Development of a Transgenic Green Fluorescent Protein Lineage Marker for Steroidogenic Factor 1

Nancy R. Stallings1, Neil A. Hanley1, Gregor Majdic, Liping Zhao, Marit Bakke and Keith L. Parker

Departments of Internal Medicine and Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857

Address all correspondence and requests for reprints to: Dr. Keith L. Parker, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8857. E-mail: keith.parker{at}utsouthwestern.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Knockout mice lacking steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a complex phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To explore further how SF-1 regulates endocrine function, we used bacterial artificial chromosome transgenesis to develop a lineage marker for SF-1-expressing cells. A genomic fragment containing 50 kb of the mouse Nr5a1 gene was used to target enhanced green fluorescent protein (eGFP) in transgenic mice. These sequences directed eGFP to multiple cell lineages that express SF-1, including steroidogenic cells of the adrenal cortex, testes, and ovaries, neurons of the ventromedial hypothalamic nucleus, and reticuloendothelial cells of the spleen. Despite the proven role of SF-1 in gonadotrope function, eGFP was not expressed in the anterior pituitary. These experiments show that 50 kb of the mouse Nr5a1 gene can target transgenic expression to multiple cell lineages that normally express SF-1. The SF-1/eGFP transgenic mice will facilitate approaches such as fluorescence-activated cell sorting of eGFP-positive cells and DNA microarray analyses to expand our understanding of the multiple actions of SF-1 in endocrine development and function.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE ORPHAN NUCLEAR receptor steroidogenic factor 1 (SF-1, also called Ad4BP) has emerged as an essential regulator of endocrine development and function. Initially identified as a tissue-specific transcriptional regulator of the cytochrome P450 steroid hydroxylases (1, 2), SF-1 now is known to play essential roles at multiple levels of the reproductive axis. The first hints of these broader roles emerged from developmental analyses of SF-1 expression in mouse embryos (3, 4, 5). In the adrenal and gonadal primordia, SF-1 was expressed from the earliest stages of organogenesis, suggesting a fundamental role in the initial differentiation of the primary steroidogenic tissues. SF-1 also was expressed in the developing pituitary primordium (6, 7) and in neurons that ultimately form the ventromedial hypothalamic nucleus (VMH; 7, 8).

Direct insights into the roles of SF-1 in vivo came from studies in knockout (KO) mice lacking SF-1 (6, 7, 8, 9, 10). The SF-1 KO mice lacked adrenal glands and gonads and therefore died shortly after birth from adrenal insufficiency. They also exhibited male-to-female sex reversal of their external and internal genitalia, impaired expression of multiple markers of pituitary gonadotropes, and structural abnormalities of the VMH. These studies established essential roles of SF-1 at multiple levels of endocrine differentiation and function, particularly with respect to reproduction. Developmental studies in SF-1 KO embryos showed that the earliest stages of gonadogenesis commenced in the absence of SF-1, but that the gonads then regressed due to apoptosis. Similarly, SF-1-expressing neurons migrated into the appropriate region of the developing diencephalon by embryonic d 17.5 (E17.5) but were no longer observed at postnatal d 1.

Because the adrenal glands, gonads, and VMH of SF-1 KO mice disappear at relatively early stages of development, it has been difficult to follow the fate of SF-1-expressing cells in SF-1 KO mice or to define the molecular basis for the loss of these cells in the absence of SF-1. In this report, we have used the strategy of bacterial artificial chromosome (BAC) transgenesis to develop a lineage marker for many cell lineages that express SF-1, providing a novel and versatile tool to study the roles of SF-1 in endocrine development and function.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of SF-1/eGFP (Enhanced Green Fluorescent Protein) Transgenic Mice
The Nr5a1 gene generates multiple transcripts via alternate promoter usage and differential splicing. In addition to SF-1, the Nr5a1 gene generates a transcript that encodes the embryonal long terminal repeat-binding protein (ELP, Ref. 11), whereas some data indicate that the predominant pituitary transcript arises from a third promoter (11, 12). In previous efforts (Parker, K., K. Morohashi, Y. Sadovsky, and L. Heckert, unpublished observations), relatively short stretches of 5'-flanking region from the mouse Nr5a1 gene failed to target reporter gene expression in transgenic mice to sites where SF-1 is expressed. These observations suggested that the proximal 5'-flanking region of SF-1 lacks element(s) that regulate expression in vivo. BAC transgenesis is emerging as a powerful approach to obtain position-independent, copy number-dependent transgenic expression (13, 14). We therefore prepared a transgene that included a 50-kb BsiW I-BsiWI I fragment from a SF-1 BAC upstream of coding sequences for eGFP and 3'-splice/transcription termination signals from bovine GH (Fig. 1Go). As described in Materials and Methods, the SF-1 sequences included approximately 45 kb of 5'-flanking region, the untranslated first exon, the first intron, and sequences from exon 2 encoding the first five amino acids. After excision from the vector, this BAC fragment was microinjected into pronuclei to generate transgenic mice. One founder with the transgene inserted at a single autosomal locus unlinked to Nr5a1 was used to generate the SF-1/eGFP transgenic line.



