Sox12 deletion in the mouse reveals nonreciprocal redundancy with the related Sox4 and Sox11 transcription factors

Abstract

The transcription factors Sox4 and Sox11 are important regulators of diverse developmental processes including heart, lung, pancreas, spleen, and B-cell development. Here we have studied the role of the related Sox12 as the third protein of the SoxC group both in vivo and in vitro. Despite widespread Sox12 expression during embryonic development, Sox12-deficient mice developed surprisingly normally, so that they were born alive, showed no gross phenotypic abnormalities, and were fertile in both sexes. Comparison with the related Sox4 and Sox11 revealed extensive overlap in the embryonic expression pattern but more uniform expression levels for Sox12, without sites of particularly high expression. All three Sox proteins furthermore exhibited comparable DNA-binding characteristics and functioned as transcriptional activators. Sox12 was, however, a relatively weak transactivator in comparison to Sox11. We conclude that Sox4 and Sox11 function redundantly with Sox12 and can compensate its loss during mouse development. Because of differences in expression levels and transactivation rates, however, functional compensation is not reciprocal.

FIG. 1.

Targeted disruption of Sox12 in mice. (A) Schematic representation of the targeting construct (top), the Sox12 wild-type locus (upper middle), and the mutant locus before Cre recombination in ES cells (lower middle) and after Cre recombination in mice (bottom). The transcribed region of the Sox12 gene is shown as a box, and flanking regions are shown as bars. Sox12 coding sequences (open reading frames [ORF]), including the position of the start codon (ATG), an intron (I) predicted in the annotated mouse genome, and neomycin resistance cassette (neo), IRES-EGFP cassette, loxP, and FRT sites, are highlighted. Tk, thymidine kinase. Regions of homology, in which recombination between wild-type locus and targeting vector occurred, are marked by crossed lines. Restriction sites for BamHI (B), BglII (G), and PstI (P) are shown as well as the locations of 5′ and 3′ probes and of primers a, b, and c for genotyping (arrowheads). (B) Southern blot analysis of DNA from ES cells before (+/+) and after (+/flox [fl]) successful homologous recombination. Correct integration of the targeting construct was verified after digestion with PstI by use of the 5′ probe and after digestion with BamHI by use of the 3′ probe. The sizes of fragments corresponding to the wild type and the targeted allele are given in kb on the left of the panels. (C) Southern blot analysis of DNA from adult wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice after Cre-mediated deletion. The loss of all sequences between the first and third loxP sites, including the complete Sox12 open reading frame, was verified after digestion with BglII by use of the 5′ probe. The sizes of fragments corresponding to the wild type and the targeted allele are given in kb on the left of the panel. (D) Genotyping PCR on DNA from adult wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. The lower band of 468 bp is indicative of the wild-type allele, the upper band of 580 bp of the Sox12 deletion allele. (E) Analysis of Sox12 expression in heads and trunks of 12.5-dpc-old wild-type (+/+) and Sox12-deficient (−/−) embryos by RT-PCR using primers specific for Sox12, GFP, and β-actin. (F) In situ hybridization of transverse sections from the trunks of wild-type (+/+) and Sox12-deficient (−/−) embryos at 12.5 dpc by use of an antisense riboprobe complementary to part of the 3′-untranslated region of Sox12.

FIG. 2.

Genotype distribution and growth parameters of Sox12-deficient mice after birth. (A) Genotype distribution in Sox12^+/− intercrosses as determined at the time of weaning. More than 15 litters were counted. Distribution is shown as the percentage of total littermates. (B) Percent gender distribution of Sox12^−/− progeny from Sox12^+/− intercrosses. (C and D) Body weights of wild-type (open circles) and homozygous Sox12-deficient (filled triangles) female (C) and male (D) littermates (≥5 for each genotype and sex) were determined during the first 50 days after birth. (E and F) Body lengths from nose to anus were measured for age-matched wild-type (open circles) and homozygous Sox12-deficient (filled triangles) female (E) and male (F) mice during the first 50 days after birth (≥5 for each genotype and sex). Data are means ± standard deviations. No statistically significant differences were detected by use of Student's t test.

FIG. 3.

RT-PCR analysis of embryonic SoxC gene expression in wild-type and Sox12-deficient embryos. (A to C) Expression of Sox12 (A), Sox11 (B), and Sox4 (C) was determined by quantitative RT-PCR for several tissues of wild-type embryos at 14.5 dpc, 16.5 dpc, and 18.5 dpc, as indicated. sp., spinal. The amounts of PCR products were normalized to the β-actin level. (D) In situ hybridization of transverse sections from wild-type (+/+) and Sox12-deficient (−/−) embryos at 12.5 dpc by use of antisense riboprobes specific for Sox4 and Sox11. Hybridization signals were colorimetrically visualized as bluish precipitates. (E and F) Expression of Sox11 (E) and Sox4 (F) in select tissues of Sox12-deficient embryos at 14.5 dpc and 16.5 dpc was analyzed by quantitative RT-PCR with normalization to the expression level of the β-actin gene and comparison to the corresponding expression in the wild type. Increased expression levels in the Sox12-deficient mutant are presented as percentages.

FIG. 4.

Determination of early embryonic Sox12 expression by in situ hybridization. Adjacent sagittal sections of wild-type embryos at 11.5 dpc (A to C) and 12.5 dpc (D to I) were hybridized with antisense riboprobes specific for Sox12 (A, D, and G), Sox11 (B, E, and H), and Sox4 (C, F, and I). Sections in panels A to C and G to I are midsagittal, while those in panels D to F are more lateral. Hybridization signals were colorimetrically visualized as bluish precipitates. Abbreviations: bm, branchial arch mesenchyme; fb, forebrain; fm, facial mesenchyme; hb, hindbrain; drg, dorsal root ganglia; gt, genital tubercle; sc, spinal cord; vc, vertebral column mesenchyme.

