Functional Equivalence of the SOX2 and SOX3 Transcription Factors in the Developing Mouse Brain and Testes
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Genetics: Early Online, published on May 17, 2017 as 10.1534/genetics.117.202549 1 Functional equivalence of the SOX2 and SOX3 transcription factors in the 2 developing mouse brain and testes 3 4 Fatwa Adikusuma1, 2, Daniel Pederick1, Dale McAninch1, James Hughes1 and Paul Thomas1, 3* 5 6 1School of Biological Sciences and The Robinson Research Institute, University of Adelaide, 7 Adelaide, SA, AUS 5005 8 2CEBIOR, Faculty of Medicine, Diponegoro University, Semarang, Indonesia, 50271 9 3South Australian Health and Medical Research Institute, Adelaide, SA, AUS, 5000 10 *Corresponding author: 11 Email: [email protected] 12 13 14 15 16 17 18 1 Copyright 2017. 19 Abstract 20 Gene duplication provides spare genetic material that evolution can craft into new 21 functions. Sox2 and Sox3 are evolutionarily-related genes with overlapping and unique sites 22 of expression during embryogenesis. It is currently unclear whether SOX2 and SOX3 have 23 identical or different functions. Here we use CRISPR/Cas9-assisted mutagenesis to perform a 24 gene-swap, replacing the Sox3 ORF with the Sox2 ORF to investigate their functional 25 equivalence in the brain and testes. We show that increased expression of SOX2 can 26 functionally replace SOX3 in the development of the infundibular recess/ventral 27 diencephalon and largely rescues pituitary gland defects that occur in Sox3 null mice. We 28 also show that ectopic expression of SOX2 in the testes functionally rescues the 29 spermatogenic defect of Sox3 null mice and restores gene expression to near normal levels. 30 Together, these in vivo data provide strong evidence that that SOX2 and SOX3 proteins are 31 functionally equivalent. 32 33 Keywords: SOXB1 genes, CRISPR/CAS9 mutagenesis, gene swap 34 35 36 37 38 2 39 Introduction 40 One of the driving forces for the evolution of complex life is the duplication of genes, 41 chromosomes or entire genomes, providing the genetic material upon which natural 42 selection can operate. Evolutionary theory predicts that having duplicated, a gene pair will 43 be relieved from selective constraints thereby enabling the accumulation of genetic 44 alterations that can alter protein function (Force et al., 1999; Lynch and Conery, 2000). The 45 consequences of this are thought to favour loss of one copy (non-functionalisation). 46 Alternatively, gene functions can be divided between the paralogues (sub-functionalisation) 47 or one copy can acquire a novel advantageous function (neo-functionalisation) while the 48 other copy retains its original function (Force et al., 1999; Lynch and Conery, 2000). Under 49 this paradigm it is expected that shared function within a given tissue will not be preserved 50 by natural selection and should therefore be lost over time. This stands in contrast to many 51 observations of genetic redundancy that have emerged in the age of molecular genetics as 52 gene deletions in seemingly important genes routinely yield no or mild phenotypes and 53 appear to be compensated for by paralogous partner genes (Wagner, 2005). Estimates 54 suggest that as many as 10-15% of mouse gene knockouts may have no or mild phenotypes 55 (Barbaric et al., 2007). What forces allow the persistence of genetic redundancy are unclear 56 but genetic robustness that acts to maintain and bolster important processes may play a 57 role (Barbaric et al., 2007; Force et al., 1999; Wagner, 2005). 58 Persistent genetic redundancy is particularly striking in the SoxB1 subfamily, which consists 59 of Sox1, Sox2 and Sox3. These genes share highly similar sequences, both within and to a 60 lesser extent outside of the DNA-binding HMG box. Several studies suggest that SOXB1 61 proteins have similar if not identical functional capabilities. For example, overexpression of 3 62 chick or mouse SoxB1 genes in chick neural tube results in inhibition of neural 63 differentiation with cells retaining a progenitor identity (Bylund et al., 2003; Graham et al., 64 2003). Similarly, mouse Sox1 and Sox3 are able to replace Sox2 for reprogramming of iPS 65 cells (Nakagawa et al., 2008). Loss of function studies also generally support functional 66 equivalence, particularly in the developing CNS where the SoxB1 genes exhibit extensive 67 overlapping expression. For example Sox3 deletion in mice results in relatively mild neural 68 defects indicating that SOX2 and/or SOX1 can compensate for the absence of SOX3 in most 69 neuroprogenitor contexts. However, one notable exception is the infundibulum, a ventral 70 evagination of the ventral diencephalon that is responsible for induction of the anterior 71 pituitary primordium (Rathke’s Pouch). Despite co-expression of Sox2 and Sox3, pituitary 72 induction and development is severely compromised in Sox2 and Sox3 single mutants 73 (Kelberman et al., 2006; Rizzoti et al., 2004). It is thought that this is due to reduced dosage 74 of SOX2 or SOX3, as opposed to unique roles of these proteins (Zhao et al., 2012). However, 75 to date, experimental approaches that distinguish between these possibilities have not been 76 published. 