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Figure 1. Strategy for Generating the SF-1/eGFP Transgene

The upper diagram shows the organization of the region of mouse chromosome 2 that includes the structural genes encoding SF-1 and germ cell nuclear factor (GCNF). A 50-kb BsiW I-BsiW I (B) BAC fragment that includes the 3'-exons of GCNF, the intergenic region (which represents the 5'-flanking region of SF-1), the untranslated first exon, first intron, and 32 nucleotides of second exon of SF-1 encoding the first five amino acids of SF-1 was digested with BsiW I and placed upstream of the eGFP coding sequences. After excision from vector sequences by digestion with PmeI, DNA was used to prepare the SF-1/eGFP transgenic mouse line as described in Materials and Methods.

 
The SF-1/eGFP Transgene Targets eGFP Expression to Cell Lineages that Express SF-1
The intrinsic fluorescence of eGFP provides a sensitive assay to determine sites where the transgenic SF-1 promoter is active. We first examined eGFP expression in adult SF-1/eGFP transgenic mice. As shown in Fig. 2Go, eGFP was expressed in the adrenal cortex (panel A), interstitial regions of the testes (panel B) and ovaries (panel C), and the VMH (panel D). Notably, eGFP was not expressed in corpora lutea (Fig. 2CGo) or the anterior pituitary gland (Stallings, N., and K. Parker, unpublished observation).



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Figure 2. Expression of the SF-1/eGFP Transgene in Adult Mice

Tissues were harvested from adult SF-1/eGFP transgenic mice, and eGFP expression was determined as described in Materials and Methods. Shown are brightfield (left) and fluorescent (right) photomicrographs. Panel A (adrenal cortex): C, cortex; M, medulla. Panel B (testis): st, seminiferous tubule. Panel C (ovary): cl, corpus luteum; af, antral follicle. Panel D (hypothalamus): A, arcuate nucleus; 3V, third ventricle. Scale bars, 100 µm.

 
The intensity of the eGFP fluorescence hampered efforts to identify individual cells that expressed the SF-1/eGFP transgene. To localize eGFP expression more precisely, we performed immunohistochemical analyses with an antiserum specific for eGFP and compared the eGFP expression pattern with that of endogenous SF-1. As shown in Fig. 3Go, these studies revealed striking correspondence of eGFP and SF-1 immunoreactivities in theca cells of the ovary (panel A), Leydig cells of the testis (panel B), the VMH (panel E), and the spleen (panel D)—a nonendocrine organ where discrete foci of reticuloendothelial cells are known to express SF-1 (15). Collectively, these studies show that the SF-1/eGFP transgene in adult mice is expressed in the adrenal cortex, gonads, VMH, and spleen, suggesting that it contains sufficient regulatory information to target gene expression to most sites where SF-1 is expressed.



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Figure 3. Immunohistochemical Visualization of eGFP and SF-1 Expression in Specific Cell Types

Tissues were harvested from SF-1/eGFP transgenic mice, and immunoreactive eGFP and SF-1 were visualized as described in Materials and Methods. Panel A, Ovary; af, antral follicle. Panel B, Testis; st, seminiferous tubule. Panel C, Spleen. Panel D, Pituitary. Panel E, VMH.