FIG. 5.

Determination of late-embryonic Sox12 expression in the trunk by in situ hybridization. Adjacent transverse sections of wild-type embryos were hybridized with antisense riboprobes specific for Sox12 (A, D, G, J, and M), Sox11 (B, E, H, K, and N), and Sox4 (C, F, I, L, and O) at 14.5 dpc (A to I) and 18.5 dpc (J to O). Sections were taken from the trunk at the level of the heart (A to C) or the kidney (D to F). Hybridization signals were colorimetrically visualized as bluish precipitates. The arrowheads in panels C and F point to hair follicles that strongly express Sox4. Magnifications from the overviews (A to F) show spinal (sp.) cord, lung, kidney, and gut at 14.5 dpc (G to I) as well as spinal cord (J to L), dorsal root ganglia (DRG), and sympathetic chain ganglia (SG) at 18.5 dpc (M to O).

FIG. 6.

Determination of late-embryonic Sox12 expression in the head by in situ hybridization. Adjacent coronal sections of wild-type embryos were hybridized with antisense riboprobes specific for Sox12 (A, D, G, J, M, P, S, and V), Sox11 (B, E, H, K, N, Q, T, and W), and Sox4 (C, F, I, L, O, R, U, and X) at 14.5 dpc (A, B, C, G, H, I, M, N, O, S, T, and U) and 18.5 dpc (D, E, F, J, K, L, P, Q, R, V, W, and X). Hybridization signals were colorimetrically visualized as bluish precipitates. Shown is a telencephalic hemisphere at low resolution (A to F) as well as high magnifications of the developing cortex (G to L), the olfactory epithelium (M to R), and the eye (S to X). The fusing palatal shelves at 14.5 dpc are marked by arrowheads (M to O).

FIG. 7.

DNA-binding activity of Sox12. (A) The radiolabeled SX site and oligonucleotides containing the S23 site from the Tubb3 promoter in the wild type (S23) or the mutant version (S23M) were incubated in electrophoretic mobility shift assays with extracts from mock-transfected HEK 293 cells (C) or HEK 293 cells ectopically expressing Sox12 as indicated. Supershifted complexes obtained after incubation with a Sox12-specific antibody are marked by an asterisk. −, no extract added. (B) A radiolabeled FXO oligonucleotide with adjacent binding sites for Sox and POU proteins was incubated with extracts from mock-transfected HEK 293 cells or from HEK 293 cells ectopically expressing Sox12, Brn2, or Oct6 as indicated. In the presence of Sox12 and a POU protein, additional complexes were obtained. The presence of both proteins in this ternary complex (TC) was verified by addition of antibodies directed against Sox12 (α-Sox12), Brn2 (α-Brn2), and Oct6 (α-Oct6). Supershifted complexes are marked by asterisks.

FIG. 8.

Transactivation capacity of Sox12. (A) Endogenous amounts of SoxC and class III POU proteins in Neuro2a and HEK 293 cells were determined by Western blotting. Extracts from HEK 293 cells transfected with corresponding plasmids served as the positive control. Extract quality was checked with an antibody directed against acetylated α-tubulin (Tub). (B to E) Transient transfections were performed in HEK 293 cells (B and D) and Neuro2a cells (C and E) with the 3×SX-luc (B and C) and 3×FXO-luc (D and E) reporter plasmids (all 375 ng) in the presence of increasing amounts of Sox12, Sox11, and Sox4 (15 ng, 50 ng, 125 ng, 375 ng, and 750 ng). All luciferase activities were determined in two independent experiments, each performed in duplicate, and results are presented as induction levels ± standard errors of the mean.

FIG. 9.

Influence of Sox12 on the transactivation capacities of other SoxC and POU proteins. Transient transfections were performed in Neuro2a cells with 3×SX-luc (A), 3×FXO-luc (B to D), or a reporter plasmid in which the luciferase gene is under control of the Tubb3 promoter (E). Fixed amounts of reporter plasmid (375 ng) were cotransfected with constant total amounts of expression plasmids (375 ng) but various contributions of Sox12 and Sox11 (ratios of 10:0, 8:2, 5:5, 2:8, and 0:10) for panels A and B as indicated below the bars, whereas they were cotransfected with various combinations of expression plasmids for Sox12, Sox11, Sox4, Brn2, and Oct6 or empty vector (400 ng each) for panels C and D. The Tubb3 luciferase reporter plasmid shown in panel E was transfected in the presence of increasing amounts of Sox12, Sox11, and Sox4 (15 ng, 50 ng, 125 ng, 375 ng, and 750 ng). All luciferase activities were determined in three independent experiments, each performed in duplicate. Luciferase activities are given as induction levels ± standard errors of the mean.

FIG. 10.

Sox12 activity during early chicken spinal cord development. Immunohistochemistry against Sox12 (D), Sox11 (E), Sox4 (F), and Tubb3 (G to L) was performed on transverse sections of chicken embryos electroporated with expression plasmids for Sox12 (A, D, G, and J), Sox11 (B, E, H, and K), or Sox4 (C, F, I, and L) 24 h (A to I) or 48 h (J to L) after the electroporation event. The electroporated side is oriented to the right and marked by GFP autofluorescence (A to C).