Investigating the maintenance of the mouse definitive adrenal cortex
The adrenal gland is an important endocrine organ, protecting the body against acute and chronic stress. The adrenal cortex consists of three morphologically and functionally distinct zones: the outer zona glomerulosa (zG), the zona fasciculata (zF), and the innermost zona reticularis (zR). In rodents, zG cells produce mineralocorticoids (mainly aldosterone), while zF cells secrete glucocorticoids (mainly corticosterone). The functions of zG and zF are defined by the mutually exclusive expression of Cyp11b2 and Cyp11b1 that encode the enzymes aldosterone synthase and 11β-hydroxylase, which catalyze the terminal reactions in the production of aldosterone and corticosterone, respectively. This thesis aims to investigate the maintenance of the definitive mouse adrenal cortex. This involves studies to identify the location of adrenal stem/progenitor cells, and the mechanisms by which differentiated adrenocortical cells are replenished in the adult mice. BrdU pulse-chase studies provided valuable information about cell division and cell fate under physiological or pathophysiological stimulations. The distribution of adrenocortical cells with nuclei stained positively for BrdU and/or Ki67 was identified. Ki67 labelling marked actively dividing cells and showed that adrenocortical cells originate at or around the zG/zF interface. BrdU labelling indicated that, following cell division, cells are displaced inwards and outwards. Acute angiotensin II treatment was shown to have no significant effects on the cell proliferation or turnover in any of the adrenocortical zones. The pathophysiological effects of long-term ACTH treatment were analyzed in a mouse model of congenital adrenal hyperplasia caused by a null mutation of Cyp11b1. Cell hypertrophy was evident in all regions of the adrenal cortex due to the impaired negative-feedback of the HPA axis. Adrenocortical cell proliferation was also increased particularly in the outer zona fasciculata at the border between zG and zF where adrenocortical stem/progenitor cells might be located. The intervening steps between cell proliferation and the final differentiation into steroidogenic zG and zF cells have yet to be discovered. A visual method of monitoring levels of Cyp11b2 and Cyp11b1would offer a convenient approach to track the stages of adult stem cell differentiation that lead to normal adrenal maintenance in vivo and in vitro. In the present study an AS-mCherry-11B-EGFP BAC construct was successfully engineered, in which Cyp11b2 and Cyp11b1 were substituted by mCherry and EGFP, respectively. This BAC construct was characterized in mouse adrenocortical Y1 cells. It was determined that EGFP faithfully recapitulated the expression of Cyp11b1. Forskolin or cAMP treatment induced a rapid cell rounding effect and caused the increased expression of EGFP transgene and endogenous Cyp11b1. An attempt was made to establish a transgenic mouse model, in which zG and zF cells would be marked with mCherry and EGFP respectively, allowing the differentiation of an adrenocortical stem cell to be traced. Following microinjection of the BAC into mouse zygotes, twoAS-mCherry-11B-EGFP transgenic founder mice were identified. Unfortunately, neither of them was able to transmit the transgene through germline, suggesting the mosaicism of transgene integration. Indeed, mosaicism of the transgenic adrenals was demonstrated by RT-PCR and immunostaining, which also revealed that the exogenous EGFP expression faithfully recapitulated the endogenous Cyp11b1 in adrenals. Although it is assumed that expression of Cyp11b2 and Cyp11b1 are mutually exclusive, zG and zF cells may have the plasticity to allow the transition from one cell type into another. The AS-mCherry-11B-EGFP BAC construct is a useful tool for studying in vitro ES cell differentiation towards the adrenocortical lineage. Transgenic AS-mCherry-11B-EGFP ES cells were successfully differentiated into mesenchymal stem cells, as identified by the expression of molecular markers for the mesenchymal lineage. It has been reported that steroidogenic factor (Sf1) can promote the differentiation of MSCs into steroidogenic cells, and Shh plays an important role in Sf1 expression and the consequent adrenal development. However, Shh treatment failed to achieve transformation of mesenchymal cells into adrenocortical cells. It is thought there might be a requirement for additional factors to combine with Shh in promoting the transdifferentiation of ESC-derived mesenchymal cells. Future studies will focus on the genetic control of Cyp11b1 and Cyp11b2 in transgenic AS-mCherry-11B-EGFP ES cells. In conclusion, the location and fate of the adrenocortical progenitor cells were demonstrated by the BrdU pulse-chase studies in different mouse models. An AS-mCherry-11B-EGFP BAC construct was generated, and used to study the mutual and differential controls of Cyp11b1 and Cyp11b2 expression in adrenocortical cells in vitro and in transgenic mice in vivo.