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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.
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