 
Although definitive conclusions are limited by our analysis of a single transgenic line, the expression patterns of SF-1 and eGFP also diverged somewhat. SF-1 immunoreactivity was clearly observed in Sertoli cells (Fig. 3BGo) and the anterior pituitary (Fig. 3DGo), whereas eGFP expression was very faint in Sertoli cells (Fig. 3BGo) and was not detected in the anterior pituitary (Fig. 3DGo). Although we observed no eGFP expression in either granulosa cells (Fig. 3AGo) or corpora lutea, the expression patterns in these sites were not discordant, as they also did not express SF-1. The apparent absence of SF-1 and eGFP expression in granulosa cells and corpora lutea is consistent with the recent observation that these ovarian cells predominantly express the orphan nuclear receptor LRH-1 rather than SF-1 (16), and also may reflect differences in the sensitivity of the immunohistochemical analyses vs. previous studies using in situ hybridization. The absence of pituitary eGFP expression in SF-1/eGFP transgenic mice is consistent with the model in which pituitary transcripts arise from a distinct promoter (11, 12) and may suggest that the 50-kb BAC fragment lacks element(s) required for expression in gonadotropes. An important caveat regarding such conclusions is that we have only analyzed a single transgenic SF-1/eGFP line.

The Developmental Profile of the SF-1/eGFP Transgene Mirrors that of SF-1
In addition to its cell specificity, the timing of SF-1 expression during development is precisely regulated. To determine whether regulatory elements in the SF-1/eGFP transgene directed temporally regulated expression, we examined eGFP expression in mouse embryos. At E9.0, SF-1 transcripts were first detected in the urogenital ridge, which contains cell lineages that form the gonads, adrenal cortex, and part of the kidney (3). Consistent with analyses of SF-1 transcripts, eGFP was expressed at E9.5 in the urogenital ridges of both male and female embryos (Fig. 4AGo and data not shown). The urogenital ridges subsequently differentiate into the indifferent gonads, which histologically are indistinguishable in males and females; at this stage (E11.5), eGFP expression again was comparable in male and female embryos (data not shown).



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Figure 4. Expression of the SF-1/eGFP Transgene in the Embryonic Gonads

Tissues were harvested from SF-1/eGFP transgenic embryos at the indicated ages, and eGFP expression was determined as described in Materials and Methods. Shown are brightfield (left) and fluorescent (right) photomicrographs. A, Male urogenital ridge at E9.5. B, Embryonic testis at E12.5. C, Embryonic testis at E14.5. D, Embryonic ovary at E12.5. E, Embryonic ovary at E14.5. Scale bars, 100 µm.

 
At E12.5–E13, the testes organize into the testicular cords, containing Sertoli cells and primordial germ cells, and the surrounding interstitial region. At this time, eGFP was expressed by Sertoli cells within the cords and by presumptive Leydig cells in the interstitial region (Fig. 4BGo). This expression pattern closely parallels previous analyses of SF-1 transcripts and is compatible with proposed essential roles of SF-1 in regulating Sertoli cell expression of anti-Müllerian hormone and Leydig cell expression of the cytochrome P450 steroid hydroxylases. As testes differentiation progressed at E14.5 (Fig. 4CGo), eGFP was expressed predominantly in the interstitial region, a pattern that persisted throughout the remainder of fetal development and postnatally. In fact, eGFP expression in Sertoli cells had largely disappeared by 3 wk after birth (data not shown) and was only equivocal in adult mice (Fig. 3BGo).

Unlike the testes, prenatal mouse ovaries progress only to the primordial follicle stage and therefore maintain a more homogenous ground-glass appearance. Consistent with this relative lack of histological differentiation, eGFP was distributed throughout the embryonic ovary at both E12.5 and E14.5 (Fig. 4Go, D and E). Although analysis of eGFP expression by fluorescence microscopy is only semiquantitative, the persistent eGFP expression in the ovary at this stage apparently differs from previous analyses of SF-1, which showed decreased expression in the ovaries after sexual differentiation (3, 4, 5). Based on other transgenic analyses in which reporter genes were used to follow the activity of promoters for transcription factors (17), one explanation is that the half-life of eGFP may exceed that of the SF-1 transcripts, such that eGFP persists after the cessation of SF-1 promoter activity. Alternatively, the 50 kb of SF-1 regulatory sequences employed here may lack an element that normally down-regulates SF-1 expression in the embryonic ovaries.

