Figures
Abstract
The Y-chromosomal gene SRY acts as the primary trigger for male sex determination in mammalian embryos. Correct regulation of SRY is critical: aberrant timing or level of Sry expression is known to disrupt testis development in mice and we hypothesize that mutations that affect regulation of human SRY may account for some of the many cases of XY gonadal dysgenesis that currently remain unexplained. However, the cis-sequences involved in regulation of Sry have not been identified, precluding a test of this hypothesis. Here, we used a transgenic mouse approach aimed at identifying mouse Sry 5′ flanking regulatory sequences within 8 kb of the Sry transcription start site (TSS). To avoid problems associated with conventional pronuclear injection of transgenes, we used a published strategy designed to yield single-copy transgene integration at a defined, transcriptionally open, autosomal locus, Col1a1. None of the Sry transgenes tested was expressed at levels compatible with activation of Sox9 or XX sex reversal. Our findings indicate either that the Col1a1 locus does not provide an appropriate context for the correct expression of Sry transgenes, or that the cis-sequences required for Sry expression in the developing gonads lie beyond 8 kb 5′ of the TSS.
Citation: Quinn A, Kashimada K, Davidson T-L, Ng ET, Chawengsaksophak K, Bowles J, et al. (2014) A Site-Specific, Single-Copy Transgenesis Strategy to Identify 5′ Regulatory Sequences of the Mouse Testis-Determining Gene Sry. PLoS ONE 9(4): e94813. https://doi.org/10.1371/journal.pone.0094813
Editor: Christoph Englert, Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), Germany
Received: February 3, 2014; Accepted: March 19, 2014; Published: April 17, 2014
Copyright: © 2014 Quinn et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Support was provided by the Australian Research Council (ARC) Discovery Grant No. DP120104534 [www.arc.gov.au] and the National Health and Medical Research Council (NHMRC) (Australia) Program Grant No. 546517 [www.nhmrc.gov.au]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The Y-chromosomal testis-determining gene SRY/Sry has a unique importance in mammalian biology, providing the key to sexual reproduction that in turn acts as the engine for genetic recombination and natural selection. Sry provides the pivot point for sex development in XY mammalian embryos by stimulating the differentiation of testes rather than ovaries from the embryonic gonadal anlagen, the genital ridges [1]. Once testes have formed, differentiation of the embryo as a male is promoted by hormones produced by the Leydig cells. In embryos where Sry is absent or non-functional the genital ridges develop as ovaries and female differentiation follows [2], [3].
More than 20 years have elapsed since SRY/Sry was discovered [4], [5], but relatively little is understood at the molecular or cellular levels regarding how it achieves its important role. The critical function of Sry is to up-regulate transcription of Sox9 in a subset of genital ridge cells [6], [7]. Once Sox9 is activated, the transcription factor it encodes appears to co-ordinate the sequence of molecular events leading to Sertoli cell differentiation [8], [9]. Sry can therefore be viewed as a switch whose activity is required only briefly, after which its work is done.
Recent studies have shed some light on the mechanism by which SRY activates Sox9 transcription. SRY protein contains an HMG DNA-binding domain that binds to regulatory elements upstream of Sox9 in conjunction with the partner factor NR5A1 (SF1) [7]. SRY activity also evidently rests on a transcriptional trans-activation domain at its C-terminus [10], [11]. Once Sox9 transcription is activated, it is sustained through a positive-feedback loop involving SOX9 protein (also an HMG transcription factor), NR5A1 and the same regulatory elements upstream of Sox9.
It has become apparent that Sry expression needs to be regulated precisely in order to activate Sox9 transcription and initiate the testis-determining pathway, rather than allowing the ovarian-determining pathway to take hold. In the mouse, Sry expression starts at 10.5 dpc in somatic cells of XY genital ridges, reaches a peak at 11.5 dpc and wanes by 12.5 dpc [12]–[16]. If Sry expression is delayed by as little as a few hours either experimentally [17] or through breeding of Y chromosomes from certain strains onto a C57BL/6 genetic background [18], [19], testis differentiation is compromised or blocked completely in favour of ovarian development. The current working model is that SRY expression in each cell must reach a threshold level within a defined window of time in order for Sox9 to be activated cell-autonomously [20]. Based on these findings it seems likely that mutations affecting human SRY expression may account for some of the many cases of XY gonadal dysgenesis that currently remain unexplained.
A handful of transcription factors have been implicated in regulating Sry expression, including WT1 [21], NR5A1 [7], GATA4/FOG2 [22], and SIX1/SIX4 [23]. In addition, insulin growth factor signalling [24], mitogen-activated protein kinase signalling [25] in conjunction with the associated stress response protein GADD45γ [26], [27], and chromatin remodelling factors such as histone demethylase JMJD1A [28] and polycomb protein CBX2 [29], have all been implicated in Sry regulation. However, the critical transcription factors and their target binding cis-regulatory sites have not been identified, clouding our understanding of how Sry is regulated. Further, Sry resides on the Y chromosome in a quicksand of genetic drift, rendering conventional bioinformatic tools used for identifying important DNA sequence motifs by cross-species comparison largely useless.
Here, we use a transgenic mouse approach aimed at identifying mouse Sry 5′ flanking regulatory sequences. In previous studies, a 14 kb genomic DNA fragment including Sry was shown to cause sex reversal in XX transgenic mice [1], [6], [11]. In addition, reporter transgenes lacking the Sry coding region but including the proximal 8 kb of the Sry 5′ region have been shown to be expressed with a similar spatial and temporal pattern to endogenous Sry [6], [30]. Based on these studies, our starting assumption was that all cis-sequences required for correct expression of Sry lie within 8 kb 5′ of the transcription start site (TSS). The strategy used here is based on Sry genomic DNA fragments that differ in their 5′ flanking sequence content, and assaying the ability of these fragments to direct male sex development in XX embryos.
