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The GRIK5 (glutamate receptor ionotropic kainate-5) gene encodes the kainate-preferring glutamate receptor subunit KA2. The GRIK5 promoter is TATA-less and GC-rich, with multiple consensus initiator sequences. Transgenic mouse lines carrying 4 kilobases of the GRIK5 5'-flanking sequence showed lacZ reporter expression predominantly in the nervous system. Reporter assays in central glial (CG-4) and non-neural cells indicated that a 1200-base pair (bp) 5'-flanking region could sustain neural cell-specific promoter activity. Transcriptional activity was associated with the formation of a transcription factor IID-containing complex on an initiator sequence located 1100 bp upstream of the first intron. In transfection studies, deletion of exonic sequences downstream of the promoter resulted in reporter gene activity that was no longer neural cell-specific. When placed downstream of the GRIK5 promoter, a 77-bp sequence from the deleted fragment completely silenced reporter expression in NIH3T3 fibroblasts while attenuating activity in CG-4 cells. Analysis of the 77-bp sequence revealed a functional SP1-binding site and a sequence resembling a neuron-restrictive silencer element. The latter sequence, however, did not display cell-specific binding of REST-like proteins. Our studies thus provide evidence for intragenic control of GRIK5 promoter activity and suggest that elements contributing to tissue-specific expression are contained within the first exon.

The avian retina and pineal gland contain autonomous circadian oscillators and photo-entrainment pathways, but the photopigment(s) that mediate entrainment have not been definitively identified. Melanopsin (Opn4) is a novel opsin involved in entrainment of circadian rhythms in mammals. Here, we report the cDNA cloning of chicken melanopsin and show its expression in retina, brain and pineal gland. Like the melanopsins identified in amphibians and mammals, chicken melanopsin is more similar to the invertebrate retinaldehyde-based photopigments than the retinaldehyde-based photopigments typically found in vertebrates. In retina, melanopsin mRNA is expressed in cells of all retinal layers. In pineal gland, expression was strong throughout the parenchyma of the gland. In brain, expression was observed in a few discrete nuclei, including the lateral septal area and medial preoptic nucleus. The retina and pineal gland showed distinct diurnal expression patterns. In pineal gland, melanopsin mRNA levels were highest at night at Zeitgeber time (ZT) 16. In contrast, transcript levels in the whole retina reached their highest levels in the early morning (ZT 0-4). Further analysis of melanopsin mRNA expression in retinal layers isolated by laser capture microdissection revealed different patterns in different layers. There was diurnal expression in all retinal layers except the ganglion cell layer, where heavy expression was localized to a small number of cells. Expression of melanopsin mRNA peaked during the daytime in the retinal pigment epithelium and inner nuclear layer but, like in the pineal, at night in the photoreceptors. Localization and regulation of melanopsin mRNA in the retina and pineal gland is consistent with the hypothesis that this novel photopigment plays a role in photic regulation of circadian function in these tissues.

We have identified an opsin, melanopsin, in photosensitive dermal melanophores of Xenopus laevis . Its deduced amino acid sequence shares greatest homology with cephalopod opsins. The predicted secondary structure of melanopsin indicates the presence of a long cytoplasmic tail with multiple putative phosphorylation sites, suggesting that this opsin’s function may be finely regulated. Melanopsin mRNA is expressed in hypothalamic sites thought to contain deep brain photoreceptors and in the iris, a structure known to be directly photosensitive in amphibians. Melanopsin message is also localized in retinal cells residing in the outermost lamina of the inner nuclear layer where horizontal cells are typically found. Its expression in retinal and nonretinal tissues suggests a role in vision and nonvisual photoreceptive tasks, such as photic control of skin pigmentation, pupillary aperture, and circadian and photoperiodic physiology.

A full-length cDNA was isolated for a thyroid hormone response gene in the metamorphosing frog intestine and shown by sequence analysis to be the frog homolog of the mammalian extracellular matrix metalloproteinase stromelysin-3 ( ST3 ). Northern hybridization indicated that ST3 gene expression is differentially activated in tadpole tissues during metamorphosis. In the small intestine, in situ hybridization localized high levels of ST3 mRNA to fibroblast-like cells during thyroid hormone-induced metamorphosis. ST3 mRNA was undetectable in the intestine prior to metamorphosis, while high levels were present at the metamorphic climax. At this time, primary intestinal epithelial cells are known to undergo cell death and replacement by secondary epithelial cells, arguing that ST3 is involved in the modification of the extracellular matrix during apoptosis. ST3 mRNA was also expressed at high levels during tadpole tail resorption, but not in premetamorphic tail or developing hindlimb, further supporting a role for ST3 when tissue remodeling is accompanied by large-scale cell death. Premetamorphic tadpoles treated with thyroid hormone showed a similar but compressed time course of ST3 gene regulation, suggesting that thyroid hormone controls ST3 gene expression during metamorphosis. In contrast, during embryogenesis, ST3 was expressed before endogenous thyroid hormone is detectable, indicating that ST3 can also be regulated independently of thyroid hormone. These findings implicate that ST3 participates in the modification of the extracellular matrix during metamorphic apoptosis, but Northern analyses using heterologous probes raise the possibility that additional matrix metalloproteinases may also be involved.

Myogenin, as well as other MyoD-related skeletal muscle-specific transcription factors, regulate a large number of skeletal muscle genes during myogenic differentiation. During later development, innervation suppresses myogenin expression in the fetal hind limb musculature. Denervation of skeletal muscle reverses the effects of the nerve, and results in the reactivation of myogenin expression, as well as of other embryonic muscle proteins. Here we report that myogenin upstream sequences confer tissue- and developmental-specific expression in transgenic mice harboring a myogenin/chloramphenicol acetyltransferase (CAT) reporter construct. Using in situ hybridization to analyze serial sections of E12.5 embryos, we found colocalization of CAT and endogenous myogenin transcripts in the primordial muscle of the head and limbs, in the intercostal muscle masses, and in the most caudal somites. Later in development, we observed that the expression of the transgene and endogenous myogenin gene continued to be restricted to skeletal muscle but decreased shortly after birth; a period that coincides with the innervation of secondary myotubes. Furthermore, denervation of the mouse hind limbs induced a 10-fold accumulation of CAT and endogenous myogenin transcripts by 1 day after sciatic nerve resection; a 25-fold increase was observed by 4 days after denervation. Interestingly, we observed that the accumulation of CAT enzyme activity lagged considerably with respect to the increase in CAT transcripts. Our results indicate that the cis-acting elements that temporally and spatially confine transcription of the gene during embryonic development, and that mediate the responses to innervation and denervation of muscle, lie within the upstream sequences analyzed in these studies.