryo (A-E,I) or into the anterior region of left LPM of a Foxh1c/- embryo (F-H,J). Electroporated expression vectors are shown in blue, while genotypes of the recipient embryos are shown in red. Six hours after electroporation, expression of Nodal (N in B,C,F) or Nodal plus Lefty1 (N+L1 in D,E,G-J) was examined by whole-mount in situ hybridization. Electroporated regions, which were confirmed by the presence of EGFP fluorescence (A), are indicated by the square brackets. Anterior views are shown in (A,B,D,F-J), whereas right lateral view is shown in C. A transverse section at the plane indicated in D is shown in E. A magnified view of the boxed region indicated in G is shown in H. Arrowheads indicate Nodal expression in the right LPM (C) and Lefty1 expression (E,H) induced by the Nodal expression vector.

View larger version (124K): [in a new window]   Fig. 6. Monitoring of Nodal activity in mouse embryos with a Nodal-responsive transgene. Expression of the Nodal-responsive transgene (n2)7-lacZ was examined in wild-type (WT) (A,B) and Foxh1c/- (C,D) embryos. In the Foxh1c/- embryo, X-gal staining in left LPM and PFP is lost whereas that in the allantois remains (C,D). Expression vectors for Nodal, EGFP and lacZ were also introduced into the left LPM of a Foxh1c/- embryo harboring the (n2)7-lacZ transgene and, 6 hours later, the embryo was stained with X-gal (E,F). The region that received the expression vectors is apparent from the EGFP fluorescence and X-gal staining in left LPM (indicated by the square brackets). X-gal-positive region in left LPM failed to expand due to the absence of Foxh1 in LPM. A magnified view of the boxed region indicated in E is shown in F. The arrowhead in F indicates X-gal staining in the PFP that was induced by Nodal. Anterior views are shown in A,C,E, whereas left lateral views are shown in B,D.

View larger version (54K): [in a new window]   Fig. 7. Model for the movement of Nodal activity during LR patterning. Three events involving Nodal are illustrated. Blue represents the domains that receive Nodal signals and in which Foxh1 is active. Red indicates the domains in which Nodal is expressed. Arrows indicate the directions of signal transfer. In the wild-type embryo (top row), Nodal produced in the node travels to left LPM and initiates Nodal expression in a small region adjacent to the node (1). At this stage, Lefty1 is expressed in a small region of PFP adjacent to the node (green). Lefty1 expression in this domain may be induced by Nodal produced in the node. (2) Nodal produced in the small region of left LPM diffuses along the anteroposterior axis in left LPM and thereby induces the expansion of Nodal expression. (3) Nodal produced in left LPM travels to the entire PFP region along the AP axis (blue), where it induces Lefty1 expression. In Foxh1c/- embryos (bottom row), Nodal produced in the node is unable to initiate Nodal expression in left LPM because of the absence of Foxh1 in LPM. As a result, Nodal expression in left LPM and Lefty1 expression in PFP are absent.

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