Previous studies also found variation in the ability of the 14 kb Sry transgene to induce XX sex reversal [1], [6], [11], most likely because in those studies it was not possible to create mice with a predetermined number of copies of the transgene, nor ensure a consistent site of transgene integration. These technical difficulties, in combination with the restricted and transient expression of Sry, have complicated the conduct and interpretation of experiments designed to locate Sry regulatory sequences.
To avoid these problems, we adopted a published strategy designed to yield single-copy transgene integration at a defined autosomal locus, Col1a1 [31]. However, we found that none of the Sry transgenes tested was expressed at levels compatible with XX sex reversal. Our experiments suggest either that the Col1a1 locus, although supposedly in an open transcriptional state, is not an appropriate context for the correct epigenetic and/or transcriptional activation of Sry transgenes, or that the assumption that the cis-sequences required for Sry transcription in the genital ridges lie within 8 kb 5′ of the TSS is incorrect.
Materials and Methods
Ethics statement
All procedures involving animals and their care conformed to institutional, state and national guidelines. This study was approved by the University of Queensland Animal Ethics Committee (Permit Numbers: IMB/076/11/ARC/NHMRC, IMB/434/09/ARC/QSG/UQ/BREED (NF) and IMB/087/10/NHRMC/ARC (NF)).
ES cells
An ES cell line (KH2) with an Frt-PGK-neo-pA-Frt-hygro-pA transgene homing cassette integrated ∼0.5 kb downstream of the 3′ untranslated region (UTR) of the Col1a1 gene was kindly provided by R. Jaenisch and K. Hochedlinger (Fig. 1) (#MES4304, Thermo Scientific Open Biosystems) [31]. KH2 is an F1 ES cell line derived from a C57BL/6 and 129svJae cross. The cassette had been inserted by homologous recombination, with correctly targeted cells selected on the basis of neomycin resistance [31]. In conjunction with a Flp recombinase, this cassette allows for site-specific Flp-Frt recombination with a targeting vector consisting of an Frt site, a gene of interest, and a PGK promoter and ATG initiation codon that enable transcription (and selection for) the hygromycin resistance gene when correctly integrated. KH2 cells were grown on hygromycin-resistant MEFs at 37°C (5% CO2) in high-glucose DMEM media (GIBCO) supplemented with 20% fetal calf serum, 1% penicillin/streptomycin, 1% β-mercaptoethanol, 1% GlutaMax, 1% non-essential amino acids and 1% sodium pyruvate.
KH2 ES cells carry an Frt-PGK-neo-pA-Frt-hygro-pA “homing” cassette at a PstI site downstream of the Col1A1 locus. Sry transgene constructs were cloned into a PGK-ATG-Frt vector, and then this was integrated into the homing site within the KH2 cells by co-electroporation with a FlpE transient expression vector (pCAGGS-FlpE-puro). Flp-Frt homologous recombination resulted in loss of the PGK-neo-pA cassette and insertion of the Sry-PGK-ATG-Frt vector bearing the transgene into the Col1A1 locus in the ES cells. Black arrowheads indicate Frt sites, black boxes indicate exons, grey arrows and italics indicate primer sites for genotyping of ES cells and mice. Amp, ampicillin resistance. Neo, neomycin resistance. Hygro, hygromycin resistance. pA, polyadenylation signal. PGKp, PGK promoter. Not drawn to scale.
Sry-PGK-ATG-Frt constructs
DNA fragments containing the Sry gene and flanking sequences were prepared from the 14.6 kb genomic clone L741 (NCBI: X67204) [5]. Five Sry fragments with 5′ regions of varying length were prepared (Fig. 2). Sry-StuI was excised from L741 by StuI digestion. Because the sequences flanking the Sry locus are palindromic, three fragments with progressively shorter 5′ arms (Sry#18, Sry#49, Sry#5) were prepared by 5′ exonuclease digestion of the Sry-StuI fragment. All five fragments were blunted by 3′ exonuclease digestion and cloned into the EcoRV site of a PGK-ATG-Frt targeting vector (#MES4490, Thermo Scientific Open Biosystems). Direction of the fragments was confirmed by direct sequencing using primers MES4490-F and MES4490-R on either side of the EcoRV site in the vector. Direction of the Sry-StuI insert was confirmed by BglII digestion because of the palindromic sequence of the Sry 5′ and 3′ flanking regions. Integrity of the 1188 bp Sry protein-coding sequence was verified for all cloned fragments by direct sequencing using primers SRY-CDS-F and SRY-CDS–R. All primers used for genotyping and sequencing are presented in Table 1, and the location of some primer sites are indicated in Figures 1 and 2.
Left panel shows the structure of the five transgene constructs integrated into the Col1a1 locus, featuring sequential deletions of the Sry 5′ region. Length of sequence 5′ to the transcriptional start site (TSS) is indicated beneath the constructs (base-pairs). Grey box, Sry coding region. PolyA, polyadenylation signal. Black arrows and italics indicate primer sites used for genotyping. Grey arrows indicate inverted repeat sequences flanking the Sry coding region. Right hand panel shows corresponding qRT-PCR analysis of Sry mRNA levels in samples (gonad and mesonephros pairs) collected at 11.5 dpc (2−ΔCt, normalized against Sdha). Error bars are 95% confidence intervals. Numbers beside bars indicate sample size (individual gonad-mesonephros pairs).