As shown in Fig. 5Go, eGFP expression during adrenal development also correlated closely with previous studies of SF-1 expression (3). The adrenal primordium emerges from a common pool of adrenogonadal precursors at about E11, forming a distinct cluster of cells within the surrounding splanchnic mesoderm (18). Before that time, strong eGFP expression was observed in the cells that comprise the dorsomedial component of the adrenogonadal precursors, which will form the adrenal cortex. As the chromaffin cell precursors migrated into the adrenal primordium from the neural crest, eGFP expression localized predominantly to the outer region of the adrenal gland. Finally, as zonation of the adrenal cortex was established at approximately E17–E18, eGFP was expressed throughout all zones of the adrenal cortex (data not shown).



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Figure 5. Expression of the SF-1/eGFP Transgene in the Embryonic Adrenal Glands

Tissues were harvested from SF-1/eGFP transgenic mice at the indicated ages, and eGFP expression was determined as described in Materials and Methods. A, E12.5. B, E14.5. Scale bars, 100 µm.

 
SF-1 transcripts also were detected at E9.5 in the ventral region of the secondary prosencephalon, the precursor of the retrochiasmatic and tuberal hypothalamus (Ikeda, Y., and K. Parker, unpublished observation). When analyzed by fluorescence microscopy, this region also expressed eGFP at E9.5, albeit at a level that made it difficult to obtain a clear-cut signal on photomicrographs (Fig. 6AGo). This expression subsequently localized to the region of the diencephalon that comprises the VMH, correlating closely with previous analyses of SF-1 transcripts within the brain (3, 8).



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Figure 6. Expression of the SF-1/eGFP Transgene in the Developing Hypothalamus and Spinal Cord

Tissues were harvested from SF-1/eGFP transgenic mice, and eGFP expression was determined as described in Materials and Methods. A, E9.5 diencephalon with brightfield (left) and fluorescent (right) photomicrographs. B, E14.5 spinal cord from control (left) and SF-1/eGFP transgenic (right) mice. Scale bars, 100 µm.

 
We also observed eGFP expression in a more caudal region of the neural floor plate immediately overlying the notochord (Fig. 6BGo). The significance of this finding remains to be determined, as neither SF-1 expression in this region nor spinal cord abnormalities in SF-1 KO mice have been reported. Intriguingly, a transgene containing 11 kb of the 5'-flanking region of Dax1 also was expressed in multiple transgenic lines in a comparable region of the posterior neural tube during embryogenesis, despite the fact that Dax1 transcripts could not be detected by ribonuclease protection assay (19). Further studies are needed to determine whether this unexpected expression directed by the promoter regions of two functionally related orphan nuclear receptors merely reflects a confluence of artifacts or whether the transgenic promoter activity in these sites reflects endogenous expression of SF-1 and Dax1 at levels below the limits of assay sensitivity.

In marked contrast to endogenous pituitary expression of SF-1, which commences at about E14, eGFP was not expressed in either the pituitary primordium or developing anterior pituitary (Stallings, N., unpublished observation). Given the crucial role of SF-1 in regulating the expression of multiple genes that comprise the gonadotrope phenotype (19), the absence of pituitary eGFP expression at any developmental stage argues that the SF-1 regulatory sequences in the SF-1/eGFP transgene lack cis-acting elements required for expression in gonadotropes. To the extent that these studies are confirmed with other transgenic lines, the identification of these putative elements will be an important goal for future studies.

The SF-1/eGFP Transgene Tracks the Fate of SF-1-Expressing Cells in SF-1 KO Mice
Serial examination of gonadal histology in SF-1 KO mice suggested that the indifferent gonads developed in the absence of SF-1 but subsequently regressed by programmed cell death (9). Based on in situ hybridization analyses of a fusion transcript in the SF-1 KO mice, SF-1-expressing neurons similarly migrated into the appropriate region of the diencephalon but then disappeared between E17 and postnatal d 1 (8). The similar expression patterns of SF-1 and eGFP (Fig. 3Go) suggested that this transgene can track the fate of SF-1-expressing cells in SF-1 KO mice. Consistent with the relatively normal appearance of the SF-1 KO indifferent gonads at E9.5, eGFP expression at this developmental stage was comparable in the urogenital ridges of wild-type and SF-1 KO mice (Stallings, N., unpublished observation). At E11.5, the indifferent gonads of SF-1 KO mice exhibited eGFP expression comparable to the expression level seen in the gonads of wild-type mice (Fig. 7AGo). Whereas eGFP expression was readily detected in the wild-type testis at E14.5, a distinct gonad was not present in the SF-1 KO embryo and only trace eGFP was observed in the corresponding urogenital region (Fig. 7BGo). These results establish that the SF-1/eGFP transgene provides a novel tool to track the fate of cells that normally express SF-1 in the gonads of SF-1 KO mice. We similarly have used this transgene to track the fate of VMH neurons in SF-1 KO mice (Davis, A., and N. Stallings, unpublished observation). Coupled with approaches such as induced gene knockouts with a floxed Nr5a1 allele (20), the SF-1/eGFP marker will permit us to follow the fate of multiple cell lineages after different physiological perturbations.



