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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.

To investigate LIM gene function in the rat cerebellar system, we analyzed expression and regulation of the rat homologue of frog Xlim-1 (rlim-1) in vivo and in cultured cells. In developing cerebellum, peak levels of rlim-1 mRNA at postnatal day 8 (p8) are coincident with the peak period of granule cell proliferation. Analysis of rlim-1 protein with a specific antibody showed that expression was also maximal at p8. In situ hybridization showed that at p8 rlim-1 mRNA was expressed in Purkinje and granule cells. Both the proliferative and the premigratory granule cells in the external germinal zone displayed high levels of rlim-1 mRNA expression. Immunocytochemical staining demonstrated that at p8 rlim-1 protein was also present in proliferative and premigratory granule cells. In adult cerebellum (p30), rlim-1 mRNA and protein expression in granule cells was strongly attenuated. The down-regulation of rlim-1 mRNA occurred in granule cells just after the time of final division, coinciding with the onset of their migration. rlim-1 protein was detected in migratory granule neurons. The developmental decrease in rlim-1 mRNA and protein found in vivo was reproduced in pure cerebellar granule cell cultures. In these cultures, granule neurons were postmitotic 1 day after plating but still displayed high levels of rlim-1 protein expression up to 3 days in vitro. Our findings indicate that 1) rlim-1 is likely to act in concert with other genes to specify granule cell fate, 2) rlim-1 expression in granule neurons is regulated autonomously, and 3) rlim-1 protein may also play an important role in granule neuron differentiation and survival. Published 2001 Wiley-Liss, Inc.

The prohormone convertase (PC2) is expressed in the mammalian central nervous system (CNS) and has been shown to play an important role in the processing of certain neuropeptide precursors and prohormones at paired basic residues. Amphibian PC2 cDNA was recently cloned for the frog Xenopus laevis, and both its sequence and its pituitary expression pattern were shown to be very similar to those of mammalian PC2. To investigate further the function of PC2 in the vertebrate CNS, we used in situ hybridization histochemistry to localize the distribution of cells expressing PC2 mRNA in the frog brain and the spinal cord. The distribution of PC2-expressing cells was also compared with that of cells expressing thyrotropin-releasing hormone (TRH) mRNA or peptide. PC2-expressing cells were detected in specific nuclei that were widely distributed in the frog CNS. In forebrain, telencephalic PC2 mRNA was found in the olfactory bulb, pallium, striatum, amygdala, and septum, and diencephalic PC2 mRNA was seen in the preoptic area, thalamus, and hypothalamus. More posteriorly, PC2 cells were localized to midbrain tegmentum, the torus semicircularis, and the optic tectum, as well as the cerebellum, brainstem, and spinal cord. Despite this wide distribution, steady-state levels of PC2 mRNA were clearly different in various brain nuclei. Regions with higher levels showed good correspondence to areas shown by others in frog to contain large numbers of neuropeptideexpressing cells, including TRH cells. On the other hand, not all brain areas with high levels of TRH mRNA had high levels of PC2 mRNA. Localization studies combining in situ hybridization and immunocytochemistry showed that, at least in optic tectum and brainstem, PC2 mRNA and pro-TRH peptide coexist. These findings suggest that pro-TRH is processed by PC2 in some, but possibly not all, brain regions. Thus, different converting enzymes may be involved in pro-TRH processing in different brain regions. © 1995 Wiley-Liss, Inc.