Site-specific recombination of Sry constructs into ES cells
Approximately 0.5–1×107 KH2 cells were electroporated with 50 µg Sry-PGK-ATG-Frt plasmid and 25 µg FlpE recombinase-expressing vector (pCAGGS-FlpE-puro, #MES4488, Thermo Scientific Open Biosystems) at 500 V and 25 µF using two pulses in a Gene Pulser Xcell Electroporation System (Bio-Rad). After 48 h growth, cells were treated with 140 µg/ml hygromycin to select for cells in which the targeting vector had been inserted by Flp-Frt recombination. Integration of Sry-PGK-ATG-Frt constructs into KH2 cells was identified by PCR amplification of a 1.5 kb product with primers SRY-PGK and COL1A1-GENO12.
Generation of chimeras and transgenic lines
Hygromycin-selected ES cells were injected into wild type C57Bl/6J donor blastocysts (10–15 ESC/blastocyst), and blastocysts were transferred into a 0.5 or 2.5 dpc pseudopregnant CD1 female. Chimeric offspring were identified by coat colour then bred to C57Bl/6 females to generate the first generation; pups in which germ line transmission had occurred were identified by genotyping for presence of the transgene. Heterozygous mice were used to establish lines for analysis.
Genotyping of transgenic offspring
Integration of the Sry transgene in offspring was determined by PCR amplification of a 261 bp product with primers SRY-GENO4 and SRY-GENO7 (for the Sry-L741 line) or a 201 bp product with primers SRY-GENO2 and SRY-GENO7 (for all other lines), using genomic DNA extracted from ear notch tissue of juveniles. Sex of juveniles was determined by examination of external reproductive anatomy. Transgenic studs were occasionally mated with transgenic females of the same line. Transgenic offspring homozygous for the Sry transgene were distinguished from heterozygotes using primers COL1A1-GENO7 and COL1A1-GENO12, which span the insertion site of the Sry-PGK-ATG-Frt construct, 3′ to the Col1a1 gene. These primers amplify a 329 bp product from the wild type allele, but do not amplify a product from Col1a1 alleles containing the inserted Sry-PGK-ATG-Frt construct (which is several kilobases in length). Thus, the 329 bp product was amplified in heterozygous transgenics but not homozygotes. A second primer pair, ROSA26-1 and ROSA26-3 [32], was included in the genotyping PCRs, to amplify a 603 bp product from the Rosa26 locus. This larger product served as a positive control for successful amplification, reducing the likelihood of incorrect genotype calls in the event of PCR failure. For each transgenic line, the entire Sry coding region was amplified, cloned and sequenced from a representative transgenic XX embryo to again verify the integrity of the protein-coding sequence.
Timed matings and dissection
Embryos were collected from timed matings of transgenic studs with outbred CD1 strain females, with noon of the day on which the mating plug was observed designated as 0.5 days post coitum (dpc). For more accurate staging, the tail somite (ts) stage of the embryo was determined by counting the number of somites posterior to the hind limb. Using this method, 10.5 dpc is approximately equivalent to 8 ts, 11.5 dpc to 18 ts, and 12.5 dpc to 30 ts [13]. For all embryos, chromosomal sex was determined by PCR genotyping with primers SX-F and SX-R [33], using genomic DNA extracted from tail tissue. From 12.5 dpc onwards, embryos were also sexed by morphological assessment of the gonads. Gonad pairs were dissected and immediately stored in RNAlater RNA stabilization solution (Invitrogen) until required for gene expression analysis by quantitative RT-PCR. For embryos at or prior to 12.5 dpc, gonads were collected with mesonephros attached, whereas gonadal tissue only was collected from embryos beyond this stage.
RNA extraction, cDNA synthesis, and quantitative real-time RT-PCR
Gonadal tissue from each embryo was processed and analyzed individually. Total RNA was extracted and subjected to DNase treatment using an RNeasy Micro kit (Qiagen) in accordance with the manufacturer's instructions, and quantified with a NanoDrop spectrophotometer (NanoDrop Technologies). cDNA was synthesized by reverse transcription with SuperScript III and random hexamers (Invitrogen), from 100 ng total RNA (for analyses of precisely-staged embryos between 8–30 ts, within a single transgenic line) or from 300 ng total RNA (for all other analyses, at 11.5 or 13.5 dpc). All qRT-PCR reactions were performed in triplicate using SYBR Green PCR master mix (Invitrogen) and 150 nM each of forward and reverse primers, and analyzed on a Viia7™ Real-Time PCR System (Invitrogen). Sry cDNA was amplified with primers 5′-TTATGGTGTGGTCCCGTGGT and 5′-GGCCTTTTTTCGGCTTCTGT [34] and Sox9 cDNA was amplified with primers 5′-AGTACCCGCATCTGCACAAC and 5′-TACTTGTAATCGGGGTGGTCT. Relative cDNA levels were determined by calculating 2−ΔCt values relative to the house-keeping gene Sdha, using primers 5′-TGTTCAGTTCCACCCCACA and 5′-TCTCCACGACACCCTTCTGT. Sdha was verified previously as a suitable normalization gene for gonadal tissue [35]. qRT-PCR data is presented as the mean 2−ΔCt value for multiple individual embryos (minimum of 3). Sample sizes are indicated in relevant figures.
