Developmental links between the fetal and adult zones of the adrenal cortex revealed by lineage tracing

Abstract

The nuclear receptor Ad4BP/SF-1 is essential for development of the adrenal cortex and the gonads, which derive from a common adrenogonadal primordium. The adrenal cortex subsequently forms morphologically distinct compartments: the inner (fetal) and outer (definitive or adult) zones. Despite considerable effort, the mechanisms that mediate the differential development of the adrenal and gonadal primordia and the fetal and adult adrenal cortices remain incompletely understood. We previously identified a fetal adrenal-specific enhancer (FAdE) in the Ad4BP/SF-1 locus that directs transgene expression to the fetal adrenal cortex and demonstrated that this enhancer is autoregulated by Ad4BP/SF-1. We now combine the FAdE with the Cre/loxP system to trace cell lineages in which the FAdE was active at some stage in development. These lineage-tracing studies establish definitively that the adult cortex derives from precursor cells in the fetal cortex in which the FAdE was activated before the organization into two distinct zones. The potential of these fetal adrenocortical cells to enter the pathway that eventuates in cells of the adult cortex disappeared by embryonic day 14.5. Thus, these studies demonstrate a direct link between the fetal and adult cortices involving a transition that must occur before a specific stage of development.

FIG. 1.

Region-specific downregulation of LacZ expression in the developing fetal adrenal cortices in Ad4BP-LacZ-FAdE Tg mice. (A) Schematic representation of the Ad4BP-LacZ-FAdE Tg construct (40). A fragment that contains 5.8 kb upstream from the initiation methionine in the second exon of Ad4BP/SF-1 was used as the basal promoter. The FAdE was placed downstream of LacZ coding sequences and a poly(A) signal. (B) Lower-power views of sections of Ad4BP-LacZ-FAdE Tg fetuses at the indicated developmental stages stained with hematoxylin and eosin to reveal adrenal morphology (bottom) or with specific antibodies for LacZ (green) and rat monoclonal antibody for Ad4BP/SF-1 (red). Superimposed images of LacZ and Ad4BP/SF-1 immunolocalization are shown. (C) Higher-power views of hematoxylin-and-eosin staining (bottom) or immunostaining (top) for LacZ and Ad4BP/SF-1. The areas enclosed by the rectangles in the immunohistochemical studies shown in panel B are enlarged. The arrows in the E15.5 and E17.5 sections indicate Ad4BP/SF-1-positive cells displaying weak LacZ staining. Fetal (f) and adult (a) zones are indicated in the E17.5 section. Bars = 100 μm (B) and 50 μm (C).

FIG. 2.

Cell lineage tracing using FAdE activity. (A) Schematic representation of the FAdE-Ad4BP-IRES-Cre Tg and expression of Cre. IRES-Cre was inserted downstream of the 5.8-kb Ad4BP/SF-1 basal promoter and the FAdE. In situ hybridization was performed with Ad4BP-LacZ-FAdE Tg fetuses (line 1) at E14.5. Cre mRNA was detected in the inner part of cortical layer of the adrenal at this stage. The area enclosed by the rectangle in the middle panel is enlarged as the right panel. Bars = 100 μm. (B) Detection of LacZ expression as a surrogate marker of Cre activity driven by the FAdE. The FAdE-Ad4BP-IRES-Cre Tg mice (lines 1 and 3) were mated with ROSA26R mice (31), and LacZ activity in the Cre/ROSA Tg fetuses (lines 1 and 3) at E11.5 was examined. An Ad4BP-LacZ-FAdE Tg fetus is shown for comparison. The LacZ staining localized to the AdP of the control mouse (arrows). While strong staining in the two Cre/ROSA Tg mice was again seen for the AdP (arrows), staining was also detected anterior to the AdP and within the gonadal primordium (arrowheads). Sections of the AdP of Cre/ROSA Tg fetuses were immunostained with the antibodies to LacZ (green) and Ad4BP/SF-1 (red). Superimposed images are shown. Bars = 50 μm. (C) Detection of endogenous expression of Ad4BP/SF-1 in the region anterior to the AdP. In situ hybridization using RNA antisense probe (left) and sense probe (middle), and immunohistochemistry with antibody for Ad4BP/SF-1 (right), were performed with wild-type fetuses at E10.5. Strong signals of Ad4BP/SF-1 were detected in the AGP (arrows). The bottom panels show enlarged views of the areas enclosed by rectangles in the top panels. As indicated (arrowheads), both Ad4BP/SF-1 transcripts and protein were expressed in the region anterior to the AGP. In situ hybridization with the Ad4BP/SF-1 sense probe showed no specific signal. The dark caudal staining in the top left panel corresponds to signal in the AGP but is seen from a dorsal view because the sample is rotated. (D) Cell lineage tracing of the fetal adrenal cortex via Cre-mediated activation of LacZ expression. LacZ expression in Cre/ROSA Tg mice (lines 1 and 3) was examined at E14.5, P13, and 2 months after birth. For comparison, age-matched Ad4BP-LacZ-FAdE mice were examined. Since the fetal adrenal (X) zone disappears at puberty in males (8, 40), male mice at P13 (prepubertal) and 2 months after birth were used. The LacZ signal in the Ad4BP-LacZ-FAdE Tg mice remains at the inner part of the adrenal cortex at P13 but disappears at 2 months after birth; in contrast, LacZ expression in the Cre/ROSA Tg mice persisted throughout the adrenal cortex at all stages examined. Go, gonad. Bars = 200 μm. (E) Schematic presentation of the FAdE-Hsp-EGFP Tg construct. EGFP was placed downstream of the basal promoter of Hsp68 and FAdE as indicated. A representative fluorescence image of the FAdE-Hsp-EGFP Tg fetus (E11.0) is shown. A cross section (middle) of the Tg fetus at the indicated region in the left panel (vertical bar) reveals that EGFP is expressed strongly in the AdP and weakly in a part of the gonadal primordium (GoP). The area enclosed by the rectangle in the middle panel is enlarged as the right panel. The region of the gonadal primordium is enclosed by a dotted line. Ao; aorta; magni., magnification. Bars = 100 μm.

