Sonic hedgehog signaling during adrenal development

Abstract

It has been speculated for a number of years that Sonic hedgehog (Shh) signaling plays an important role in adrenal development. Over the past two years several reports have described the expression and function of Shh pathway genes in the adrenal cortex, using primarily mouse models. The key findings are that Shh signals produced by a population of partially differentiated cortical cells located in the outer cortex/zona glomerulosa are received by non-cortical mesenchymal cells located predominantly in the overlying capsule. This signal is required for growth of both the capsule and the cortex, but not for cortical zonation or steroidogenic cell differentiation. Using molecular genetic tools to define the adrenocortical cell lineages that are descended from both Shh signaling and receiving cells, both capsule and cortical cells were found to have properties of adrenocortical stem and/or progenitor cells. Here we place these observations within the context of prior studies on adrenal development, postnatal adrenal maintenance and adrenocortical stem/progenitor cell lineages.

Figure 1. Summary of the major stages of murine adrenal development

(A) Cells expressing Sf-1 delaminate from the dorsal coelomic epithelium (ce) and invade the overlying mesonephric mesenchyme (m). The adrenal anlagen (a) forms from cells that migrate dorsomedially, while the gonadal anlagen precursors (g) settle dorsolaterally. The adrenal primordium is invaded by migratory sympathoadrenal neural crest cells (nc) that will form the neuroendocrine medulla. Subsequently the gland becomes encapsulated by mesenchymal cells. (B) As the gland grows the cortical cells sort into a central medulla and outer cortex. The cortex is composed of fetal adrenal cells that are established before the outer definitive adrenal population. (C) The cortical cells segregate into functional zones. Some ZG cells do not completely differentiate, although they do express Sf-1 and some steroidogenic biosynthetic pathway enzymes. Fully differentiated steroidogenic ZG cells express Cyp11b2, while differentiated ZF cells express Cyp11b1. (D) Transverse section of 11.5 dpc mouse embryo immunostained for expression of Sf-1 (green) and PECAM, which marks endothelial cells (red), and counterstained for DNA (blue). By this age the adrenal (a) and gonadal (g) primordia are clearly segregated, and the adrenal is embedded in the mesonephric mesenchyme (m). (E) Whole mount ventral view of an Sf-1-Cre^Tg; R26^mT/mG 12.5 dpc embryo in which Cre-mediated recombination has induced GFP reporter expression (green) in the Sf-1 lineage in the adrenal (a) and gonadal (g) anlagen. All non-recombined cells express tdTomato, and are labeled in red. (F) Whole mount ventral view of an Sf-1-Cre^Tg; R26^mT/mG 14.5 dpc embryo. By this age the metanephric kidneys (k) have grown adjacent to the adrenal glands. (G) Section of adult adrenal gland coimmunostained for expression of Cyp11b2 (green) and Cyp11b1 (red). Note that some immature ZG cells do not express Cyp11b2. Scale bar: 10 microns. Abbreviations: D, dorsal; V, ventral; M, medial; L, lateral; n, notochord; nt, neural tube; nc, neural crest; d, dorsal aorta; im, intermediate mesoderm; m, mesonephric mesenchyme; ce, coelomic epithelium; agp, adrenogonadal primordium; a, adrenal; g, gonad; k, kidney; c, capsule; zg, zona glomerulosa; zf, zona fasciculata; x, X-zone; m, medulla. Methods: All experiments with live animals were performed according to Columbia University Institutional Animal Care and Use Committee guidelines. Mice were maintained on a mixed Sw.B6.129 background and fed standard laboratory chow. Sf-1-Cre; R26-mT/mG mice were generated from Sf1-cre^Tg(Hi) (Kim et al., 2008) and R26^mT/mG (Muzumdar et al., 2007) parents. Noon of the day of vaginal plug detection was considered 0.5 dpc. Tissue was collected and immunostained as described (King et al., 2009).

Figure 2. PHS mice have adrenal glands. Tissue was collected from embryonic wild type (+/+) or Gli3^Δ699/Δ699 mutant (Böse et al., 2002) (Δ699/Δ699) mice and the adrenal glands examined

(A,B) Representative whole mount images of tissue dissected from 16.5 dpc embryos. Adrenal glands of similar size and shape (yellow arrowheads) are visible in both control (A) and mutant (B) embryos, although the mutant kidneys (K) are dysmorphic or absent. (C,D) Representative multichannel fluorescence images of control (C,C’) and mutant (D,D’)18.5 dpc adrenal tissue. Similar staining patterns of endogenous biotin, which labels all cortical cells with steroidogenic potential, and tyrosine hydroxylase (TH), which labels neuroendocrine cells of the medulla, are present in adrenals of both genotypes. Boxed regions enlarged in lower panels (C’,D’). Scale bars: 100 microns. Methods: All live animal procedures were approved and performed according to University of Duisberg-Essen institutional guidelines. Mice were maintained on a mixed 129.B6 background and fed standard laboratory chow. Gli3^Δ699/+ mice (Böse et al., 2002) were intercrossed to generate mutant and control sibling embryos. Noon on the day of vaginal plug detection was designated 0.5 dpc. Cryosectioned tissue was stained as described (Paul and Laufer, 2011).

Figure 3. Gene expression, signaling and lineage relationships between the adrenal capsule and cortex

(A) Shh is expressed by clusters of incompletely differentiated Sf-1 positive cells in the ZG (pale green), and signals (red arrow) to Gli1 positive cells in the overlying inner capsule (yellow). Both Wnt and FGF signaling are required for adrenal development and maintenance, and potentially expression of these ligands in the capsule depends on Shh. These ligands might signal (orange arrows) to the Shh expressing progenitor cells and/or the Cyp11b2 expressing cells (light green) in the ZG, all of which potentially express beta catenin, Fgfr2 and Fgfr4. (B) Lineage tracing studies indicate that Gli1 positive but Sf-1 negative cells in the capsule or non-capsule mesenchyme can convert to Sf-1 positive steroidogenic lineages during development of the gland (black arrows) (King et al., 2009; Huang et al., 2010), at least in part via a Shh expressing intermediate (King et al., 2009). Shh expressing cells differentiate to become both Cyp11b1 and Cyp11b2 positive, but not Gli1 positive cells (white arrows).