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Figure 7. The SF-1/eGFP Transgene Tracks the Fate of SF-1-Expressing Cells in SF-1 KO Mice

Tissues were prepared from wild-type (right panels) and SF-1 KO (left panels) mice carrying the SF-1/eGFP transgene, and eGFP expression was determined as described in Materials and Methods. Shown are fluorescent photomicrographs of the genital ridge or gonads at the indicated times. A, Male genital ridge at E11.5. B, Male gonad at E14.5. Scale bars, 100 µm.

 
Fluorescence-Activated Cell Sorting (FACS) Selectively Enriches SF-1-Expressing Cells
Previous studies with other eGFP transgenes have used FACS to segregate eGFP-positive and eGFP-negative cells (21, 22). To illustrate the utility of the SF-1/eGFP transgene for similar studies, testes and adrenal glands from SF-1/eGFP transgenic mice were harvested, digested to produce single cell suspensions, and resolved into eGFP-positive and eGFP-negative populations by FACS as described in Materials and Methods. To assess the success of the purification, we compared gene expression profiles in the positive and negative pools. As expected (Fig. 8AGo), FACS of the testes cell population positively enriched for SF-1, while diminishing considerably transcripts for the postmeiotic germ cell marker Meg-1 (top). Similarly, the eGFP-positive pool of adrenal cells was enriched for the expression of both SF-1 and its target gene cholesterol side-chain cleavage enzyme (Fig. 8BGo). These results validate the utility of the SF-1/eGFP transgene to enrich distinct populations of cells that express or do not express SF-1 from complex tissues.



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Figure 8. FACS Enrichment of eGFP-Positive and eGFP-Negative Cells

Testes (3 wk and 6 wk of age) and adrenal glands (newborn) from SF-1/eGFP transgenic mice were dissected, pooled, and digested with collagenase to produce single cell suspensions as described in Materials and Methods. Cells were resolved into positive and negative pools by FACS, and RNA samples from the positive and negative pools were used in semiquantitative RT-PCR assays to detect SF-1, cholesterol side chain cleavage enzyme, Meg-1, and glyceraldehyde phosphate dehydrogenase as described in Materials and Methods.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this report, we used BAC transgenesis to place the eGFP reporter gene under the control of regulatory sequences from the Nr5a1 locus encoding SF-1, thereby targeting eGFP to many sites that express SF-1. An important caveat is that we have assessed only one transgenic line, such that the observed expression pattern may result in part from the specific integration site. Despite this limitation, there are striking parallels between the tissue-specific and developmental profiles of eGFP expression defined here and previous studies examining the expression of SF-1 transcripts and/or protein. Moreover, BAC transgenesis is emerging as a powerful tool to obtain position-independent, copy number-dependent transgene expression (13, 14), at least partly assuaging concerns about artifactual results. Finally, irrespective of the molecular mechanisms responsible for its expression, the SF-1/eGFP transgene described here will permit selective cell enrichment from many SF-1-expressing tissues (e.g. the adrenal cortex, Leydig/theca cells, and VMH neurons) as well as developmental lineage tracing in these sites, thus providing a versatile new tool for studying the roles of SF-1.

Previous transfection analyses of relatively short stretches of the SF-1 5'-flanking region identified several elements that regulate SF-1 promoter activity in cultured cell lines. Of particular note, an E box motif at approximately -80 regulates promoter activity in a variety of SF-1-expressing cell lines, including Y1 adrenocortical cells, {alpha}T3 gonadotropes, and Sertoli cells (23, 24, 25, 26, 27). Although the specific protein(s) that interact with this element are not fully defined, one important activator is the basic helix-loop-helix transcription factor, upstream stimulatory factor 1 (25). It also has been proposed that SF-1 binds a nuclear receptor half-site in its own first intron to autoregulate its transcription (28).

