Expression of steroidogenic factors 1 and 2 in normal human pancreas

Orphan nuclear receptor steroidogenic factor-1 (SF-1) is crucial for development and function of steroidogenic organs. The steroidogenic factor-2 (SF-2) is an essential factor involved in cholesterol transfer and activation of promoters of steroidogenic enzymes CYP11A1, CYP17 and Steroidogenic Acute Regulatory Protein (StAR). We have previously demonstrated steroidogenic activity in pancreatic tissue. The aim of this study was to investigate the presence of SF-1 and SF-2 in human pancreas. Total RNA was extracted from normal male (five) and female (five) samples, obtained from the organs donor program. RT-PCR approach was used to analyze the expression of SF-1 and SF-2. Immunohistochemical analysis was performed for SF-1. The bands of expression were present in both male and female samples, although differential expression was observed. For both factors, the signal detected was more evident in males than in females. A similar pattern was present in the immunohistochemical study. Normal human pancreas expresses SF-1 and SF-2 factors similarly to ovary and adrenals. A distinctive characteristic is the sexually dimorphic expression of these factors. Our data provide evidence suggesting that the pancreas achieves steroidogenic activity supporting the presence of gender- and location-related differences in the expression of these steroidogenic factors.     

Cre‐mediated transgene activation in the developing and adult mouse brain

Summary: The neuron‐specific rat enolase (NSE) promoter was employed to establish transgenic mice expressing Cre recombinase in the central nervous system. Founders were crossed with dormant lacZ indicator mice and specificity as well as efficiency of Cre‐mediated transgene activation was determined by PCR and/or X‐gal staining. Whereas most transgenic lines exhibited Cre activity in early development resulting in widespread Cre activity, one line (NSE‐Cre26) expressed high levels of Cre in the developing and adult brain. With the exception of kidney, which showed occasionally low level of Cre activity, Cre recombination in double transgenics was restricted to the nervous system. Whole‐mount X‐gal staining of 9.5 dpc embryos indicated Cre‐mediated lacZ expression in forebrain, hindbrain, and along the midbrain flexure. A similar expression pattern was observed during later stages of embryogenesis (11.5–13.5 dpc). In adult mice, Cre recombinase was expressed in cerebral cortex and cerebellum and high levels of Cre‐mediated lacZ expression were observed in hippocampus, cortex, and septum. The NSE‐Cre26 transgenic mouse line thus provides a useful tool to specifically overexpress and/or inactivate genes in the developing and adult brain. genesis 31:118–125, 2001. © 2001 Wiley‐Liss, Inc.     

Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function

Knockout (KO) mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a compound endocrine phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To inactivate a conditional SF-1 allele in the gonads, we targeted the expression of Cre recombinase with a knock-in allele of the anti-Müllerian hormone type 2 receptor locus. In testes, Cre was expressed in Leydig cells. The testes of adult gonad-specific SF-1 KO mice remained at the level of the bladder and were markedly hypoplastic, due at least partly to impaired spermatogenesis. Histological abnormalities of the testes were seen from early developmental stages and were associated with markedly decreased Leydig cell expression of two essential components of testosterone biosynthesis, Cyp11a and the steroidogenic acute regulatory protein. In females, the anti-Müllerian hormone type 2 receptor-Cre allele directed Cre expression to granulosa cells. Although wild-type and SF-1 KO ovaries were indistinguishable during embryogenesis and at birth, adult females were sterile and their ovaries lacked corpora lutea and contained hemorrhagic cysts resembling those in estrogen receptor alpha and aromatase KO mice. Collectively, these studies establish definitively that SF-1 expression in the gonads is essential for normal reproductive development and function.     

Transgenic mice engineered to target Cre/loxP-mediated DNA recombination into catecholaminergic neurons

To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We crossbred TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of "floxed" genes in catecholaminergic neurons.     

Surfactant protein A promoter directs the expression of Cre recombinase in brain microvascular endothelial cells of transgenic mice.

Brain microvascular endothelial cells (ECs) have unique characteristics distinguished from peripheral ECs and play important roles in blood-brain barrier (BBB). To investigate the physiological control of the brain ECs, we generated a transgenic mouse line in which the expression of Cre recombinase was driven by the promoter of the mouse surfactant protein A (SP-A) gene. The Cre activity was detected in blood vessels of brain, alveolar type II cells of lung and epithelium of gland stomach. In brain ECs, the Cre activity started at embryonic day 11.5, indicating that the subpopulation of ECs in brain could be molecularly defined at early embryonic stages. The use of SP-A-Cre mice should facilitate analysis of gene function in the brain ECs.