Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on April 28, 2004

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Human Molecular Genetics, 2004, Vol. 13, No. 12 1213-1218
DOI: 10.1093/hmg/ddh141
Human Molecular Genetics, Vol. 13, No. 12 © Oxford University Press 2004; all rights reserved

Long-range activation of Sox9 in Odd Sex (Ods) mice

Yangjun Qin¹, Ling-kun Kong², Christophe Poirier¹, Cavatina Truong¹, Paul A. Overbeek² and Colin E. Bishop^1,3,*

¹Department of Obstetrics and Gynecology, ²Department of Molecular and Cellular Biology and ³Department of Molecular and Human Genetics, Baylor College of Medicine, 6550 Fannin Street, Houston, TX 77030, USA

Received January 16, 2004; Accepted April 15, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
The Odd Sex mouse mutation arose in a transgenic line of mice carrying a tyrosinase minigene driven by the dopachrome tautomerase (Dct) promoter region. The minigene integrated 0.98 Mb upstream of Sox9 and was accompanied by a deletion of 134 kb. This mutation causes female to male sex reversal in XX Ods/+ mice, and a characteristic eye phenotype of microphthalmia with cataracts in all mice carrying the transgene. Ods causes sex reversal in the absence of Sry by upregulating Sox9 expression and maintaining a male pattern of Sox9 expression in XX Ods/+ embryonic gonads. This expression, which begins at E11.5, triggers downstream events leading to the formation of a testis. We report here that the 134 kb deletion, in itself, is insufficient to cause sex reversal. We demonstrate that in Ods, the Dct promoter is capable of acting over a distance of 1 Mb to induce inappropriate expression of Sox9 in the retinal pigmented epithelium of the eye, causing the observed microphthalmia. In addition, it induces Sox9 expression in the melanocytes where it causes pigmentation defects. We propose that Ods sex reversal is due to the Dct promoter element interacting with gonad-specific enhancer elements to produce the observed male pattern expression of Sox9 in the embryonic gonads.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Sex determination is a consequence of the commitment of the indifferent fetal gonad to develop as an ovary or a testis. This involves precursor cell differentiation into Sertoli cells in males or granulosa cells in females (1,2). In mice, this is triggered by the transient expression of the Y-located Sry gene (3), which leads to proliferation of coelomic epithelial cells from which the Sertoli cells are derived (4). Further development of the male gonad into a functional testis is dependent on Sertoli cell signaling, mesenchymal proliferation, mesonephric cell migration and the establishment of other testicular cell types (1–8).

At the molecular level, a number of key genes involved in the sex determining pathway have been identified, although the precise way in which they interact to form a testis is yet to be determined. Several genes have been shown to exert their effect on gonad formation by working upstream of Sry; M33, Sf1, Wt1, Lim1, Emx1, Lhx9 and Gata4/Fog2 are all required for the correct formation of the bipotential gonad (7,9–12). Targeting these genes in mice leads to complete or partial failure of the gonad to develop. It is therefore difficult, at present, to evaluate whether they have an additional role downstream of Sry.

The transcription factor, Sox9 (Sry-like HMG box9), is upregulated in the male mouse gonad, and extinguished in the female, at the time of sex determination (E11.5) (13,14). It represents one of the earliest markers of Sertoli cell differentiation (7). In humans, 75% of XY human campomelic dysplasia (CD) patients carrying mutations in SOX9 show overt gonadal dysgenesis, whereas ovarian development in XX CD patients is unaffected (15). In addition, duplication of SOX9 has been reported to lead to sex reversal in an XX proband (16). In mice, female to male sex reversal has been reported in the Wt1–Sox9 transgenic mice (17) and in the Odd Sex (Ods) transgenic insertional mutant, which constitutively expresses Sox9 in XX embryonic gonads (18). In the case of Odd Sex, sex reversal is also accompanied by a characteristic eye phenotype of microphthalmia with cataracts. We have previously put forward the hypothesis that the 134 kb deletion, caused by the tyrosinase minigene insertion, has removed gonad-specific regulatory elements allowing Sox9 expression in the XX Ods/+ gonad, leading to male development. In this paper, we refute this hypothesis and show that the deletion, in itself, is insufficient to cause sex reversal. We propose that gonad-specific expression is due to a long-range interaction between the transgenic Dct promoter and Sox9 upstream enhancer/control elements.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
In the Odd Sex mouse, two copies of a Dct–tyrosinase transgene (Dct–TyBS) have integrated into distal chromosome 11, 0.98 Mb proximal to Sox9, causing a deletion of 134 kb (Fig. 1A) (18). A detailed examination of the deleted sequence failed to identify any obvious genes or transcriptional elements that had been affected. It has been reported that the HMG motif of Sox9 can functionally substitute for that of Sry when expressed as a chimeric, Sry/Sox9 transgene (19). We have previously shown that in E11.5–14.5 XX Ods/+ gonads, the spatio–temporal expression pattern of Sox9 is indistinguishable from that of normal XY males (18). In order to address the possibility that a precocious expression of Sox9 in the XX Ods/+ gonad could be causing sex reversal by substituting for Sry, we examined its expression in E10.5 gonads using in situ hybridization. Even after prolonged exposure we were unable to detect its expression at this time point, making this explanation unlikely (data not shown).