Despite considerable effort, however, the molecular basis for the tissue-specific expression of SF-1 remains poorly understood. The lack of success in previous transgenic targeting studies suggests that the proximal promoter of the Nr5a1 gene lacks an enhancer element necessary for high-level expression in vivo. To the extent that the expression of the SF-1/eGFP BAC transgene described here accurately mirrors expression of the endogenous gene, the high levels of eGFP expression in multiple cell lineages suggests that the 50-kb fragment includes one or more regulatory elements that are lacking in the proximal promoter regions used previously. The 50-kb region that suffices to target gene expression to the appropriate sites in vivo provides a point of departure to map specific elements that regulate the cell-specific and developmental expression of SF-1. Presumably, additional sequences are required for transgenic expression of SF-1 in gonadotropes, which exhibit a distinct dichotomy between the clear-cut expression of SF-1 and the absence of eGFP (Fig. 3DGo).

The ability to follow SF-1-expressing cells nondestructively also provides a powerful tool to monitor their fate in organ explants. Elegant studies have followed the fate of GnRH neurons in hypothalamic slices from mouse embryos (29); the SF-1/eGFP marker will allow us to follow the fate of SF-1-expressing neurons that ultimately form the VMH in similar slice preparations. Organ explants using cultured gonads and mesonephroi likewise have yielded important insights into cell migration and cell-cell interactions during gonadogenesis (30, 31, 32). We envision that the ability to identify SF-1-expressing cells with the eGFP tag will markedly facilitate the analysis of cell migration and fate in gonadal explants.

In addition to cell fate mapping, the SF-1/eGFP transgene has considerable potential utility for resolving SF-1-positive and SF-1-negative cells from heterogeneous mixtures such as the primary steroidogenic organs and the hypothalamus. Support for this approach is provided by our successful enrichment of SF-1-positive cells from testes and adrenal glands (Fig. 8Go). The ability to isolate SF-1-expressing cells from complex structures such as the VMH or urogenital ridge should markedly facilitate approaches such as DNA chip microarrays or subtractive cloning to identify novel SF-1 target genes at different sites of expression.

In summary, we describe here the first successful transgenic targeting of a reporter gene with regulatory sequences from the mouse Nr5a1 gene encoding SF-1. Using this transgene, we track the fate of cells that normally express SF-1 in SF-1 KO gonads and demonstrate the use of the SF-1/eGFP marker to select SF-1-expressing cells from single cell suspensions derived from the adrenal cortex. This SF-1/eGFP transgene provides novel insights into the regulation of SF-1 and will be an invaluable resource by which to expand our understanding of the multiple roles of SF-1 in endocrine development and function.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of the SF-1/eGFP Transgenic Mice
All experiments involving mice were approved by the Institutional Animal Care Research Advisory Committee at University of Texas Southwestern. Full details of the cloning strategy for the SF-1/eGFP transgene are available from the authors upon request. Briefly, the transgene contained a 50-kb BsiW I-BsiW I fragment of the SF-1 BAC (Research Genetics, Inc., St. Louis, MO) that included approximately 45 kb of 5'-flanking region from the mouse Nr5a1 gene, the noncoding first exon, the first intron, and 32 nucleotides from the second exon. As a result of manipulations involved in the cloning strategy, the transgene encodes a protein containing the first five amino acids from SF-1 (Met-Asp-Tyr-Ser-Tyr) and an additional Ala generated by the cloning strategy fused in frame to the initiator Met from eGFP (CLONTECH Laboratories, Inc., Palo Alto, CA). It should be noted that the same residues found in the SF-1 coding sequence are also contained within ELP, such that this transgene potentially could also direct eGFP expression to sites where ELP is expressed. The 3'-splice/polyadenylation signals from bovine GH were amplified by PCR and then inserted 3' of the eGFP coding sequence. The proper orientation of the SF-1/eGFP transgene was verified by DNA sequence analysis, and its ability to encode functional eGFP was verified by stable transfection of Y1 mouse adrenocortical tumor cells and fluorescence microscopy (Hanley, N., unpublished observation). The transgene was excised from the pBeLoBAC11 vector by digestion with PmeI and resolved by preparative pulsed field gel electrophoresis in a 1% Seaplaque agarose gel. Successful resolution of the transgene fragment from the vector was confirmed by pulsed field gel electrophoresis analysis.

