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Biol Reprod. 2009 May; 80(5): 905–912.
Prepublished online 2009 January 21. doi:  10.1095/biolreprod.107.067322
PMCID: PMC2804840

Extra-Germ Cell Expression of Mouse Nuclear Receptor Subfamily 6, Group A, Member 1 (NR6A1)1

Abstract

Nuclear receptor subfamily 6, group A, member 1 (NR6A1) is an orphan member of the nuclear receptor superfamily and is required for normal mouse embryonic development. In adult mice, NR6A1 is predominantly expressed in spermatogenic cells and growing oocytes of the gonads and has a role in female reproduction by modulating the transcription of the oocyte-specific genes bone morphogenetic protein 15 (Bmp15) and growth differentiation factor 9 (Gdf9). In our goal to further understand the functional role of NR6A1 during postnatal development, we generated a Nr6a1:beta-galactosidase (LacZ) knockin reporter (Nr6a1LacZ/+) mouse line in which the Nr6a1:LacZ fusion gene was expressed and then characterized Nr6a1 expression in these reporter mice by performing LacZ staining. Our RT-PCR analyses showed that Nr6a1 was expressed in a variety of somatic tissues (e.g., oviduct and lung) other than gonads of normal adult mice. In adult Nr6a1LacZ/+ mice, robust LacZ staining was observed in the gametes of gonads. Strong positive LacZ staining was also observed in the sperm of the epididymis, epithelial cells of the oviduct, and bronchioles within the lung. In adult Nr6a1LacZ/+ mice, positive LacZ staining was observed in other somatic tissues, including hippocampus, cerebral cortex, cerebellum, and thalamus of brain; pars intermedia and pars anterior of pituitary; parathyroid; and islet of pancreas. NR6A1 expression in sperm within the epididymis, epithelial cells in the oviduct, and bronchioles of the lung was further confirmed by immunohistochemical studies. Nr6a1 is expressed not only in the germ cells of mouse gonads but also in a variety of somatic tissues, including epididymis, oviduct, brain, and pituitary. The extra-germ cell expression of NR6A1 makes it a less attractive contraceptive.

Keywords: brain, epididymis, gametogenesis, gene regulation, islet, knockin, LacZ staining, lung, mouse, NR6A1, ovary, oviduct, parathyroid, pituitary, sperm, spermatogenesis, steroid hormone receptors, testis

INTRODUCTION

Receptor subfamily 6, group A, member 1 (NR6A1) is an orphan member of the nuclear receptor superfamily [1, 2]. NR6A1 functions as a transcription factor that binds to a direct repeat of the consensus sequence AGGTCA with 0-bp spacing between the half sites (DR0) element to repress gene transcription in vitro and in vivo [1, 35]. It is expressed in early mouse embryos after the onset of gastrulation [6, 7] and is essential for normal embryonic development [7, 8], for repression of Pou5f1 (also known as Oct4) expression in somatic cells of early mouse embryos [5, 7], and for embryonic stem (ES) cell and neuronal cell differentiation [9, 10]. The DNA-binding domain (DBD) of NR6A1 is essential for the function of NR6A1 during embryonic development and for Pou5f1 repression [11]. In adult vertebrates, Nr6a1 is predominantly expressed in the gonads of several species, including mouse, rat, and human [1, 1215]. In murine testis, Nr6a1 is predominantly expressed in postmeiotic round spermatids at both the mRNA and protein levels [1, 12, 16], while in human testis it is expressed in pachytene spermatocytes [14]. In the testis, it is thought that NR6A1 functions as a transcription factor to regulate the transcription of postmeiotic genes such as protamines [3, 17, 18], possibly affecting male fertility. In the ovary, Nr6a1 is expressed in oocytes in mouse, Xenopus, and zebra fish [1, 12, 19, 20]. In mouse ovary, Nr6a1 is exclusively expressed in the oocytes of primary, secondary, and preovulatory follicles but not primordial follicles at both the mRNA and protein levels [1, 12, 16]. NR6A1 is also present in ovulated oocytes and preimplantation embryos [16]. Our recent studies of oocyte-specific Nr6a1 knockout mice disclosed the important role of NR6A1 in female reproduction by regulating the expression of bone morphogenetic protein 15 (Bmp15) and growth differentiation factor 9 (Gdf9) in oocytes [21]. The germ cell expression of NR6A1 and its membership in the nuclear receptor superfamily, which makes it a natural small-molecule target, increase its potential as a contraceptive target, at least in the male. However, little is known about the expression and function of NR6A1 in adult mouse tissues other than gonads that may contribute to undesired adverse effects.

