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First published online 27 April 2005
doi: 10.1242/dev.01844

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Ssdp1 regulates head morphogenesis of mouse embryos by activating the Lim1-Ldb1 complex

¹ Laboratory for Embryonic Induction, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
² Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
³ Department of Neurology, Graduate School of Medicine, Osaka University, D-4, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
⁴ Laboratory for Animal Resources and Genetic Engineering (LARGE), RIKEN Center for Developmental Biology, 2-2-3 Minatojimaminamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
⁵ Division of Bioscience, Graduate School of Environmental Earth Science, Hokkaido University, North 10 West 8, Sapporo 060-0810, Japan
⁶ Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA
⁷ Laboratory for Genome Exploration Research Group, Genomic Sciences Center, RIKEN, Yokohama, Kanagawa 230-0045, Japan
⁸ Laboratory of Mammalian Genes and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
⁹ Department of Molecular Genetics, The University of Texas M. D. Anderson Cancer Center, Huston, TX 77030, USA
¹⁰ Center for Advanced Science and Technology, Hokkaido University, North 10 West 8, Sapporo 060-0810, Japan

View larger version (56K): [in a new window]   Fig. 1. Gross phenotype of headshrinker mutants. (A-H) External views (A,B) and skeletal specimens (C-H) of wild-type (A,C,E,G) and headshrinker (hsk) mutant (B,D,F,H) P0 neonates. Higher magnification views of the head region of C and D (C',D'), sternum (E,F) and vertebral bones (G,H). Arrowheads and asterisks in E,F indicate asymmetrical attachment of ribs to the sternum, and anteroposterior dislocation of the sternum, respectively; arrowheads in H indicate bifurcation and/or fusion of ribs. (I-P) The external morphologies of wild-type (I-L) and hsk mutant embryos (M-P). The arrowhead in N indicates the abnormality in the anterior neural folds. (Q) Phenotypic variability of hsk mutants. Sections of wild-type (R) and hsk mutant (S) embryos; asterisks indicate somites. c1, atlas; e, exoccipital bone; f, frontal bone; i, interparietal bone; mn, mandible; mx, maxilla; n, nasal bone; nt, neural tube; p, parietal bone; pm, premaxilla; s, supraoccipital bone. Scale bar: 5 mm in C,D; 1 mm in C',D'; and 20 µm in R,S.

View larger version (62K): [in a new window]   Fig. 2. Identification of the transgene insertion site in headshrinker mutants. (A,C) Chromosomal FISH showing the single transgene insertion site at Chr4 C5-C6 (arrow). (B,D) Hoechst 33258 staining of the chromosomes shown in A and C. (C,D) Close up of the transgene insertion site. (E) A physical map of the area surrounding the transgene insertion site. The long horizontal bar at the center represents Chr4, and the positions along the chromosome are given in Mb above the bar. The short arrows below the bar and the numbers indicate the positions of genes and their accession numbers, respectively. The upper bar marked with thinner vertical lines shows the detailed structure of the Ssdp1 gene. The vertical lines represent exons. Four copies of the transgenes were inserted in the fourth intron of Ssdp1. P1, P2 and P3 indicate the positions of the PCR primers used for genotyping. (F,H,J,L) Whole-mount in situ hybridization of Ssdp1. Left side view of ES stage (F), LB stage (H), E9.0 (L) and frontal view of E8.0 (J) embryos. (G,I,K) Transverse sections of embryos shown in F,H and J, respectively (approximate position of sections are indicated by bars in F,H,J). (G',I',K') Higher magnifications of G,I,K, respectively. ADE, anterior definitive endoderm; AVE, anterior visceral endoderm; al, allantois; de, definitive endoderm; ec, ectoderm; ep, epiblast (thickness indicated by a bar); m, mesoderm; pp, prechordal plate; ps, primitive streak (indicated by dotted lines in F,H); ve, visceral endoderm (thickness indicated by arrowheads). Dashed lines in G',I',K' indicate positions of AVE, ADE and pp, respectively. (M) In situ hybridization of Ssdp1 on a section of E9.0 embryo. The hybridization signals appeared brown. (N) Whole-mount in situ hybridization showing the reduced expression of Ssdp1 in hsk homozygous embryos. Embryos were grouped according to their genotypes, and were subjected to whole-mount in situ hybridization, performed at the same time in different wells. Similar results were obtained by two independent experiments. Scale bars: 200 µm in L,N; 100 µm in F,H,I,J,K,M; 20 µm in G,G',I',K'. (O) Northern blot analysis of E9.5 RNA showing expression of Ssdp1 RNA in hsk homozygous embryos at a reduced level. (P) Relative expression levels of the genes surrounding the transgene insertion site in hsk mutants compared with wild type. Values shown represent the means and standard errors of the relative expression levels. The number on each bar indicates the number of samples analyzed. The expression level of Ssdp1 in hsk heterozygotes (*P<0.01) and homozygotes (**P<0.001) was reduced.

View larger version (49K): [in a new window]   Fig. 3. Rescue of the Ssdp1hsk/hsk mutant phenotype by the Ssdp1 transgene. (A) Structure of the Ssdp1 transgene (TG). (B-D) Lateral view of E9.5 embryos obtained by the cross of TG#37/Ssdp1+/hsk or TG#141/Ssdp1+/hsk with Ssdp1+/hsk mice. (B) Ssdp1hsk/hsk embryo. (C) Completely rescued Ssdp1hsk/hsk embryo carrying TG#37. (D) Partially rescued Ssdp1hsk/hsk embryo carrying TG#141. Scale bar: 400 µm for B-D. (E) TG#37 completely rescues the mutant phenotype. (F) The expression level of Ssdp1 in two TG lines. Values shown represent the means and standard errors of the relative expression levels of the sum of endogenous and transgenic Ssdp1 RNA, as quantified by RT-PCR. The number on each bar indicates the number of samples analyzed.