Section immunofluorescence
Whole embryos were collected at 11.5 dpc and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C overnight, washed three times with PBS at 4°C, then dehydrated and embedded in paraffin. Section immunofluorescence was performed on 7 µm-thick sagittal sections as described previously [16]. Rabbit anti-SRY (1∶100) [16] and goat anti-rabbit Alexa Fluor 488 (1∶200, Invitrogen) were used as primary and secondary antibodies, respectively. Images were captured using a BX51 fluorescence microscope fitted with a DP70 camera (Olympus), and compiled using the associated software (Olympus) and Adobe Photoshop CS6 version 16.0 (Adobe) software.
Results
We created five transgenic constructs containing the mouse Sry coding region (1.2 kb) with sequential deletions of the 5′ flanking sequence; Sry-L741 (8.0 kb of 5′ sequence), Sry-StuI (4.1 kb), Sry#18 (2.1 kb), Sry#49 (1.3 kb), and Sry#5 (0.3 kb) (Fig. 2). All constructs also included 3′ flanking sequences for Sry, encompassing both the minor and major polyadenylation sites [13], [14] (L741, 5.2 kb of 3′ sequence; all others, 4.2 kb). Constructs were integrated in single copy at the same autosomal locus (3′ to the Col1a1 gene) by homologous recombination in ES cells bearing a homing cassette at that location [31]. We established transgenic mouse lines from blastocysts injected with the ES cells, then analysed the lines for XX sex reversal and for level and timing of Sry transgene expression, in an attempt to identify cis-sequences required for correct regulation of Sry.
Genotype and phenotype analysis of transgenic offspring
Breeding lines for each of the five Sry transgenes were maintained by mating of transgenic males with wild type CD1 females. To identify any instances of XX sex reversal, a total of 495 juvenile offspring of these matings (n = 82 for Sry-L741, n = 81 for Sry-StuI, n = 123 for Sry#18, n = 131 for Sry#49, n = 78 for Sry#5) were sexed and genotyped to determine both chromosomal sex (XX or XY) and presence or absence of an Sry transgene (Table 2). The ratios of transgenic to wild type (251∶244) and XX to XY (249∶246) offspring were both very close to 1∶1. None of 113 XX Sry-transgenic offspring developed as male, indicating that all five variants of the Sry transgenes in single-copy at the Col1a1 locus were unable to induce XX sex reversal. Moreover, no indications of sex reversal were observed in the gross structure of dissected gonads from 13.5 dpc XX embryos transgenic for Sry-L741, Sry#18 or Sry#49.
To test whether increased dosage of Sry-L741 or Sry#49 were able to induce XX sex reversal, transgenic males were mated with transgenic females of the same line to generate XX offspring homozygous for the transgene (at an expected proportion of 0.125 XX offspring/litter). Three of 19 embryos examined at 13.5 dpc were determined by genotyping as XX and homozygous for Sry-L741, and four of 30 embryos examined at 13.5 dpc were XX and homozygous for Sry#49. No indications of female to male sex reversal were observed in the gross gonadal morphology of the XX transgenic homozygotes for either line.
Transgene expression analysis for gonads at 11.5 dpc
For all five transgenic lines, gonadal expression of Sry and Sox9 was analysed by qRT-PCR at 11.5 dpc, the time point when endogenous Sry expression reaches its maximum in wild type XY embryos. In XX gonads, the Sry#49 transgene showed markedly higher Sry mRNA expression than the four other transgenes, all of which showed very low expression (Fig. 2). Although Sry#49 was the most strongly expressed of the five transgene constructs in XX gonads, the total level of Sry mRNA was approximately half that found in wild type XY gonads at 11.5 dpc (Fig. 3A). Analysis of total Sry mRNA levels in XY gonads, comprising both endogenous Sry and transgenic Sry mRNA, similarly revealed that Sry#49 produced the highest expression for the five transgenic lines (Fig. 3A). The total Sry mRNA level in XY Sry#49 gonads was strikingly higher than for XY wild type gonads, and presumably reflected the combined effect of endogenous Sry mRNA and Sry#49 mRNA. Total Sry mRNA levels for XY Sry-L741, Sry-StuI, Sry#18 and Sry#5 gonads did not differ significantly from wild type XY levels.
(A) Total Sry mRNA expression for wild type and transgenic XX (black) and XY (white) embryos. (B) Sox9 mRNA expression for the same embryos. Relative expression is measured as 2−ΔCt values, normalized against Sdha. Numbers above bars indicate sample size (individual gonad-mesonephros pairs). Error bars are 95% confidence intervals. *P<0.01, **P<0.001, Student's unpaired t-test.
There was no apparent effect of Sry transgene expression on the level of Sox9 transcription in the XX transgenic gonads at 11.5 dpc, even for Sry#49, the line exhibiting the highest transgene expression (Fig. 3B). For all XX transgenic lines, Sox9 mRNA expression did not differ significantly from the basal level of Sox9 expression in XX wild type gonads, and was far less than for XY gonads. Similarly, expression of the Sry transgenes in XY gonads did not cause any further increase in Sox9 expression above the level for XY wild type gonads at 11.5 dpc. The failure of the Sry transgenes to upregulate Sox9 in the gonads evidently underlies the lack of XX sex reversal in the transgenic lines.
Given the lack of Sox9 upregulation and lack of XX sex reversal observed for all five transgenic lines, subsequent molecular analyses were restricted to Sry-L741, the transgene bearing the longest Sry 5′ region sequence, and Sry#49, the transgene producing the highest level of Sry transcription.