To further understand the functional role of NR6A1 during postnatal development, we generated Nr6a1:β-galactosidase (LacZ) knockin reporter (Nr6a1LacZ) mice and then characterized Nr6a1 expression in these adult reporter mice by performing LacZ and/or immunohistochemical staining. It was found that Nr6a1 not only is expressed in the germ cells of mouse gonads, as its previous name (germ cell nuclear factor) suggests, but also is expressed in a variety of somatic tissues such as oviduct, lung, brain, and pituitary. Results of this study suggest that NR6A1 is not a germ cell-specific factor and likely has a functional role in somatic tissues, including other reproductive organs besides the gonads.

MATERIALS AND METHODS

ES Cell Culture

Nr6a1LacZ/+ ES cells with an insertion of a gene trap vector (LacZ/Neo cassette) into intron 2 of the Nr6a1 allele were obtained from BayGenomics (University of California at Davis). These ES cells were cultured in ES cell media containing 400 μg/ml of G418 (Invitrogen, Carlsbad, CA) on irradiated SNL feeder cells (a mouse fibroblast STO cell line transformed with neomycin resistance and murine leukemia inhibitory factor genes [22]) as described previously [23]. The ES cells were incubated with 10−6 M all-trans-retinoic acid (RA) (Sigma, St. Louis, MO) to induce differentiation and Nr6a1 expression.

Southern Blot Analysis

Genomic DNA from Nr6a1LacZ/+ ES cells was isolated as described previously [23] and then digested with BstE I or Sac I. Digested DNA fragments were then separated on a 0.7% agarose gel and transferred to a nylon membrane. The membrane was then hybridized with the Neo probe [11] using a QuickHyb (Stratagene, La Jolla, CA) hybridization buffer according to the manufacturer's protocol.

Generation of Nr6a1LacZ/+ Mice and Mouse Genotyping

Nr6a1LacZ/+ ES cells were expanded in ES cell media and injected into C57BL/6 blastocysts and then transferred into pseudopregnant mothers to generate male chimeras. Male chimeric mice with agouti color ranging from 50% to 100% were obtained and then bred with female C57BL/6 mice to obtain Nr6a1LacZ/+ mice. Mouse tail genomic DNA was extracted and genotyped by PCR as described previously [7, 24]. The nucleotide sequences of PCR primers for mouse genotyping are as follows: Nr6a1+ (5′-CAGTGCTGACTTATCCATG-3′ and 5′-TTCCTGTTCATGCCCATCT-3′ [239 bp]) and Nr6a1LacZ (neoprimers 5′-TCGATGCGATGTTTCGCTT-3′ and 5′-ATATGGGATCGGCCATTGA-3′ [416 bp]) and LacZ primers (5′-CAGACGATGGTGCAGGATAT-3′ and 5′-ATACAGCGCGTCGTGATTAG-3′ [300 bp]). All animal studies were conducted according to ethical guidelines and were approved in advance by the Animal Welfare and User Committee of Baylor College of Medicine.

RNA Isolation and RT-PCR Analysis

Total RNA from various mouse tissues was isolated using Trizol reagent (Invitrogen). The RT-PCR analyses using specific primers for Nr6a1 (the DBD region) and Actb were performed as described previously [21]. The nucleotide sequences of PCR primers to detect the Nr6a1:LacZ fusion transcript in Nr6a1LacZ embryos are 5′-ACTACGAAGGCGCAAGTCAT-3′ (Nr6a1 exon 1-F) and 5′-ATACAGCGCGTCGTGATTAG-3′ (LacZ-R) (1.7-kb PCR products).

LacZ Staining and Immunohistochemical Studies

LacZ staining of RA-induced ES cells, embryos, or tissue samples with X-Gal (Specialty Media, Phillipsburg, NJ) was performed according to the manufacturer's protocol. Immunohistochemical studies were performed on paraffin-embedded or frozen tissue sections using NR6A1 antibodies as described previously [16].

