Functional Equivalence of the SOX2 and SOX3 Transcription Factors in the Developing Mouse Brain and Testes

Abstract

Gene duplication provides spare genetic material that evolution can craft into new functions. Sox2 and Sox3 are evolutionarily related genes with overlapping and unique sites of expression during embryogenesis. It is currently unclear whether SOX2 and SOX3 have identical or different functions. Here, we use CRISPR/Cas9-assisted mutagenesis to perform a gene-swap, replacing the Sox3 ORF with the Sox2 ORF to investigate their functional equivalence in the brain and testes. We show that increased expression of SOX2 can functionally replace SOX3 in the development of the infundibular recess/ventral diencephalon, and largely rescues pituitary gland defects that occur in Sox3 null mice. We also show that ectopic expression of SOX2 in the testes functionally rescues the spermatogenic defect of Sox3 null mice, and restores gene expression to near normal levels. Together, these in vivo data provide strong evidence that SOX2 and SOX3 proteins are functionally equivalent.

Keywords: CRISPR/CAS9 mutagenesis; SOXB1 genes; gene swap.

Figure 1

Generation of Sox3^Sox2KI and Sox3^KO mice with CRISPR mutagenesis. (A) Schematic showing the strategy for generation of the Sox3^Sox2KI mice. (B, C) PCR screening of founder mice. (D) Sox2 transcript levels were measured in 11.5 dpc brains by qPCR with primers located in the Sox2 ORF. (E) qPCR showing expression from the endogenous Sox2 locus using primers located in the 3′UTR. (F) Transcription from the Sox3 locus was reduced in Sox3^Sox2KI/Y as determined by qPCR with primers located in the Sox3 3′UTR. (G) SOX3 and SOX2 coimmunostaining in the ventral diencephalon of 12.5 dpc Sox3^Sox2KI/+ embryos. Asterisks indicate SOX3 positive cells, and arrowheads indicate SOX3 negative cells. Quantification of SOX2 staining intensity in SOX3 positive and SOX3 negative cells (n = 3 embryos).

Figure 2

Increased expression of Sox2 can rescue Sox3 null pituitary defects. (A) Adult pituitaries showing dorsal displacement of the neural lobe in Sox3^KO/Y mice. (B) Haemotoxylin and eosin staining of 8-week-old pituitary coronal sections. Asterisk highlights abnormal clefting. Arrowhead indicates hypoplastic anterior lobe. (C) Phase contrast images of 12.5 dpc developing pituitaries (saggital sections). (D) Quantification of infundibulum width of 12.5 dpc embryos (n = 3). One-way ANOVA using Tukey’s Multiple comparison test. Mean ± SEM.

Figure 3

SOX2 regulates SOX3 target genes in the mouse testes. (A) Sox2 qPCR analysis in 2 week Sox3^+/Y and Sox3^Sox2KI/Y testes [Student’s two tailed unpaired t-tests (****<0.0001)]. (B) qPCR analysis of Sox3 3′ UTR in 2 week Sox3^+/Y and Sox3^Sox2KI/Y testes [Student’s two-tailed unpaired t-tests (**<0.01)]. (C) SOX2 and SOX3 immunostaining on Sox3^+/Y, Sox3^KO/Y and Sox3^Sox2KI/Y 4-week-old testes. (D) Microarray analysis was performed on 2 week testes from Sox3^+/Y, Sox3^KO/Y and Sox3^Sox2KI/Y, and two-way comparisons were performed between Sox3^+/Y and either Sox3^Sox2KI/Y or Sox3^KO/Y. Genes presented were significantly different between Sox3^+/Y and Sox3^KO/Y testes (Step-up P value <0.05). * <0.05 step-up P value between Sox3^KO/Y and Sox3^Sox2KI/Y. (E) qPCR validation of SOX2 regulation of SOX3 target genes in 2 week testes. ANOVA multiple comparisons with Bonferroni’s correction were performed using Graphpad Prism (****<0.0001, ***<0.001, **<0.01, *<0.05, ns, not significant).

Figure 4

SOX2 can functionally replace SOX3 in the testes. (A) Gross morphology of Sox3^+/Y, Sox3^KO/Y and Sox3^Sox2KI/Y 4-week testes. (B) Quantification of testes weight at 4 and 8 weeks old [at least 18 testes were weighed for each genotype, and results were compared using one way ANOVA multiple comparisons with Bonferroni’s correction (****<0.0001, ns, not significant)]. (C) DAPI staining of Sox3^+/Y, Sox3^KO/Y and Sox3^Sox2KI 4 week old testes. Asterisks indicate empty tubules. (D) Sperm counts from 8-week Sox3^+/Y, Sox3^KO/Y and Sox3^Sox2KI/Y epididymis [at least 14 samples were counted for each genotype, and results were compared using one way ANOVA multiple comparisons with Bonferroni’s correction (****<0.0001, **<0.01 ns, not significant)].