Nuclear hormone receptor signaling in the developing CNS : studies on the retinoid receptors RAR and RXR, and the orphan receptors NURR1, NOR1 and NGFI-B
Sammanfattning: The nuclear hormone receptor superfamily comprises more than sixty members, including receptors for steroids, thyroid hormone, vitamin D and retinoids. Many nuclear receptors function as ligand-induced transcription factors that upregulate expression of specific target genes. RAR (retinoic acid receptor) and RXR (retinoid X receptor) bind and mediate the physiological effects of retinoids (vitamin A derivatives), primarily as RXR-RAR heterodimers. In addition, RXR is a crucial dimeric partner for a number of nuclear receptors. This has raised the question of whether RXR is actively involved in retinoid signaling in vivo, or merely is a silent heterodimerization partner for RAR. Also included in the superfamily are receptors that are homologous, but whose ligands and functions are unidentified, and are referred to as orphan receptors. One subgroup consists of the related orphan receptors NURR1, NOR1 and NGFI-B, that act as constitutively active monomers. In addition, NURR1 and NGFI-B can efficiently promote vitamin A signaling when heterodimerized with RXR. The biological significance of these activities and the understanding of the in vivo functions has been limited. It has been difficult to analyze nuclear receptor activity in vivo, mainly because nuclear receptors bind small lipophilic and diffusible molecules, and are subsequently involved in endo-para- or autocrine nuclear receptor signaling. Due to their lack of known ligands, the functions of orphan receptors have been even more difficult to explore. The present study aimed at understanding the following aspects of nuclear receptor signaling, focusing on the developing CNS: - To provide a stringent in vivo methodology whereby nuclear receptor activation can be assessed in situ. - To analyze retinoid signaling and specifically to examine the role of RXR in vivo. - To investigate the functions of the orphan receptors NURR1, NOR I and NGFI-B. An effector-reporter assay was established in transgenic mice, where a reporter (bacterial lacZ or green fluorescent protein) gene is induced by activated nuclear receptor effector protein. By this system RAR and RXR activation was analyzed. Both RAR and RXR showed similar but temporally distinct activation patterns in vivo, demonstrating that RXR is a bona fide retinoid receptor. The expression patterns of the reporter transgenes indicated that retinoid signaling is involved in the differentiation of limb-innervating motor neurons in the embryonic spinal cord. In a subsequent experiment, the potential of a feedback inducible version of the effector-reporter assay was examined. Analysis of RAR activation revealed established as well as potentially novel regions of retinoid signaling in embryonic tissues. Analysis of NURR1, NOR1 and NGFI-B demonstrated both distinct and common properties. In contrast to NURR1 and NGFI-B, NOR I fails to heterodimerize with RXR and to mediate retinoid signaling. Furthermore, Nurr1 and Nor1 are expressed in the prenatal CNS, in both unique and ovelapping patterns, whereas NGFI-B is mainly detected postnatally. In addition, NURR1 was found to be a very early expression marker in the developing midbrain. The biological importance of this was investigated in a gene targeting study. Nurr1 null mutant mice die during the first day of life and display an absence of midbrain dopaminergic neurons, thus establishing NURR1 as a critical factor in dopamine cell differentiation.
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