How neurons diversify in developing brain to produce discrete cell fates in their appropriate regions remains a fundamental question. Embryonic Xenopus was previously used to identify juxtaposed embryonic cells that first express proopiomelanocortin mRNA in forebrain and pituitary, supporting the idea that this neuropeptide phenotype is induced locally. (Hayes and Loh, 1990, Development 110:747–757). To begin to examine how a more widespread population of forebrain cells is set up, the present focus is on the thyrotropin-releasing hormone (TRH) phenotype. Serial section in situ hybridization histochemistry produced the unexpected finding that the adult-like TRH system spanning forebrain and comprising over six different telencephalic and diencephalic nuclei, is preceded by an embryonic TRH cell population that is initially localized and then highly regionalized in the area from which the adult pattern develops. Thus, the first TRH cells, detected in vivo after 35 h (stage 29/30), were confined to discrete anterior or posterior bilateral clusters in embryonic forebrain or hindbrain. Thereafter, the TRH cell clusters in diencephelon, but not hindbrain, expanded to form rows, extending anteriorly into telencephalon and bifurcating posteriorly around the infundibulum. By 80 h (stage 42), after extensive brain morphogenesis, these forebrain rows showed regional differences in levels of TRH and mRNA corresponding to the specific brain nuclei that have been shown to contain TRH cells in adult. These findings show that subsets of phenotype-specific forebrain cell first form a regionalized neuronal cell fate before distinct brain nuclei form. This is turn points to the testable hypothesis in Xenopus that certain neuronal cell fates in forebrain may be dictated by cell lineage or local induction. 1994 John Wiley & Sons, Inc.

To investigate, at the molecular level, the remodeling of small intestine during amphibian metamorphosis, a subtractive hybridization approach was used to identify genes that are differentially regulated by thyroid hormone. A frog cDNA was isolated from Xenopus laevis and determined to be the gene encoding the intestinal fatty acid-binding protein (IFABP) based on its high sequence hormology to the previously cloned mammalian IFABP gene. Northern blot analyses and in situ hybridization histochemistry also showed that, like the mammalian IFABP genes, frog IFABP gene expression is restricted to the intestinal epithelium. Xenopus embryos express detectable IFABP mRNA at stage 33/34, suggesting that intestinal epithelial cells differentiate well before feeding begins at stage 45. Moreover, during metamorphosis, levels of IFABP mRNA were gradually down-regulatod over a period of about 20 days between stages 54 and 62, reaching a minimum at metamorphic climax, after which they were reelevated as the secondary epithelium forms. This reduction in IFABP gene expression could be reproduced in only 3 days by treating premetamorphic tadpoles with thyroid hormone. Our findings also show that this effect, while likely to be indirect, takes place before overt morphological changes are evident in primary epithelial cells. Thus, the down-regulation of IFABP mRNA is one of the early molecular events preceding epithelial cell death during intestinal remodeling.

The Xenopus LIM class homeobox gene Xlim-3 was identified initially as a fragment isolated by polymerase chain reaction cloning with an embryonic cDNA library as template (Taira et al., 1992, Genes Dev. 6, 356-366). cDNA clones representing most of the Xlim-3 mRNA were isolated from an adult brain library. The predicted Xlim-3 protein contains two copies of the LIM domain, a homeodomain, and a C-terminal region rich in proline, glycine, and serine. RNA blot hybridization showed that Xlim-3 mRNA is detected in dorsal regions at neural tube and tailbud stages and in adults predominantly in the pituitary gland and weakly in the eye and brain. Whole mount in situ hybridization revealed that Xlim-3 mRNA is first detectable at the neural plate stage in the stomodeal-hypophyseal (pituitary) anlage and in the neural plate where labeled cells were found adjacent to the forming floor plate. In situ hybridization analysis on serial sections at later stages showed that embryonic Xlim-3 expression persists in the pituitary and pineal, as well as in some cells of the retina, hindbrain, and spinal cord. In the retina, Xlim-3 mRNA was only detected in a distinct sublamina of the inner nuclear layer, but not in dividing cells of ciliary margin. This discrete manner of Xlim-3 expression, especially persistent expression in the pituitary (before morphogenesis of the gland to adult), supports a role in the specification and maintenance of differentiation of distinct neuronal and neuroendocrine tissues.

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.

