Expression and distribution of differentiation and proliferation markers during mouse adrenal development

Abstract

ACTH and N-terminal POMC have been implicated as important modulators of adrenal development and function. While ACTH signals through its cognate receptor the melanocortin 2 receptor (MC2-R) and induces steroidogenesis, N-terminal POMC is thought to be cleaved by the Adrenal secretory Protease (AsP) into a mitogenic active peptide. In an attempt to characterize expression patterns of proliferating and steroidogenic active cells during adrenocortical development, we investigated the expression of proliferation (BrdU incorporation) and differentiation (3-beta-hydroxysteroid dehydrogenase, 3β-HSD) markers together with MC2-R expression within the developing adrenal cortex at E9.5, E11.5, E13.5, E15.5, E17.5 and 2 weeks post partum. To co-localize the expression patterns of these gene products confocal microscopy imaging was performed after double stained immunohistochemistry (IHC) or combined in situ hybridization (ISH)/IHC. In addition, MC2-R, 3βHSD and AsP expression was assessed by means of RT-PCR. While RT-PCR demonstrated increasing MC2-R mRNA levels from E11.5 until birth, highest AsP expression was detected at E13.5 with an decrease in expression levels thereafter. Interestingly, AsP expression levels were paralleled by the number of BrdU positive cells within the adrenal cortex. At E13.5 BrdU positive cells started to assemble in a subcapsular cell layer possibly representing the stem cell layer of the adult adrenal cortex. In contrast, 3βHSD IHC and MC2–3 ISH revealed expression throughout the developing adrenal cortex. While only a small percentage of proliferating cells expressed 3β-HSD, confocal microscopy revealed co-expression of MC2-R mRNA and 3β-HSD immunoreactivity in the majority of cells. These findings suggest that the expression of the MC2-R is associated with a non-proliferating but differentiated phenotype. Thus, MC2-R expression might be involved in the induction from a proliferating into a differentiated cellular phenotype. Ongoing experiments aim at the co-localization of AsP and BrdU.