RESULTS

Successful Germline Transmission of the Nr6a1LacZ Knockin Allele

To validate the correct insertion of the gene trap LacZ/Neo cassette into the Nr6a1 allele (Fig. 1A), we performed Southern blot analyses using Neo probes. As shown in Figure 1B, single 13.5-kb and 7-kb bands were detected by Neo probes in BstE II- and Sac I-digested genomic DNA from Nr6a1LacZ/+ ES cells, respectively. These results suggest the correct insertion of the LacZ/Neo cassette into intron 2 of the Nr6a1 gene. Because NR6A1 can be induced by RA during ES cell differentiation [9], we performed LacZ staining of RA-treated ES cells. As shown in Figure 1C, there was no positive LacZ staining in undifferentiated Nr6a1LacZ/+ ES cells. In contrast, robust LacZ staining signals were observed in RA-treated Nr6a1LacZ/+ ES cells. Breeding of chimeric males from microinjection of Nr6a1LacZ/+ ES cells into recipient blastocysts with wild-type C57BL/6 females produced agouti pups. Genotypic analyses using Neo and LacZ primers showed successful germline transmission of the Nr6a1LacZ allele (Fig. 1D).

FIG. 1.
Germline transmission of the Nr6a1LacZ knockin allele and expression of the Nr6a1:LacZ fusion gene in Nr6a1LacZ/+ mice. A) Schematic representation of the gene trap Nr6a1LacZ allele, with the gene trap vector inserted into the intron between exons 2 and ...

Expression of Nr6a1:LacZ Fusion Gene in Nr6a1LacZ/+ Mice

To determine whether the expression of LacZ transcript is driven by endogenous Nr6a1 promoter, we performed RT-PCR analysis of Nr6a1LacZ/+ embryos at Embryonic Days (E) 8.5–9.5 using a forward primer recognizing exon 1 of Nr6a1 and a reverse primer recognizing the LacZ gene (Fig. 1E). As shown in Figure 1F, a 1.7-kb band was detected in Nr6a1LacZ/+ embryos but not in control wild-type littermates.

Embryonic Lethality of Nr6a1LacZ/LacZ Mice and LacZ Staining of Nr6a1LacZ/+ Embryos at E7.5

Nr6a1LacZ/LacZ mice were embryonic lethal at E9.5 (data not shown). Protrusion of the tail bud outside of the yolk sac was observed in Nr6a1LacZ/LacZ embryos at E8.5 but not in control wild-type littermates (Fig. 2, A and B). There was positive LacZ staining throughout the Nr6a1LacZ/+ embryos at E7.5 but not in control littermates (Fig. 2, C and D). Because of embryonic lethality of Nr6a1LacZ/LacZ mice, Nr6a1LacZ/+ mice were used to catalog Nr6a1 expression in adult tissues.

FIG. 2.
Gross morphology (A and B) and LacZ staining (C and D) of mouse embryos. A and B) Protruded tail bud (outside of the yolk sac) in Nr6a1LacZ/LacZ embryos at E8.5 (D) but not in Nr6a1+/+ control littermate (C). Arrow in B indicates the bulge caused by posterior ...

Nr6a1 Expression in Adult Tissues from Wild-Type Mice by RT-PCR Analyses

The RT-PCR analyses were performed to gain information about the expression pattern of Nr6a1 in a variety of adult tissues from normal wild-type mice. As shown in Figure 3, a specific Nr6a1 PCR product was detected in all tissue tested except the uterus. There were more abundant Nr6a1 PCR products in the testis, ovary, oviduct, and lung than in other tissues tested such as brain, pituitary, parathyroid, and pancreas.

FIG. 3.
The RT-PCR analyses showing Nr6a1 expression in a variety of somatic tissues from adult wild-type mice. Note the relative high expression in the lung, oviduct, and gonads.

Nr6a1 Expression in the Germ Cells of Adult Mouse Gonads

Previous studies [1, 12, 13, 16, 25, 26] have shown that Nr6a1 is predominantly expressed in adult mouse gonads, more specifically in the germ cells within the gonads. To validate this observation, we performed LacZ staining of frozen testicular sections and whole-mount ovaries. There was no positive LacZ staining in wild-type testes or ovaries (data not shown). In contrast, positive staining was observed in adult testes and ovaries of Nr6a1LacZ/+ mice (Fig. 4). In Nr6a1LacZ/+ testes, positive signals were observed in spermatogenic cells within seminiferous tubules but not in interstitial cells. Within seminiferous tubules, robust signals were observed in postmeiotic round and elongating spermatids (Fig. 4A). No staining was observed in spermatogonia, and weak or close to no staining was observed in pachytene spermatocytes of Nr6a1LacZ/+ mice. In Nr6a1LacZ/+ ovaries, positive LacZ staining was observed in the oocytes of primary follicles (data not shown) and follicles at later follicular stages (Fig. 4B) but not in granulosa and thecal cells within the follicle and stromal cells. These results confirm that NR6A1 is a germ cell-specific factor in adult gonads.