View larger version (22K): [in this window] [in a new window]   Figure 1. Re-creation of a deletion similar to that in the original Ods mutation. (A) Schematic representation of the Ods locus showing position of the transgene, accompanying deletion, map of targeting vectors and surrounding homology (vertical dotted lines). (B) Structure of doubly targeted locus showing position of duplicated and gap repaired DNA, orientation of LoxP sites (red arrows) and screening primers (black arrows). (C) Final structure of the locus after deletion of DNA between the LoxP sites with CRE recombinase. The 11.133 deletion is 1 kb smaller than the original 134 kb in Ods, and contains the tyrosinase, HPRT and agouti minigenes.

 
One possible mechanism that we have proposed for the sex reversal phenotype is that the deletion has removed long-range, gonad-specific regulatory element(s) that mediates the repression of Sox9 in XX fetal gonads. In order to investigate this hypothesis we used LoxP/Cre mediated chromosome engineering in ES cells to generate mice carrying a similar deletion event (Fig. 1A–C). As shown in Figure 1C, the deletion (termed 11.133) as 1 kb smaller than the original 134 kb, and introduced a single copy of the tyrosinase minigene in the same orientation found in Ods. In this case the gene was driven by its own promoter, rather than the Dct promoter used in the original Odd Sex mice. A light pigmentation could be seen in the eyes of mice carrying 11.133, indicating the tyrosinase gene was active, but no effect on sex determination, fertility or eye phenotype was evident, even when the deletion was bred to a homozygous state. Histology of E12.5 and adult gonads was examined and also found to be normal. The presence of the complete deletion was confirmed by PCR testing DNA from 11.133 homozygous mice with eight primer pairs spread evenly throughout the deletion (Fig. 2 and Table 1). All primers failed to amplify Ods/Ods and 11.133/11.133 DNA, whereas they produced the expected band sizes in heterozygous Ods/+ DNA. Mice carrying 11.133 were initially generated on the 129/sv inbred background. To rule out any effects of genetic background on sex reversal (20), 129/sv 11.133 mice were backcrossed to FVB/N for three generations. Again no effect on sex or eye phenotype was detected.

View larger version (66K): [in this window] [in a new window]   Figure 2. Confirmation of the 11.133 deletion in homozygous mice. No amplification of loci within the deletion was seen in either homozygous Ods/Ods or 11.133/11.133 mice. In contrast all loci were amplified in heterozygous Ods/+ DNA.

 

View this table: [in this window] [in a new window]   Table 1. Sequence of primers used in this study

 
These data imply that the deletion of 134 kb of DNA, and any putative regulatory elements it may contain, cannot alone be the cause of sex reversal in the Odd Sex mutant. They also rule out a simple change in local chromatin conformation as an explanation. In order to address the possibility that there may be some small undetected rearrangements in the known Sox9 control region in Ods mice, 100 kb of DNA immediately upstream of Sox9 was PCR amplified in 10 kb fragments from homozygous XX Ods/Ods and control XX FVB/N DNA. After cloning, it was subjected to restriction enzyme fingerprinting. No evidence was found for any rearrangements, suggesting that Ods sex reversal is a result of the transgenic insertion rather than a secondary rearrangement (data not shown).

Both the original and the engineered 11.133 deletion contained a functional tyrosinase minigene. RT–PCR analysis of RNA derived from E12.5 XX Ods/+ or XY 11.133 gonads showed that tyrosinase itself was not expressed, ruling out a role of this gene in the sex reversal. A significant difference between the two strains was the substitution of the Dct promoter with an endogenous tyrosinase promoter in the latter. This raised the possibility that the mechanism involves a long-range interaction between the Dct promoter and Sox9. To test this possibility we constructed two transgenic mouse lines carrying a SOX9 minigene driven by the Dct promoter. (Although we used a human SOX9 construct, it is 97% identical and 100% similar at the amino acid level to that of the mouse.) Again, no evidence of sex reversal was seen in either line of XX Dct-SOX9 transgenic mice. In contrast, the characteristic Odd Sex eye phenotype of iris coloboma, a severe developmental defect in the anterior segment of eye and cataract, was clearly regenerated in the two independent lines (Fig. 3). In normal retinal pigmented epithelial (RPE) cells Sox9 is turned on at E10.5 and downregulated at E15.5. In the Ods/+ mice Sox9 and the transgenic Dct-SOX9 mice, an identical spatio–temporal expression pattern of Sox9 can be seen. It is not extinguished at E15.5 and its continued expression correlates with the development of the eye phenotype at E17.5 (unpublished data).