Sry protein expression analysis of transgenic gonads
Immunofluorescent antibody staining of sectioned embryos failed to detect Sry protein expression in XX Sry-L741 gonads at 11.5 dpc, or in XX Sry#49 gonads at 11.5 and 12.5 dpc, but did identify Sry expression along the entire length of a control XY wild type gonad at 11.5 dpc (Fig. 4). This result is consistent with the very low mRNA levels for XX transgenic gonads revealed by qRT-PCR, and suggests the level of Sry protein per cell, even for XX Sry#49 gonads, was probably too low to be detectable by immunofluorescence. The sequence of the transgenic Sry coding region in each of the transgenic lines was also verified as being intact, eliminating the possibility that introduced mutations had caused protein instability and/or loss of function. It is possible that Sry protein was not expressed at all, but based on the apparently very low mRNA expression per cell, we consider it likely that Sry protein levels were simply below a threshold for detection by immunofluorescence.
Expression was visualised by immunofluorescent staining of sagittal embryo sections. Dashed outline indicates gonad tissue. (A) XY wild type embryo at 11.5 dpc. (B) XX Sry#49 embryo at 11.5 dpc. (C) XX Sry#49 embryo at 12.5 dpc. (D) XX Sry-L741 embryo at 11.5 dpc. Scale bar, 100 µm.
Temporal transgene expression analysis
None of the five transgenes were evidently transcribed at a level sufficient to upregulate Sox9 expression and effect XX sex reversal, but a further question remained over the ability of the transgenes to emulate the temporal expression pattern of endogenous Sry, which is switched on at approximately 10.5 dpc, peaks at 11.5 dpc, and ceases at 12.5 dpc (Fig. 5A). To test this, we compared transgene expression in XX gonads with Sry expression in XY wild type gonads from precisely staged embryos between 10.5 and 12.5 dpc, using wild type/transgenic littermates whenever possible. Sry#49 was examined most thoroughly because it produced the highest level of Sry mRNA expression of the five transgenes, enabling the temporal pattern of expression to be established clearly (Fig. 5B). A smaller sample of precisely staged Sry-L741 gonads was also analysed, since this transgene bears the longest Sry promoter sequence of the variants. A few gonads from the later stage of 13.5 dpc were included in this analysis for both the Sry#49 and Sry-L741 lines.
qRT-PCR (2−ΔCt, normalized against Sdha) was performed for gonad-mesonephros pairs from precisely staged embryos between 10.5 dpc (∼8 ts) and 12.5 dpc (∼30 ts), and for gonad pairs at 13.5 dpc. Each circle represents a single sample. (A) Wild type XY (white) and XX (black) gonads. Dashed line shows curve of fit generated by non-linear regression for XY wild type gonads (5–30 ts, 6th order polynomial function, least squares fit, R2 = 0.7012). (B) XX Sry#49 (white), XY Sry#49 (grey), and XX Sry-L741 (black) gonads. XY Sry#49 gonads are included at 13.5 dpc only, as endogenous Sry expression would mask transgenic Sry expression at the earlier time points. Dashed line shows curve of fit generated by non-linear regression for XX Sry#49 gonads (5–30 ts, 6th order polynomial function, least squares fit, R2 = 0.2081).
Sry#49 displayed roughly equivalent expression in gonads from the earliest stages examined (6–7 ts) right through to the latest stage (13.5 dpc) - that is, prior to and well after the window of endogenous Sry expression - and did not peak at 11.5 dpc. A single gonad pair at 18 ts gave a strikingly higher expression value for Sry#49, but this sample appeared to be an outlier amongst many other gonads analysed at 17–19 ts. If any pattern could be discerned for Sry#49, it was possibly a subtle increase in gonad expression as time progressed. The few Sry-L741 gonads analysed produced much lower Sry expression than Sry#49 gonads, in agreement with the previous analysis at 11.5 dpc (Fig. 2). Sry-L741 also showed no indication of a temporal pattern reminiscent of endogenous Sry, most notably because expression remained roughly consistent between 11.5 and 13.5 dpc.
Non-gonadal transgene expression
In addition to the defined temporal window, endogenous Sry expression is also largely restricted during embryogenesis to the precursor Sertoli cells of the developing gonads. To determine if the Sry transgenes emulated the tissue-specificity of endogenous Sry, expression of Sry#49 mRNA was compared for gonad, liver and brain tissue from XX and XY embryos at 13.5 dpc (Fig. 6A). Expression did not differ significantly between the sexes, or between the gonad and liver, but was slightly higher for gonad compared with brain tissue (P<0.05). Expression of the Sry-L741 transgene was similarly compared for gonad, liver and brain tissue from XX embryos at 11.5 dpc (Fig. 6B; XY wild type Sry expression shown for comparison). Expression of the Sry-L741 transgene was very low in all tissue types, and did not differ significantly between tissues. For both transgenes examined, expression was clearly not restricted to gonad tissue, in contrast to endogenous Sry.
Analyses were performed by qRT-PCR (2−ΔCt, normalized against Sdha). (A) Expression of Sry mRNA in XX Sry#49 (black) and XY Sry#49 (white) tissues at 13.5 dpc. (B) Expression of Sry mRNA in XX Sry-L741 (black) and XY wild type (white) tissues at 11.5 dpc. Error bars are 95% confidence intervals. Numbers above bars indicate sample size.
These data suggest that Sry transgenes from the Col1a1 locus are transcribed in most, if not all cell types. If this is the case, then the level of Sry mRNA expression for XX Sry#49 gonads at 11.5 dpc (the most strongly-expressed transgene; Fig. 2A) may reflect the total amount for all gonad (and mesonephros) cells combined, such that the amount of Sry mRNA per precursor supporting cell is very low compared with the equivalent cells in the XY wild type gonad at 11.5 dpc. This situation likely explains why there is insufficient Sry expression per cell in Sry#49 gonads to upregulate Sox9 and induce XX sex reversal. It would also be consistent with the failure to detect SRY protein in the XX Sry#49 gonads by IF staining: the amount of transgenic SRY protein per cell may be too low to be detectable.