FIG. 4.
Nr6a1 expression in adult mouse gonads indicated by LacZ staining on the sections of testis (A) and ovary (B) of adult Nr6a1LacZ/+ mice.

Nr6a1 Expression in the Sperm of Epididymis and Epithelial Cells Within the Oviduct

It has been reported from several laboratories that NR6A1 is expressed in epithelium cells and spermatozoa within the epididymis [2729]. To validate this observation, we performed LacZ staining of frozen epididymal sections from adult Nr6a1LacZ/+ and wild-type littermate mice (Fig. 5, A and B). As shown in Figure 5A, weak LacZ staining was observed in epididymal sections in wild-type control mice, which is likely because of the expression of the mouse LacZ gene in that tissue. In Nr6a1LacZ/+ mice, robust positive LacZ staining was observed in sperm within the epididymal tubules, whereas the staining in epithelial cells was similar to that in control wild-type littermates (Fig. 5B). To further validate NR6A1 protein expression in the epididymis, we performed immunohistochemical studies of frozen tissues using specific NR6A1 antibodies generated in our laboratory [16]. There was no positive signal detected by preimmune serum IgG in epididymal sections (Fig. 5C). However, positive NR6A1 protein expression was observed in sperm, but not in other cells, within the epididymis (Fig. 5D). Our results suggest that NR6A1 is present in sperm within the epididymis.

FIG. 5.
Nr6a1 expression in the sperm within the epididymis (A–D) and epithelial cells in the oviduct (E and F). A and B) LacZ staining of frozen epididymal sections from wild-type (A) and Nr6a1LacZ/+ (B) littermates. C and D) Immunohistochemical studies ...

Similarly, LacZ staining and immunohistochemical studies were performed to determine NR6A1 expression in the oviduct and uterus. As shown in Figure 5E, positive LacZ staining was observed in epithelial cells within the oviduct of adult Nr6a1LacZ/+ mice but not in control mice (data not shown). Immunohistochemical studies showed positive NR6A1 staining in epithelial cells within the oviduct by anti-NR6A1 antibodies (Fig. 5F) but not by IgG (data not shown). These results suggest that NR6A1 is expressed in the oviduct. In contrast, no positive LacZ or NR6A1 staining was observed in the uterus of adult Nr6a1LacZ/+ mice (data not shown).

Nr6a1 Expression in the Bronchioles Within Adult Lung

To test whether Nr6a1 is expressed in adult lung, LacZ staining was performed on frozen lung sections from Nr6a1LacZ/+ mice and control wild-type littermates. No positive blue staining was observed in the bronchioles or alveoli of wild-type mice (data not shown). However, positive LacZ signals were observed in the bronchial epithelium of Nr6a1LacZ/+ mice (Fig. 6A). To further confirm NR6A1 expression, immunohistochemical studies were performed on frozen lung sections from wild-type mice. There was no NR6A1 protein signal detected by preimmune serum IgG (data not shown). However, strong positive NR6A1 protein signals were detected in bronchial epithelial cells by anti-NR6A1 antibodies (Fig. 6B).

FIG. 6.
Nr6a1 expression in the bronchioles of adult mouse lung indicated by LacZ staining (A) of frozen lung sections from Nr6a1LacZ/+ mice and by immunohistochemical studies (B) of NR6A1 on frozen lung sections from adult wild-type mice using anti-NR6A1 antibodies. ...

Nr6a1 Expression in the Hippocampus, Cerebral Cortex, Cerebellum, and Thalamus

LacZ staining of frozen brain sections from adult Nr6a1LacZ/+ and control wild-type mice was performed to determine Nr6a1 expression. No positive LacZ staining was observed in brain sections from wild-type mice (data not shown). However, positive LacZ signals were observed in the hippocampus, cerebral cortex, thalamus, and cerebellum of adult Nr6a1LacZ/+ mouse brains (Fig. 7). More specifically, positive LacZ signals were observed primarily in cornu ammonis regions of the hippocampus (Fig. 7, A and D), layers II–IV of the neocortex (Fig. 7, A and C), and Purkinje cells of the cerebellum (Fig. 7F) in Nr6a1LacZ/+ mice.