FIG. 3.

Restricted window in which the FAdE-active fetal adrenocortical cells can develop into adult adrenal cortex. (A) Schematic representation of the FAdE-Ad4BP-Cre-ERT2 Tg construct. Cre-ERT2 is placed downstream of the FAdE and the 5.8-kb promoter fragment of Ad4BP/SF-1. (B) Monitoring Cre-ERT2 activity through LacZ activity. The FAdE-Ad4BP-Cre-ERT2 Tg mice (lines 4, 5, and 7) were mated with ROSA26R mice, pregnant mice were treated with tamoxifen at E10.5, and the lacZ activity of the Cre-ERT2/ROSA Tg fetuses was examined at E14.5. For comparison, the LacZ expression for an Ad4BP-LacZ-FAdE Tg fetus at E14.5 is shown (left). Whole-abdomen views of the urogenital region are shown at the top (the control for the whole-abdomen view is shown in Fig. 2D), while enlarged views and sections of the adrenal gland are shown at the middle and bottom panels, respectively. LacZ is expressed throughout the adrenal cortices in fetuses from the three Cre-ERT2/ROSA Tg lines but only in the inner part of the cortex of the Ad4BP-LacZ-FAdE Tg fetus. Ad, adrenal gland; Go, gonad. Bars = 100 μm. (C) Restricted potential of the FAdE-active fetal adrenocortical cells to develop into the adult adrenal cortex. The Cre-ERT2/ROSA Tg fetuses (line 4) were treated with tamoxifen, thereby activating Cre-ERT2 at E11.5, E12.5, E13.5, or E14.5, and then subjected to LacZ staining at E14.5 or E15.5 as indicated. Whole-abdomen views are shown at the top, while enlarged views of the adrenal gland are shown at the bottom. The fetal adrenal glands are indicated by dotted lines. (D) Cell lineage tracing of the FAdE-active fetal adrenocortical cells in adult mice. The Cre-ERT2/ROSA Tg fetuses were treated with tamoxifen at E11.5, E14.5, or P6, and then LacZ expression in the adrenal glands of males was examined 2 months after birth. LacZ-positive cells were observed only in the mouse treated with tamoxifen at E11.5. Bars = 500 μm.

FIG. 4.

Proliferation of FAdE-positive cells at early stages of adrenal development. Cell proliferation in Ad4BP-LacZ-FAdE Tg mice was examined using BrdU incorporation at E11.5, E12.5, and E13.5. Shown are sections stained with anti-BrdU (green) and anti-LacZ (red) antibodies. Data are expressed as percentages of LacZ-positive cells that were also positive for BrdU. Means of proliferating and total cell numbers are shown below along with standard deviations. Bars = 50 μm.

FIG. 5.

Expression of steroidogenic enzymes in the developing adrenal glands. Three steroidogenic enzymes, 3β-Hsd, Cyp11a1, and Cyp21, were analyzed by combined in situ hybridization (blue) and immunofluorescence for LacZ (green). Cross sections of the adrenal gland were prepared from the Ad4BP-LacZ-FAdE Tg fetuses at E14.5. Expression of each marker is shown at the left as indicated, with the merged picture shown at the right. The areas enclosed by the rectangles are enlarged at the far right. Arrows indicate cells that express LacZ but not the steroidogenic markers. Bars = 100 μm.

FIG. 6.

Expression of Ad4BP/SF-1 and Dax-1 in the developing gonads and adrenal glands. (A) Differential expression of Dax-1 in the developing gonads and adrenal glands. Cross sections of the urogenital region were prepared from the Ad4BP-LacZ-FAdE Tg fetuses at E10.0, E10.5, E11.5, and E12.5 and used for immunofluorescence analysis for Ad4BP/SF-1 (green) and Dax-1 (red). Expression of Ad4BP/SF-1 is shown at the top, while superimposed images of Dax-1 and Ad4BP/SF-1 immunostaining are shown at the bottom. GoP, gonadal primordium. Bars = 50 μm. (B) Expression of LacZ and Dax-1 in the adrenal glands of the Ad4BP-LacZ-FAdE Tg mice at E13.5, E17.5, and P9. Sections of the adrenal gland were subjected to immunofluorescent detection of LacZ (green) and Dax-1 (red). The areas enclosed by the rectangles are enlarged at the bottom. Arrows indicate cells expressing Dax-1 which have decreased expression of LacZ. Bars = 50 μm. (C) Stimulation of FAdE activity by Ad4BP/SF-1 and inhibitory effect of Dax-1. The mouse Ad4BP/SF-1 and Dax-1 expression constructs were cotransfected into HEK293T cells with reporter tk-luciferase (tk-luc) plasmids that either lacked or contained the FAdE as indicated. The relative luciferase activities were normalized with β-galactoside (β-Gal) activity and are shown as means ± standard deviations from triplicate transfections. CMV, cytomegalovirus promoter.