The number of optic synapses in the half tectum of goldfish was counted by using an improved HRP-labeling protocol and a columnar sampling method that spanned the entire optic innervation layer, S-SO-SFGS. It was previously found by using this procedure in intact tectum that the normal number of optic synapses was regenerated by 30 days and maintained thereafter even in the absence of impulse activity. This suggested that the number of synapses in this system was intrinsically fixed. In order to examine whether this limit was imposed by optic fibers or by target cells, optic synapses were counted in surgically halved tecta which received compressed optic projections consisting of regenerating optic fibers from the entire retina. We reasoned that if synapse number is a function of the number of afferents, then there should be twice the normal number of optic synapses per column; on the other hand, if their number is fixed by target, then their number per column should be normal. We found that the number of optic (labeled) synapses was normal in sample columns from fish at 70 days and 160 days after optic nerve crush. Thus, retinal ganglion cells, on average, formed half as many synapses on the half tectum compared to intact tectum, indicating the number of optic synapses was limited by the tectum. The number of nonoptic (unlabeled) synapses was also found to be normal. By contrast, the S-SO-SFGS was found to be 88–103% thicker compared to normal fish, apparently because of a 20-fold increase in the number of optic fibers. As a result, the density of synapses was about half normal in half tecta, and so, in contrast to synapse number, synaptic density is not constrained during regeneration. We infer from these data that optic fibers compete for limited numbers of postsynaptic sites during regeneration and suggest that this competition promotes neural map refinement and the various plasticities described for this projection.

The distribution of normal and regenerating retinal fibers and synapses was studied on tectum in goldfish by light (LM) and electron microscopy (EM). Since labeling of the early regenerating fibers was previously reported to be difficult, a new “cold-fill” HRP labeling protocol was developed, which labeled regenerating optic fibers and terminals on tectum as early as 14 days after nerve crush when they first arrive on tectum. In order to characterize the laminar distribution of optic afferents in normal fish and in fish regenerating for 14–240 days, EM photomontages of areas 14 μm wide by 160 μm deep through the HRP-labeled primary optic innervation layer (S-SO-SFGS) were constructed. The time points in regeneration that were examined spanned the period in which others have shown that an initially diffuse retinotopic map becomes spatially restricted. At the LM level regenerating optic fibers were restricted to the optic lamina. They reinnervated tectum in an anterior to posterior sequence as previously seen with autoradiography. In addition, at 14 days, some “pioneer” optic fascicles were found to have already grown to posterior tectum where they gave rise to branches with boutonlike terminations and growth-cone-like processes. From the ultrastructural analysis it was clear that optic fibers and terminals observed strict laminar boundaries as they partitioned themselves in the optic laminae (S, SO and SFGS) in both normal and regenerating fish. The behavior of optic fibers was lamina specific with respect to synapse formation and the orientation of fiber outgrowth. As early as 14 days regeneration, optic fibers made synapses onto the four types of postsynaptic profiles observed in normal fish. Numerous optic terminals were labeled at 14 days, and there appeared to be no waiting period between fiber ingrowth to the SO and synapse formation in the S and SFGS. At 14–60 days, atypical synaptic contacts which appear to be nascent synapses were made by labeled optic fibers in fascicles and by growth-cone-like processes. By 21–30 days, the density of optic terminals was high and there were many more fasciculated optic fibers in the SFGS than normal as late as 240 days. These findings suggest that optic fiber lamination is highly constrained by tectal cues, that fibers rapidly regenerate many synaptic terminals before retinotopic map refinement is complete, and that fibers have a strong affinity for each other.

Selected optic fibers were surgically deflected from one tectum onto the opposite host tectum which was denervated by eye enucleation. At 6–8 months, deflected fibers were labeled with horseradish peroxidase and the retinotopically inappropriate part of tectum was examined using electron microscopy. Numerous (labeled) optic synapses were found in the primary optic innervation layer of the ‘wrong’ part of tectum but they were about half the normal density. The number and density of non-optic synapses was not found to be affected. These findins indicate optic fibers compete with each other but not with non-optic fibers for synaptic sites in tectum.