View larger version (30K): [in this window] [in a new window]   Figure 3. Eye phenotype of Dct-Sox9 and XX Ods/+ mice. A similar phenotype of microphthalmia with cataracts was seen in both Dct-SOX9 transgenic lines [only transgenic line (a) shown] and XX Ods/+ mice when compared with a normal wild-type control. Long arrows indicate the iris and short arrows the lens.

 
As the transgenic mice were made in a pigmented strain of FVB (pFVB), rather than in the more usual albino strain, it gave us the opportunity to observe any pigmentation defects. As can be seen in Figure 4A, both transgenic Dct-SOX9 lines showed distinctive white spots and light coat color indicative of pigmentation defects. In order to test whether this was also the case with Odd Sex mice, they were backcrossed for three generations to the black C57BL/6 strain. As can be seen in Figure 4B, transferring Ods to a pigmented background revealed typical pigmentation defects, similar to those found in the Dct-SOX9 transgenic lines. This suggests that in Ods and the Dct-SOX9 lines, inappropriate expression of Sox9 interferes with the normal development of the melanoblast lineage.

View larger version (101K): [in this window] [in a new window]   Figure 4. Pigmentation defects in Dct-SOX9 and Odd Sex mice. (A) Distinctive white spots and light coat color and eye phenotype is evident in both Dct-SOX9 transgenic lines compared with the original PFVB parental line (center). (B) Large areas of white spotting can be seen in the Ods/Ods homozygotes with rather less spotting in the Ods/+ heterozygotes compared with the wild-type littermate (far left).

 
These data support the concept that the Dct promoter can modify Sox9 expression over a distance of 980 kb in Ods mice. In the case of the Ods eye and pigmentation phenotypes, the action is probably a direct promoter effect, as Dct is known to direct expression to the eye and the melanocytes (21). The absence of the sex reversal phenotype in XX Dct-SOX9 transgenic mice indicates that in the original Ods mice, the influence of Dct on Sox9 must be indirect, possibly acting through gonad-specific Sox9 cis-enhancer element(s) to achieve target specificity. It is conceivable that a specific chromosomal conformation is also necessary for such interactions to occur. In the case of Ods the deletion may be required to sufficiently change the local conformation of chromatin, bringing the Dct promoter close enough to the Sox9 enhancer region, to interact with it.

Action over such a distance, although rare, is not unknown. DNA rearrangements located over 100 kb upstream of the Steel (Sl) locus have been shown to deregulate its expression and cause female sterility (22). Disruption of control elements located over several hundred kilobases upstream of SOX9 are known to cause CD with sex reversal in humans (23). Using mouse/human sequence comparisons, Loots et al. (24) located transcriptional control elements 10 and 125 kb upstream of the interleukins IL4 and IL5, respectively. However, human/mouse comparisons are not so useful when the overall sequence conservation is high, as is the case upstream of Sox9. By comparing to the genome of a more distantly related species, the puffer fish (Fugu), Nobrega et al. (25,26) were able to identify nine functional enhancers of the DACH gene, one of which was located 1 Mb from the transcription start point. In order to similarly identify putative regulatory elements of Sox9, we used pip-plot genome alignment and ECR browser tools (LLNL www.dcode.org) to compare 1 Mb of mouse (mm4), human (hg16) and chicken (gg2) upstream sequence. Apart from Sox9, no conserved genes were detected in this region. However, using relatively stringent alignment parameters (minimum 200 bp length and 90% identity), and excluding the Ods deletion and the Sox9 gene itself, over 60 mouse/human evolutionary conserved sequences (ECS) elements were detected. In contrast, only four mouse/chicken ECS elements were present (Fig. 5), all of which mapped into mouse/human ECS elements. The fifth mouse/chicken ECS element, located at 55.7 kb in the deleted sequence, is not conserved in humans. All four ECS elements contain potential SRY binding sites, but due to the redundancy of site sequence itself, the significance of this finding is unclear. Thus, these four mouse/human/chicken ECS elements, which are located between 390–800 kb upstream of Sox9, may represent potential regulatory elements which could be influenced by the Dct promoter to produce gonad-specific expression of Sox9, and consequent sex reversal in XX Ods/+ mice.