Discussion
More than two decades have passed since SRY was established as the testis-determining gene in humans and mice, but the specific cis-sequences required for the correct regulation of this pivotal sex-determining gene are still unidentified. In this study, we set out to delineate the 5′ sequences required for correct spatiotemporal regulation of Sry in the mouse by employing a site-specific, targeted transgenic strategy designed to avoid the limitations associated with conventional transgenesis. We created five transgenic lines bearing a single-copy of an Sry transgene, with sequentially deleted promoter regions ranging from 8 kb (the original L741 sequence) to only 0.3 kb of the region 5′ to the transcriptional start site. Unexpectedly, all five Sry transgenes failed to induce sex reversal in XX embryos. Our analyses indicated that the Sry transgenes were expressed in all cell types, but the level of expression in precursor supporting cells of the developing gonadal ridge was too low to upregulate Sox9 expression and induce XX male development.
Our present findings contrast with those of our previous study, in which we found that a series of Sry transgenes, bearing sequentially deleted 5′ promoter regions ranging from 8 kb (L741) to only 57 bp (relative to the TSS), were all capable of inducing XX sex reversal when randomly integrated into the genome by pronuclear injection [36]. Moreover, in that study, there was no reduction in the frequency of sex reversal with sequentially shortened constructs. We assumed that any differences between the 5′ regions in their ability to facilitate transactivation of the Sry transgene were masked by the position effects typically associated with transgenesis by pronuclear injection [37]–[39]. Such effects arise because transgenes are randomly integrated at one or multiple unknown locations within the genome, and in variable copy number - sometimes up to several hundred copies arrayed in tandem. Both copy number and genomic location contribute to variable and unpredictable expression levels; this precluded any meaningful quantitative analysis of Sry expression from the deletion constructs. In contrast, the targeted transgenic strategy used in the current study was expected to produce consistent transgene expression levels, allowing us to accurately assay the activity and tissue-specificity of different promoter constructs in vivo. The lack of XX sex reversal for all five constructs analysed in the present study was unexpected; at the very least, we anticipated that the L741 transgene would induce sex reversal. We suggest two possible explanations for the discrepancy: (1) the Col1a1 site was not suitable as a genomic location for examining transcriptional activation of Sry; or (2) the cis-sequences necessary for Sry regulation lie beyond the 8 kb of 5′ sequence in L741.
Regarding the first possibility, we adopted this gene targeting strategy based on a published report that demonstrated its effectiveness for an EGFP transgene [31]. The Col1a1 locus was chosen for the original study because it had been shown to support high transgene expression even in cell types not normally expressing Col1a1 [40]. In that investigation, the transgene was inserted into a targeting vector that additionally included a tetracycline-inducible promoter. In our laboratory, we have successfully used this tetracycline-inducible system to target and induce expression of a Cyp26b1 transgene at the Col1a1 integration site [41]. In the case of Sry, however, we used a targeting vector that lacked the tetracycline-inducible promoter because we aimed to identify important regulatory sequences by promoter deletion analysis. Despite this difference, there is no immediately apparent explanation why the Col1a1 site might be unsuitable for proper transcription of the Sry transgenes. The fact that Sry#49 showed strong mRNA expression relative to the other transgenes, in both XX and XY embryos, suggests that the Col1a1 integration site was accessible to transcriptional machinery to some degree; otherwise, all five transgenes would have been expressed at an equally low level.
If the integration site did not hinder Sry expression, an alternative explanation for our data is that the cis-sequences necessary for stage and tissue-specific activation of Sry to a level sufficient for Sox9 upregulation were absent from all five transgenic constructs. If that were so, such sequences must lie beyond the 8 kb of 5′ sequence (or outside the entire 14.6 kb fragment) of L741. This assumes that expression of randomly integrated Sry transgenes in previous studies [1], [6], [11] was driven by the fortuitous presence of functional regulatory sequences at the integration sites. Consistent with this assumption, one study found that XX sex reversal could be induced by an Sry transgene in which the entire Sry 5′ region was replaced by the weak basal promoter of the Hsp70.3 gene [42]. However, the conclusion that L741 lacks the essential cis-regulatory sequences is difficult to reconcile with previous transgenic and in vitro studies of Sry regulation.
Firstly, Albrecht and Eicher (2001) analysed an EGFP reporter transgene under the control of the L741 5′ promoter region (lacking only 0.5 kb of the 8 kb sequence at the 5′ end). SryEGFP mRNA was expressed in mouse genital ridges at a low level at 10.5 dpc, increased at 11.5 dpc, and thereafter decreased by 15.5 dpc, and diminished further by postnatal days 1 and 28. EGFP protein was expressed specifically in gonadal somatic cells co-expressing WT1 and GATA4, appeared in the centre of the gonadal ridge at about 11.0 dpc, and spread to 70–80% of the ridge by 11.5 dpc. Thus the SryEGFP transgene was expressed in the same cell lineage and with almost the same stage-specificity as endogenous Sry. It differed only in that the transgene was not totally transcriptionally silenced after 12.5 dpc, which the authors attributed to the lack of Sry 3′ flanking sequence in the transgene.
In a later study, Sekido et al. (2004) analysed two reporter variants of an L741 transgene, one bearing a Myc tag immediately 3′ to the Sry coding region and another in which the HMG and bridge domains were replaced by the human placental alkaline phosphatase gene. Expression of both SryMyc and SryhPLAP was gonad-specific and began after 10.5 dpc, peaked at 11.5 dpc, and ceased by 12.5 dpc (both constructs included Sry 3′ sequences, unlike SryEGFP). Further, SryhPLAP expression was shown to occur only in XY gonadal cells fated to become Sertoli cells.