View larger version (70K): [in a new window]   Fig. 4. Expression of anterior patterning markers in Ssdp1hsk/hsk mutants. In situ hybridization showing gene expression in the anterior visceral endoderm (AVE) (A-D), anterior definitive endoderm (ADE) (E-H) and the prechordal plate (I-P) in wild-type and Ssdp1hsk/hsk mutants. Wild-type (A) and mutant (B, n=5/5) expression of Lefty1; wild-type (C) and mutant (D, n=7/7) expression of Lim1; wild-type (E) and mutant (F, n=4/4) expression of Hhex; wild-type (G,I) and mutant (H, n=4/4; J, n=2/9) expression of Cerl; wild-type (K) and mutant (L, n=0/6) expression of Dkk1, wild type (M) and mutant (N, n=0/6) expression of Foxa2, and wild-type (O) and mutant (P, n=0/3) expression of Gsc. n, number of embryos expressing the gene/number of embryos analyzed. Broken lines in A-D and E-H indicate the AVE and ADE, respectively. Arrows in I-P indicate the prechordal plate. (Q-T) Sagittal section of wild-type (Q,S) and mutant (R,T) embryos stained with Hematoxylin and Eosin. Scale bars: in D, 100 µm for A-D; in H, 100 µm for E-H; in P, 200 µm for I-P; in R, 200 µm for Q,R; in T, 200 µm for S,T.

View larger version (102K): [in a new window]   Fig. 5. Head defects in Ssdp1hsk/hsk mutants. In situ hybridization showing the expression of various regional markers in wild-type and Ssdp1hsk/hsk embryos. Wild-type (A,E) and mutant Six3 expression (B, n=2/5; F, n=0/8); wild-type (C,G) and mutant (D, n=7/7; H, n=1/5) Otx2 expression; wild-type (I) and mutant (J, n=0/2) Pax6 expression; and wild-type (K) and mutant (L, n=2/3) En2 expression. (M,N) Fgf8 was expressed in the commissural plate (arrowhead) and MHB (black arrow) in wild-type embryos (M), while MHB expression in Ssdp1hsk/hsk mutants was observed at the anterior tip (black arrow in N, n=6/10). Expression of Krox20 was unaffected (M,N, white arrows). (O,P) Wnt1 expression in the MHB (O; arrow) was observed at the anterior tip in some Ssdp1hsk/hsk mutants (arrow in P, n=3/5). Scale bars: in D, 200 µm for A-D; in P, 400 µm for E-P.

View larger version (43K): [in a new window]   Fig. 6. Apoptosis and cell proliferation in Ssdp1hsk/hsk mutants. Sections showing the distribution of apoptotic (A,B) and proliferating (E,F) cells in the head region of wild-type (A,E) and Ssdp1hsk/hsk mutant (B,F) embryos. (D) Scale bar in F, 400 µm for A,B,E,F. (C,D,G,H) Quantitation of apoptotic and proliferating cells in various tissues of wild type and Ssdp1hsk/hsk mutants at E8.0 and E8.5. Values shown represent the means and standard errors of the percentages of the apoptotic (C,D) or proliferating (G,H) cells to total cells. Black and white bars represent wild-type and Ssdp1hsk/hsk mutant embryos, respectively. The number above each bar indicates the number of samples analyzed. The tissues with significant differences between wild-type and mutant are indicated (*P<0.05, **P<0.01). mesen, mesenchyme.

View larger version (46K): [in a new window]   Fig. 7. Ssdp1 acts as a coactivator component of a Lim1-Ldb1-Ssdp1 complex. (A) E8.0 Lim1-lacZ knock-in embryo stained for ß-galactosidase activity. (B) Cross-section of the embryo shown in A; approximate sectioning position is indicated in A. (C) Whole-mount in situ hybridization of Ldb1 in E8.0 embryo. (D) Cross-section of embryo shown in C. Scale bars in C,D: 200 µm. (E) Schematic representation of the effector and the reporter plasmid used in the transfection assay described in F. (F) The fusion protein comprised of the GAL4 DNA-binding domain and Ssdp1 activated reporter gene expression in a dose-dependent manner. (G) Schematic representation of the effectors and a reporter used in the transfection assays described in H and I. (H) Effects of Lim1, Ldb1 and Ssdp1 on the Gsc promoter. (I) Effects of varying Ssdp1 concentration on the activity of Lim1- and Ldb1-mediated reporter gene expression. Values are the means and standard errors of duplicate experiments. (J) A model for the action of Ssdp1. Ssdp1, Ldb1 and Lim1 constitute a ternary complex and regulate genes expressed in the prechordal plate.

View larger version (69K): [in a new window]   Fig. 8 Genetic interactions of Ssdp1 with Lim1 and Ldb1. (A-D) E9.5 embryos obtained by crossing Ssdp1+/hsk and Lim1+/- mice. (A) Wild-type embryo. (B-D) Double heterozygotes, which can be classified into three types: I, normal (B, n=6); II, microcephaly (C, n=5); III, dwarfism (D, n=4). (E-G) E9.5 embryos obtained by crossing Ssdp1+/hsk and Ldb1+/- mice. (E) Wild-type embryo. (F,G) Double heterozygotes showing no apparent abnormalities (F, n=7) or varying degrees of abnormality (G, n=6). Scale bar: 400 µm.