View larger version (13K): [in this window] [in a new window]   Figure 5. Pip-Plot comparison of 1 Mb of sequence upstream of mouse and chicken Sox9. Evolutionary conserved sequences (ECS) are indicated by red bars with boxes above, green boxes below represent repetitive elements, and percent identity is indicated at left. Analysis parameters were a minimum of 200 bp in length and 90% identity. The position of the Ods deletion and Dct–Tyr minigene insertion is indicated in the left, and the position of the conserved Sox9 can be seen in the right (blue). Apart from Sox9, no conserved genes are seen, but four mouse/human/chicken ECS elements were identified, outside the Ods deletion and Sox9 itself.

 

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 
Gene targeting
In the Odd Sex mouse, two copies of the Dct–Tyrosinase transgene (Dct–TyBS) have integrated on distal chromosome 11, 0.98 Mb proximal to Sox9 (Fig. 1A), accompanied by a 134 kb deletion. Two gene-targeting steps were used to regenerate this deletion according to the sequence at the transgenic breakpoints. For the first step, a 9.3 kb targeting clone was selected from a 5'HPRT library (27) using a 550 bp PCR fragment, amplified by primers SQA1F and SQA2R, which define the 5' transgenic integration site as shown (Fig. 1A). AflII was used to create a 4.1 kb gap in the clone giving homologous arms of 4.1 and 0.85 kb. After transfection into Hprt deficient ES cells (AB2.1), they were selected with G418. Correctly targeted clones, having repaired the gap, were identified using primers 5GAPR and 53527, which give a 1.1 kb product. Out of 196 clones 10 were identified in this way. In the second targeting step, a 7.6 kb clone was isolated from a 3'HPRT library using a 230 bp PCR fragment amplified by primers SQ6R and SQ5F, which identify the 3' end of the transgenic insertion (Fig. 2A). After gapping with AflII, homologous arms of 1.6 and 2.2 kb were generated. A single clone, selected from the first targeting step, was transfected with this construct, and selected using puromycin. Homologous targeting events were identified using primers 3AMP and SQ5F, which give a 1.6 kb product. Out of 196 clones 12 were identified. The structure of the double-targeted locus with duplication of homologous DNA and gap repair is shown in Figure 2B. Clones were transiently transfected with a Cre expressing plasmid (pOG231) and those that had correctly deleted all DNA between the loxP sites, reconstructing the HPRT gene, were selected for in HAT medium. The deletion was confirmed by the presence of the specific deletion breakpoints, identified with primers 5GAPR/53527 and 3AMP/SQ5F and by the absence of DNA between the LoxP sites using 5BZP/BZP-del and SQ6R/3Puro as shown in Figure 2B. The arrangement of the final targeted locus is shown in Figure 1C. A 133 kb deletion was created which contains a single copy of the HPRT, agouti and tyrosinase minigenes. The tyrosinase cDNA is driven by a 2 kb fragment of its own promoter and is transcribed in the same orientation as the original Dct–Ty in Odd Sex. Sequences of all primers are shown in Table 1.

Mouse breeding and transgenesis
A SOX9 minigene construct was made by linking the mouse Dct promoter to the human SOX9 cDNA. This was then injected into the pronuclei of one cell PFVB/K (C/C, a/a) non-agouti embryos to produce Dct-SOX9 transgenic mice. Two independent expressing lines were established, termed Dct-SOX9a and Dct-SOX9b.

An FVB XY Ods/+ male was bred to C57BL/6 females and the resulting F1 XY Ods/+ males backcrossed to C57BL/6 females. The presence of the Y and Ods was detected using Y-specific and tyrosinase minigene primers as described previously. In the N3 generation, several fertile female XX Ods/+ mice were detected [due to the segregation of Ods suppressor loci (REF)] and intercrossed to their XY Ods/+ littermates. In this way, we were able to identify heterozygous XX Ods/+ and homozygous XX Ods/Ods mice on a pigmented, mainly C57BL/6 background.

	ACKNOWLEDGEMENTS

 
We would like to thank Ms Thersa Ty for excellent technical assistance. This work was supported by grants from the March of Dimes Birth Defects Foundation and the NIH (to C.E.B.).

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +1 7137988221; Fax: +1 7137985074; Email: bishop{at}bcm.tmc.edu

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TOP ABSTRACT INTRODUCTION RESULTS AND DISCUSSION MATERIALS AND METHODS REFERENCES

 

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