Finally, in an in vitro LacZ reporter study, Ito et al. (2005) transfected Sry-Cre deletion constructs into primary cultures of gonad, brain and liver cells from CAG/loxP/CAT/loxP/LacZ transgenic mice. Constructs with Sry promoters ranging in length from 4.1 kb to 0.5 kb all induced LacZ expression in gonad cells from 11.5 dpc, but not in brain or liver cells, or gonad cells from 13.5 dpc. This tissue and stage-specificity disappeared for the shortest Sry promoter of 0.4 kb; LacZ expression occurred in all the cell types from 11.5 dpc and in gonad cells from 13.5 dpc.
Together, these studies suggest that the 14.6 kb L741 fragment contains the necessary cis-sequences for correct spatiotemporal regulation of Sry. The conspicuous difference between the previous L741 reports and our study, where L741 (and four shorter variants) did not emulate Sry expression, is that we integrated a single copy of each transgene into a defined autosomal locus. We conclude that the most likely explanation for our observations is that the Col1a1 integration site was inappropriate for our purpose of testing Sry promoter deletion constructs. It remains to be determined why this integration site might have been unsuitable; a possible explanation is that the epigenetic conformation of this site is not accessible to transcription factors necessary for Sry regulation. Further studies may benefit from targeting different transgene integration sites.
Despite the lack of XX sex reversal or correct spatiotemporal Sry expression in any of the five transgenic lines, the strikingly higher Sry mRNA expression observed for XX Sry#49 gonads relative to the four other XX lines may still provide clues to the location of important 5′ regulatory sequences. This result suggests that: (a) the 1044 bp between the 5′ ends of Sry#49 and Sry#5 (−1313 to −269 relative to the TSS) may contain cis-sequences that strongly transactivate Sry; and (b) the 782 bp sequence between the 5′ ends of Sry#18 and Sry#49 (−2095 to −1313 relative to the TSS) may contain cis-sequences that have a repressive effect on Sry transcription. These possibilities require further investigation; rather than targeted integration of Sry transgenes, an alternative approach to elucidating Sry regulation might be to employ the newly emerged nuclease-based genome-editing technologies such as TALENS or CRISPR/Cas-mediated genome engineering to directly modify the 5′ and 3′ flanking sequences of Sry in its native Y chromosome location.
Acknowledgments
We thank Drs Rudolf Jaenisch (MIT) and Konrad Hochedlinger (Harvard) for the gift of KH2 cells. We also thank Christina Harris for technical assistance.
Author Contributions
Conceived and designed the experiments: AQ KK JB PK. Performed the experiments: AQ KK TD EN KC. Analyzed the data: AQ KK. Wrote the paper: AQ KK JB PK.
References
- 1. Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R (1991) Male development of chromosomally female mice transgenic for Sry. Nature 351: 117–121.
- 2. Hawkins J, Taylor A, Berta P, Levilliers J, Van der Auwera B, et al. (1992) Mutational analysis of SRY: nonsense and missense mutations in XY sex reversal. Hum Genet 88: 471–474.
- 3. Kato T, Miyata K, Sonobe M, Yamashita S, Tamano M, et al. (2013) Production of Sry knockout mouse using TALEN via oocyte injection. Scientific Reports 3: 1–8.
- 4. Sinclair A, Berta P, Palmer M, Hawkins J, Griffiths B, et al. (1990) A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346: 240–244.
- 5. Gubbay J, Collignon J, Koopman P, Capel B, Economou A, et al. (1990) A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 346: 245–250.
- 6. Sekido R, Bar I, Narvaez V, Penny G, Lovell-Badge R (2004) SOX9 is up-regulated by the transient expression of SRY specifically in Sertoli cell precursors. Dev Biol 274: 271–279.
- 7. Sekido R, Lovell-Badge R (2008) Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer. Nature 453: 930–934.
- 8. Vidal V, Chaboissier M-C, de Rooij DG, Schedl A (2001) Sox9 induces testis development in XX transgenic mice. Nat Genet 28: 216–217.
- 9. Chaboissier MC, Kobayashi A, Vidal VI, Lutzkendorf S, van de Kant HJ, et al. (2004) Functional analysis of Sox8 and Sox9 during sex determination in the mouse. Development 131: 1891–1901.
- 10. Dubin RA, Ostrer H (1994) Sry is a transcriptional activator. Mol Endocrinol 8: 1182–1192.
- 11. Bowles J, Cooper L, Berkman J, Koopman P (1999) Sry requires a CAG repeat domain for male sex determination in Mus musculus. Nat Genet 22: 405–408.
- 12. Koopman P, Munsterberg A, Capel B, Vivian N, Lovell-Badge R (1990) Expression of a candidate sex-determining gene during mouse testis differentiation. Nature 348: 450–452.
- 13. Hacker A, Capel B, Goodfellow P, Lovell-Badge R (1995) Expression of Sry, the mouse sex determining gene. Development 121: 1603–1614.
- 14. Jeske YW, Bowles J, Greenfield A, Koopman P (1995) Expression of a linear Sry transcript in the mouse genital ridge. Nat Genet 10: 480–482.
- 15. Bullejos M, Koopman P (2001) Spatially dynamic expression of Sry in mouse genital ridges. Dev Dyn 221: 201–205.
- 16. Wilhelm D, Martinson F, Bradford S, Wilson MJ, Combes AN, et al. (2005) Sertoli cell differentiation is induced both cell-autonomously and through prostaglandin signaling during mammalian sex determination. Dev Biol 287: 111–124.