FIG. 7.
Nr6a1 expression in the hippocampus, cortex, thalamus, and cerebellum of adult mouse brain. Frozen brain sections from Nr6a1LacZ/+ mice were subjected to LacZ staining. A) Hippocampus and cortex. B) Cortex and hippocampus. C) High magnification of the ...

Nr6a1 Expression in Pituitary, Parathyroid, Pancreas, and Other Tissues

There was no LacZ staining in pituitary, parathyroid, thyroid, or pancreas of wild-type mice (data not shown). However, positive LacZ staining signals were observed in the anterior region and pars intermedia of pituitary, parathyroid, and islet of Langerhans in Nr6a1LacZ/+ mice (Fig. 8). Very weak and spotty LacZ staining was also observed in heart, liver, and kidney from adult Nr6a1LacZ/+ mice (data not shown). However, there was no positive LacZ staining in spleen, thyroid, or thymus of adult wild-type or Nr6a1LacZ/+ mice (data not shown).

FIG. 8.
Nr6a1 expression in the pituitary (A), parathyroid (B), and pancreas (C) indicated by positive LacZ staining in tissues from adult Nr6a1LacZ/+ mice.

DISCUSSION

We report herein the successful generation of Nr6a1:LacZ reporter mice and the characterization of Nr6a1 expression in adult tissues. Our Southern blot analyses of gene trap Nr6a1 ES cells suggest that a single LacZ/Neo cassette is correctly inserted into the Nr6a1 allele. Induced LacZ staining in RA-treated Nr6a1 gene trap ES cells is consistent with previous observations of induction of endogenous Nr6a1 expression by RA in ES cells [5, 9]. Expression of the Nr6a1:LacZ fusion gene in Nr6aLacZ/+ mice (Fig. 1F) suggests that the transcription of the LacZ gene is driven by endogenous Nr6a1 promoter. Homozygous mice generated from these gene trap Nr6a1 ES cells died in midgestation and displayed tail buds that protruded outside of the yolk sac (Fig. 2), similar to Nr6a1 null mutant mice and Nr6a1 DBD deletion mice [7, 11]. LacZ staining was detected throughout the embryo at E7.5 and at a stage just before repression of Pou5f1 expression, similar to previous findings [5]. Furthermore, we observed positive LacZ staining in spermatogenic cells within the testis and in the oocytes within the follicle in adult ovary of Nr6a1LacZ/+ mice (Fig. 4). The germ cell-specific expression of the LacZ reporter gene is consistent with endogenous Nr6a1 expression observed in many laboratories, including ours [1, 12, 13, 16, 25, 26]. Collectively, we have successfully generated a Nr6a1:LacZ reporter mouse model in which LacZ expression mimics endogenous Nr6a1 expression.

Although the functional role of NR6A1 in adult ovary has been characterized in oocyte-specific Nr6a1 knockout mice [21], its role in spermatogenic cells within the testis remains to be characterized. Using individually generated NR6A1 antibodies, it has been shown that NR6A1 is present in spermatogonia [29], XY bodies of primary spermatocytes [30], and pachytene spermatocytes [29]. In our study, there were no positive LacZ staining signals in spermatogonia, and very weak LacZ staining was observed in pachytene spermatocytes (Fig. 4B). These results are consistent with our previous immunohistochemical findings using our NR6A1 antibodies [16]. Thus, we believe that NR6A1 is mainly expressed in postmeiotic spermatids, with a weak expression in pachytene spermatocytes, but not in spermatogonia or XY bodies of primary spermatocytes within the testis. NR6A1 has been shown to bind to DR0 elements in the promoters of postmeiotic genes protamine 1 and 2 and to repress their promoter activities in cultured cells [3, 17, 18]. Protamines are required for normal spermatogenesis and male fertility [3133]. Thus, NR6A1 likely has a role in regulation of the transcription of postmeiotic genes (e.g., protamines) within spermatogenic cells, possibly affecting male fertility.

In the epididymis, NR6A1 protein has been detected not only in the spermatozoa [29] but also in the epididymal cells (e.g., principal, halo, and clear cells) [27, 28]. Although we did not observe specific Nr6a1 expression in epididymal epithelial cells as reported by other laboratories [27, 28], strong positive LacZ staining was observed in sperm within the epididymal tubules of adult Nr6a1LacZ/+ mice (Fig. 5B). These results were further validated by the use of specific NR6A1 antibodies (Fig. 5D). These analyses suggest that NR6A1 is a paternal protein factor that may be involved in fertilization and early embryonic development.