77 Restricted zones of SoxB1 expression outside of the nervous system have also been 78 described, many of which are in stem/progenitor cells of developing organs. For example, 79 Sox3 is uniquely expressed in the spermatogonial stem/progenitor cells of the postnatal 80 testes (Raverot et al., 2005; Rizzoti et al., 2004). Consistent with a model of limited sub- 81 functionalisation, more severe phenotypes occur in knockout mice at sites of unique 82 expression. For example, Sox3 null mice have spermatogenic defects likely due to the 83 absence of Sox1 and Sox2 (Raverot et al., 2005). However, it is not known whether SoxB1 84 genes are functionally interchangeable at these unique zones of expression. 4 85 86 Herein we describe an in vivo gene swap experiment in which Sox3 open reading frame 87 (ORF) was deleted and replaced with Sox2 ORF to investigate their functional similarities. 88 We show that SOX2 can functionally replace SOX3 in both the developing pituitary and 89 testes, thereby rescuing phenotypes associated with SOX3-null mice. 90 91 92 Materials and Methods 93 Generation of CRISPR/Cas9 modified mice 94 CRISPR gRNAs were designed either side of the Sox3 ORF ( 5’-CCTGATGCGTTCTCTCGAGC-3’ 95 and 5’-GACAGTTACGGCCAAACTTT-3’) using CRISPR Design tool (crispr.mit.edu) and 96 generated according to the protocol described in (Wang et al., 2013). gRNA IVT was 97 performed using HiScribe™ T7 Quick High Yield RNA Synthesis Kit. Cas9 mRNA was 98 generated by IVT using mMESSAGE mMACHINE® T7 ULTRA Transcription Kit (Ambion) from 99 pCMV/T7-hCas9 (Toolgen) digested with Xho1. gRNAs and Cas9 mRNA were purified using 100 MEGAclear™ Transcription Clean-Up Kit (Ambion). Our previously published Sox3 targeting 101 vector (Hughes et al., 2013) was modified to replace Sox3 ORF with Sox2 ORF. Cas9 mRNA 102 (100 ng/uL), gRNAs (50 ng/uL each) and donor plasmid (200 ng/uL) were injected into the 103 cytoplasm of C57BL/6N zygotes using a FemtoJet microinjector, transferred to pseudo 104 pregnant recipients and allowed to develop to term. Homology directed repair from the 105 vector resulted in the Sox3Sox2KI mice carrying a neomycin resistance cassette 1 kb 106 downstream from the Sox2-KI stop codon. The Sox3Sox2KI mice also contain a 2 bp deletion in 5 107 the 5’UTR at the upstream gRNA site and a 1 bp in the 3’UTR at the downstream gRNA site, 108 presumably as a result of CRISPR/Cas9 re-cutting after homology directed repair. 109 110 Microarray analysis 111 Microarray expression profiling was performed using Affymetrix GeneChip Mouse Gene 1.0 112 ST Arrays on three Sox3 null and three Sox3Sox2KI 2 week testes. A total of six wild type age 113 matched samples were included comprising two groups of three matched to the same 114 genetic background as the Sox3 null and Sox3Sox2KI samples. Two way-ANOVA, using batch as 115 a factor, was used to identify the significantly regulated genes. ANOVA was performed 116 comparing to matched WT samples and comparing to pooled WT samples with similar 117 results. We have presented data comparing two pooled WT samples. 118 119 Sperm counting 120 Cauda epididymis were isolated and minced in 1 mL of 37 0C DMEM media. Sperm were 121 allowed to disperse for 10-15 minutes at 37 0C. 10 uL of the resuspension was diluted with 122 10 uL of 1 M Tris pH 9.5 solution to immobilise sperm before counting with a 123 hemocytometer. 124 125 Data availability 6 126 Microarray data has been submitted to Gene Expression Omnibus (GEO) with accession 127 number GSE96805. All other reagents can be made available upon request. Supporting data 128 can be found in supplemental data (File S1). 129 130 131 Results 132 Generation of Sox3Sox2KI mice using CRISPR/Cas9 mutagenesis 133 To investigate the functional redundancy within the SoxB1 subgroup we replaced the Sox3 134 ORF with that of Sox2 while leaving the remaining native Sox3 flanking sequences including 135 the promoter and untranslated regions (UTR) intact. This mouse model, which we refer to as 136 Sox3Sox2KI, therefore lacks SOX3 and expresses SOX2 from the Sox3 locus on the X- 137 chromosome. To generate Sox3Sox2KI mice, we initially modified an existing Sox3 KO 138 targeting construct (Hughes et al., 2013; Rizzoti et al., 2004) by replacing the Sox3 ORF with 139 Sox2. Attempts to generate Sox3Sox2KI mice by conventional gene targeting in mouse ES cells 140 failed to produce any chimeras despite multiple rounds of injections using germline 141 competent cells (data not shown). To circumvent this issue, we employed CRISPR/Cas9 142 technology to generate Sox3Sox2KI mice by zygotic injection of Cas9 mRNA as well as gRNA 143 pairs targeting either side of the Sox3 ORF and the donor (targeting) plasmid (Figure 1A).