- 17. Hiramatsu R, Matoba S, Kanai-Azuma M, Tsunekawa N, Katoh-Fukui Y, et al. (2009) A critical time window of Sry action in gonadal sex determination in mice. Development 136: 129–138.
- 18. Eicher EM, Washburn LL, Whitney JB 3rd, Morrow KE (1982) Mus poschiavinus Y chromosome in the C57BL/6J murine genome causes sex reversal. Science 217: 535–537.
- 19. Bullejos M, Koopman P (2005) Delayed Sry and Sox9 expression in developing mouse gonads underlies B6-YDOM sex reversal. Dev Biol 278: 473–481.
- 20. Kashimada K, Koopman P (2010) Sry: the master switch in mammalian sex determination. Development 137: 3921–3930.
- 21. Bradford ST, Wilhelm D, Bandiera R, Vidal V, Schedl A, et al. (2009) A cell-autonomous role for WT1 in regulating Sry in vivo. Hum Mol Genet 18: 3429–3438.
- 22. Tevosian SG, Albrecht KH, Crispino JD, Fujiwara Y, Eicher EM, et al. (2002) Gonadal differentiation, sex determination and normal Sry expression in mice require direct interaction between transcription partners GATA4 and FOG2. Development 129: 4627–4634.
- 23. Fujimoto Y, Tanaka SS, Yamaguchi YL, Kobayashi H, Kuroki S, et al. (2013) Homeoproteins SIX1 and SIX4 regulate male sex determination and mouse gonadal development. Dev Cell 26: 416–430.
- 24. Pitetti J-L, Calvel P, Romero Y, Conne B, Truong V, et al. (2013) Insulin and IGF1 receptors are essential for XX and XY gonadal differentiation and adrenal development in mice. PLoS Genet 9: e1003160.
- 25. Bogani D, Siggers P, Brixey R, Warr N, Beddow S, et al. (2009) Loss of mitogen-activated protein kinase kinase kinase 4 (MAP3K4) reveals a requirement for MAPK signalling in mouse sex determination. PLoS Biol 7: e1000196.
- 26. Gierl MS, Gruhn WH, von Seggern A, Maltry N, Niehrs C (2012) GADD45G Functions in Male Sex Determination by Promoting p38 Signaling and Sry Expression. Dev Cell 23: 1–11.
- 27. Warr N, Carre G-A, Siggers P, Faleato JV, Brixey R, et al. (2012) Gadd45γ and Map3k4 Interactions Regulate Mouse Testis Determination via p38 MAPK-Mediated Control of Sry Expression. Dev Cell 23: 1020–1031.
- 28. Kuroki S, Matoba S, Akiyoshi M, Matsumura Y, Miyachi H, et al. (2013) Epigenetic regulation of mouse sex determination by the histone demethylase Jmjd1a. Science 341: 1106–1109.
- 29. Katoh-Fukui Y, Tsuchiya R, Shiroishi T, Nakahara Y, Hashimoto N, et al. (1998) Male-to-female sex reversal in M33 mutant mice. Nature 393: 688–692.
- 30. Albrecht KH, Eicher EM (2001) Evidence that Sry is expressed in pre-Sertoli cells and Sertoli and granulosa cells have a common precursor. Dev Biol 240: 92–107.
- 31. Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R (2006) Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis 44: 23–28.
- 32. Soriano P (1999) Generalized LacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21: 70–71.
- 33. McFarlane L, Truong V, Palmer J, Wilhelm D (2013) Novel PCR Assay for Determining the Genetic Sex of Mice. Sex Dev 7: 207–211.
- 34. Bouma GJ, Hart GT, Washburn LL, Recknagel AK, Eicher EM (2004) Using real time RT-PCR analysis to determine multiple gene expression patterns during XX and XY mouse fetal gonad development. Gene Expr Patterns 5: 141–149.
- 35. Svingen T, Spiller C, Kashimada K, Harley V, Koopman P (2009) Identification of suitable normalizing genes for quantitative real-time RT-PCR analysis of gene expression in fetal mouse gonads. Sex Dev 3: 194–204.
- 36. Koopman P, Bullejos M, Bowles J (2001) Regulation of male sexual development by Sry and Sox9. J Exp Zool 290: 463–474.
- 37. Jasin M, Moynahan ME, Richardson C (1996) Targeted transgenesis. Proc Natl Acad Sci U S A 93: 8804.
- 38. Wirth D, Gama-Norton L, Riemer P, Sandhu U, Schucht R, et al. (2007) Road to precision: recombinase-based targeting technologies for genome engineering. Curr Opin Biotech 18: 411–419.
- 39. Ohtsuka M, Ogiwara S, Miura H, Mizutani A, Warita T, et al. (2010) Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgene expression. Nucleic Acids Res 38: e198.
- 40. McCreath K, Howcroft J, Campbell K, Colman nA, Schnieke A, et al. (2000) Production of gene-targeted sheep by nuclear transfer from cultured somatic cells. Nature 405: 1066–1069.
- 41. Bowles J, Secker G, Nguyen C, Kazenwadel J, Truong V, et al. (2014) Control of retinoid levels by CYP26B1 is important for lymphatic vascular development in the mouse embryo. Dev Biol 386: 25–33.
- 42. Kidokoro T, Matoba S, Hiramatsu R, Fujisawa M, Kanai-Azuma M, et al. (2005) Influence on spatiotemporal patterns of a male-specific Sox9 activation by ectopic Sry expression during early phases of testis differentiation in mice. Dev Biol 278: 511–525.