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. 1998 Mar 15;12(6):776-81.
doi: 10.1101/gad.12.6.776.

Sox1 directly regulates the gamma-crystallin genes and is essential for lens development in mice

S Nishiguchi  1 , H Wood, H Kondoh, R Lovell-Badge, V Episkopou
Affiliations

Sox1 directly regulates the gamma-crystallin genes and is essential for lens development in mice

S Nishiguchi et al. Genes Dev. .

Abstract

gamma-Crystallins are major structural components of the lens fiber cells in amphibians and mammals. Many dominant inherited cataracts in humans and mice have been shown to map within the gamma-crystallin gene cluster. Several transcription factors, including PAX6 and SOX proteins, have been suggested as candidates for crystallin gene regulation. Here we show that the targeted deletion of Sox1 in mice causes microphthalmia and cataract. Mutant lens fiber cells fail to elongate, probably as a result of an almost complete absence of gamma-crystallins. It appears that the direct interaction of the SOX1 protein with a promoter element conserved in all gamma-crystallin genes is responsible for their expression.

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Figures

Figure 1
Figure 1
Targeted disruption of the Sox1 gene. (A) The structure of the wild-type allele, targeting vector, and targeted allele are shown together with the restriction sites. The open box represents Sox1 coding sequence. The sizes of DNA fragments from the wild-type and mutated allele detected by the 5′ and 3′ probes are indicated. The positions of the PCR primers for genotyping are shown as arrows above the gene. (B) BamHI; (R) EcoRI. (B) Southern blot analysis of BamHI-digested DNA from ES cell clones using 5′ and 3′ probes, (Lanes 1,3) Wild-type ES cells; (lane 2) targeted ES cells. (C) PCR analysis of offspring derived from a mating of mice heterozygous for Sox1 deletion.
Figure 1
Figure 1
Targeted disruption of the Sox1 gene. (A) The structure of the wild-type allele, targeting vector, and targeted allele are shown together with the restriction sites. The open box represents Sox1 coding sequence. The sizes of DNA fragments from the wild-type and mutated allele detected by the 5′ and 3′ probes are indicated. The positions of the PCR primers for genotyping are shown as arrows above the gene. (B) BamHI; (R) EcoRI. (B) Southern blot analysis of BamHI-digested DNA from ES cell clones using 5′ and 3′ probes, (Lanes 1,3) Wild-type ES cells; (lane 2) targeted ES cells. (C) PCR analysis of offspring derived from a mating of mice heterozygous for Sox1 deletion.
Figure 2
Figure 2
Histological analysis and cellular proliferation of wild-type and mutant lens. Hematoxylin and eosin staining of embryonic lens: (A,C,E) Wild-type lens, (B,D,F) mutant lens at 12.5 dpc (A,B), 15.5 dpc (C,D), and postnatal day zero (P0) (E,F). Loss of Sox1 causes impaired posterior lens fiber cell elongation and small, hollow lens. The nuclei in the mutant lens fiber cells are located closer to the retinal side at 12.5 dpc, whereas at P0, they are located closer to the cavity of the lens. BrdU incorporation assays: (G,I) Wild-type lens; (H,J,K) mutant lens at 12.5 dpc (G,H) and 15.5 dpc (I–K). Arrows indicate BrdU-positive nuclei in the lens fiber cells. Bar, 50 μm in A, B, G, and H; 100 μm in C and D; 140 μm in E and F. 120 μm in I and J; 50 μm in K.
Figure 3
Figure 3
Immunofluorescent analysis of the wild-type lens. Eye sections at 10.5 (A,B), 12.5 (C,D), and 15.5 (E,F) dpc. (A,C,E) SOX1 and (B,D,F) SOX2 expression. The left of each panel shows the DAPI nuclear counterstain (blue) with SOX protein expression patterns (green); the right shows protein expression only. SOX1 protein can be detected in the nuclei of the presumptive lens fibers later during day 10 of development (not shown). SOX2 protein is detected in the nuclei of the cells of both the optic cup and the lens pit at 10.5 dpc (B). At 12.5 dpc, SOX1 is present in the nuclei of the developing lens fibers and the anterior epithelium (C); anti-SOX2 predominantly stains the cytoplasm of the lens fibers (D), although the protein is sometimes detected in the nuclei of the less differentiated lens fiber cells around the equatorial region and in the anterior epithelium (not shown). At 15.5 dpc in the lens, SOX1 is still detected in fiber cell nuclei (E), but SOX2 protein is absent (F). (oc) Optic cup; (lp) lens pit; (ae) anterior epithelium. Bar, 50 μm.
Figure 4
Figure 4
Crystallin gene-expression study of wild-type and mutant lens. RT–PCR analysis of hprt, αA-, αB-, βA3/A1-, γA-, γB-, γC-, γD-, γE-, and γF-crystallins at 12.5 and 15.5 dpc. αB-Crystallin expression in the Sox1 mutant eye at 15.5 dpc is up-regulated possibly due to hypertonic stress (Dasgupta et al. 1992).
Figure 5
Figure 5
Binding of SOX1 protein to the mouse γA-crystallin promoter. (A) Complementary strand of SOX binding consensus sequence, wild-type (γA), and mutated (γAM) oligonucleotide sequence from the γA-crystallin promoter that was used for EMSA, promoter–sequence alignment of the six mouse γ-crystallin genes. Sequences identical to the consensus SOX binding site are shown in boldface type. The substituted nucleotides in γAM are underlined. Nucleotide positions of the γF-crystallin gene relative to the transcription start site are shown. (B) EMSA with recombinant SOX1 protein. γA oligonucleotide probe incubated without recombinant protein (in vitro transcription/translation reaction mixture incubated without DNA template) (lane 1) or with SOX1 recombinant protein (lanes 2–7). Assays were done in the presence of nonspecific competitor (lane 2), nonradioactive γA oligonucleotide (lane 3), nonradioactive mutated γAM oligonucleotide (lane 4), normal rabbit serum (NRS) (lane 5), SOX1 rabbit antiserum abFB43 (lane 6), or ab791 (lane 7) as shown. (→) The position of SOX1 complex.
Figure 5
Figure 5
Binding of SOX1 protein to the mouse γA-crystallin promoter. (A) Complementary strand of SOX binding consensus sequence, wild-type (γA), and mutated (γAM) oligonucleotide sequence from the γA-crystallin promoter that was used for EMSA, promoter–sequence alignment of the six mouse γ-crystallin genes. Sequences identical to the consensus SOX binding site are shown in boldface type. The substituted nucleotides in γAM are underlined. Nucleotide positions of the γF-crystallin gene relative to the transcription start site are shown. (B) EMSA with recombinant SOX1 protein. γA oligonucleotide probe incubated without recombinant protein (in vitro transcription/translation reaction mixture incubated without DNA template) (lane 1) or with SOX1 recombinant protein (lanes 2–7). Assays were done in the presence of nonspecific competitor (lane 2), nonradioactive γA oligonucleotide (lane 3), nonradioactive mutated γAM oligonucleotide (lane 4), normal rabbit serum (NRS) (lane 5), SOX1 rabbit antiserum abFB43 (lane 6), or ab791 (lane 7) as shown. (→) The position of SOX1 complex.
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