In contrast to the well-investigated Nr6a1 expression in the gonad and the embryo, little is known about Nr6a1 expression in other tissues in adulthood. Previous studies [1, 6, 25, 3436] have shown that low levels of Nr6a1 transcript or protein are detected in several other adult tissues such as brain, lung, and pancreas. In fact, we found that Nr6a1 is expressed in a number of somatic mouse tissues, as demonstrated by positive LacZ staining in adult Nr6a1LacZ/+ mice. In the brain, Nr6a1 transcript and protein have been detected in the neuronal cells of all brain regions, including the granular layer of the dentate gyrus, the cerebral cortex (forebrain), granule and Purkinje cells of the cerebellum (hindbrain), pyramidal neurons of the hippocampus, the thalamus, cerebellar nuclei, and the pons region [37]. Consistent with that study, we found positive LacZ staining in the cerebral cortex, Purkinje cells of the cerebellum, hippocampus (mainly in cornu ammonis regions), and thalamus of adult brain of Nr6a1LacZ/+ mice (Fig. 7). These expression results indicate that NR6A1 has a role in neurogenesis and are supported in part by recent studies of NR6A1 during neuronal cell differentiation in cultured cells [10] and embryonic midbrain development in Nr6a1 null mutant mice [8]. Besides brain, our studies demonstrated that NR6A1 is expressed in other somatic tissues/cells, including epithelial cells within the oviduct (Fig. 5, E and F) and bronchioles of the lung (Fig. 6), anterior and intermediate pituitary, parathyroid gland, and islet of Langerhans within the pancreas in adult mice (Fig. 8). Broad expression of NR6A1 in adult tissues among Nr6a1:LacZ reporter mice is consistent with the Nr6a1 expression pattern observed in our RT-PCR analyses (Fig. 3), although it is not completely in agreement with the lack of expressed sequence tags for Nr6a1 in tissues such as epididymis, oviduct, lung, and pituitary (http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Mm.439703). The expression of NR6A1 in other somatic tissues suggests that NR6A1 may function in biological processes other than reproduction and neurogenesis. In some tissues in which we observed robust LacZ staining such as the parathyroid and the islet of Langerhans, we failed to detect NR6A1 protein expression by immunohistochemistry. This may be indicative of isoform-specific expression undetected by our antibody. However, it is also possible that Nr6Aa1 expression is inhibited. Indeed, there is a miRNA (MIR181) encoded within intron 4 of the Nr6a1 gene that targets an element in the 3′-untranslated region of Nr6a1; expression of MIR181 in these tissues may prevent expression of NR6A1 protein [38]. Further studies using marker genes for specific type(s) of cells within the bronchioles and oviduct, various regions of the brain, anterior pituitary, pars intermedia, parathyroid gland, and islet of pancreas in Nr6a1LacZ/+ mice will pinpoint the exact type(s) of cells that expresses NR6A1. Most important, conditional Nr6a1 knockout using the Cre/loxP system [39] will be needed to reveal the functional role of NR6A1 in those NR6A1-expressing tissues (e.g., brain, pituitary, lung, and oviduct). Regardless, our results suggest that NR6A1 is neither a germ cell-specific factor nor a neuronal cell-specific factor. The extra-germ cell expression of NR6A1 makes it a less attractive target for the development of contraceptives, which is compounded by the embryonic lethality of Nr6a1 null mice that suggests NR6A1 ligands would have teratogenic effects much like retinoids.

In conclusion, we successfully generated a Nr6a1:LacZ reporter mouse model in which the LacZ reporter gene mimics endogenous Nr6a1 expression. By performing LacZ staining on these mouse tissues, we found that Nr6a1 is expressed not only in the germ cells of mouse gonads but also in a variety of somatic tissues such as oviduct, lung, brain, pituitary, parathyroid, and pancreas. These expression studies will facilitate identification of the functional role of NR6A1 during postnatal development. In addition, the Nr6a1:LacZ reporter mouse line generated herein will be an excellent tool for future characterization of the cell lineage and fate of NR6A1-expressing cells (e.g., differentiated ES cells) during early embryonic development, as well as for future identification of factors involved in the regulation of NR6A1 expression in specific tissues or cells.

Acknowledgments

We thank Dr. Francesco J. DeMayo and the Transgenic Core Laboratory, Baylor College of Medicine, for ES cell microinjection.

Footnotes

1Supported by NIH grants DK73524 and HD32878 (to A.J.C.). Z.J.L. is a junior investigator supported in part by NIH COBRE grant 5P20 RR017702.

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