The SF-1 KO mice were generated and genotyped as previously described (9). SF-1/eGFP transgenic mice were produced by the National Institute of Child Health and Human Development Core Transgenic Facility at the University of Alabama, Birmingham. Mice carrying the SF-1/eGFP transgene were identified initially by dot blot analysis with an 800-bp NcoI/XbaI fragment of eGFP. Thereafter, mice were genotyped by genomic PCR with the following primers: forward (5'-CACCATCTTCTTCAAGGACGAC-3') and reverse (5'-GAATGACACCTACTCAGACAATGC-3').

Transgenic male and female mice were paired in cages at 1800 h, and 1200 h of the morning on which the copulatory plug was detected was designated d 0.5 of gestation (E0.5). After the mothers were anesthetized, the embryos were harvested by cesarean section. In all cases, the ages of the embryos were confirmed according to the external criteria described by Kaufman (33), and the sex of embryos was determined by PCR assays for the Sry gene as described previously (9).

Visualization of eGFP and SF-1 Expression
Embryos were harvested from the mother and fixed en bloc in 4% paraformaldehyde, whereas postnatal mice were anesthetized and then perfused with 4% paraformaldehyde. Embryos or tissues were embedded in Tissue Tek (Sakura, Tokyo, Japan), frozen in liquid nitrogen, and stored at -80 C until sections were cut on a cryostat (20 µm for brain and 15 µm for other tissues). Expression of eGFP was visualized using a Optiphot microscope (Nikon, Melville, NY) equipped with a UV light source and filters for fluorescein visualization. The same sections were then analyzed for tissue histology by drying overnight at 37 C, followed by staining/counterstaining with hematoxylin/eosin.

Immunohistochemical analyses were preformed as follows. Mice were anesthetized and then perfused with 4% paraformaldehyde, postfixed for 12–16 h, transferred to 70% ethanol, and then embedded in paraffin using standard procedures. Sections (7 µm) were cut on a microtome and dried overnight at 37 C. An antiserum specific for eGFP was purchased from Novus (Littleton, CO) and used for immunohistochemical detection of eGFP at a 1:2500 dilution according to the supplier’s recommendations. The rabbit polyclonal antiserum against bovine SF-1 was a generous gift from Dr. Ken Morohashi and was used as described previously (4).

Enrichment of SF-1-Positive Cells by Fluorescence-Activated Cell Sorting (FACS)
Testes were dissected from SF-1/eGFP transgenic mice at 3 and 6 wk of age, and adrenal glands were isolated from adult mice. Single cell suspensions were prepared by collagenase digestion, and eGFP-positive and eGFP-negative cells were resolved by FACS essentially as described (22). RNA was prepared using the Trizol reagent and then was used for semiquantitative RT-PCR assays as described (20). Previously described conditions and primer sets were used to detect SF-1, the cholesterol side-chain cleavage enzyme, and glyceraldehyde phosphate dehydrogenase (20), and the postmeitoic germ cell marker Meg-1 was measured as described (34).


    ACKNOWLEDGMENTS
 
We thank Ken Morohashi, Yoel Sadovsky, and Leslie Heckert for communicating unpublished data; Stuart Tobet, Robert Hammer, Steve Young, Nat Heintz, Tom Sato, and Toshiyuki Motoike for helpful discussions; Angela Mobley and the University of Texas Southwestern Cell Sorting Core for assistance with FACS analysis; and Dr. Carl Pinkert for injecting the BAC transgene.


    FOOTNOTES
 
This work was supported by a UK Department of Health Clinician Scientist Fellowship (N.A.H.) and NIH Grants 5T32HD-07190 (N.R.S.), DK-54480, and DK-54028.

1 N.R.S. and N.A.H. contributed equally to this work. Back

Abbreviations: BAC, Bacterial artificial chromosome; E12.5, E14.5, E17.5, embryonic d 12.5, 14.5, 17.5, respectively; eGFP, enhanced green fluorescent protein; ELP, embryonal long terminal repeat-binding protein; FACS, fluorescence-activated cell sorting; KO, knockout; SF-1, steroidogenic factor 1; VMH, ventromedial hypothalamic nucleus.

Received for publication January 3, 2002. Accepted for publication July 9, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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