Isolation and characterization of a candidate gene for autosomal-dominant polycystic kidney disease.
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Animals↗
Biomedical subjects
M Schalling
Publications and source records attributed to M Schalling.
Insulin-like growth factor I shifts from promoting cell division to potentiating maturation during neuronal differentiation.
SH-SY5Y neuroblastoma cells undergo neuronal differentiation and their proliferation is inhibited when they are treated with phorbol 12-myristate 13-acetate (PMA). Insulin and insulin-like growth factor I (IGF-I) are mitogens for the nontreated SH-SY5Y cells, whereas the proliferative response to such factor stimulation is lost upon differentiation, in spite of the fact that the receptors for insulin and IGF-I remain expressed and functional in the differentiated cells. Here we show that the PMA-induced differentiation of SH-SY5Y cells grown in a serum-free medium is strongly potentiated by nanomolar concentrations of IGF-I, as judged by morphology and markers for neuronal differentiation--e.g., neuropeptide tyrosine and growth-associated protein 43. Also, insulin and IGF-II potentiated the phorbol ester-induced differentiation, although less efficiently than IGF-I. Using blocking anti-receptor antibodies, it could be shown that the differentiation induced by these factors, in combination with PMA, was primarily mediated through the IGF-I receptor.
Blotting, Northern↗
Analysis of a murine male germ cell-specific transcript that encodes a putative zinc finger protein.
A cDNA species, corresponding to a gene with testis-specific expression (TSGA), was isolated from a testis cDNA library. The temporal and spatial expression of TSGA was studied by in situ hybridization as well as RNA filter hybridization. In tissue sections, the TSGA sequence was confined to cells within the seminiferous tubules. For filter hybridization, RNA was isolated from testis of prepubertal rats of different ages as well as from enriched populations of various germ cell types. It was found that TSGA is expressed only in male germ cells and that the steady-state level of TSGA transcripts reaches a maximum during the meiotic and the postmeiotic stages of germ cell development, suggesting a meiotic or postmeiotic function for the encoded protein. TSGA encodes a putative protein having 1,214 amino acids and contains a zinc finger, a structure that previously has been shown to mediate binding to nucleic acids.
Amino Acid Sequence↗
Calcitonin gene-related peptide and its mRNA in pulmonary neuroendocrine cells and ganglia.
The occurrence of calcitonin gene-related peptide (CGRP) and it's mRNA was studied in lungs of rats and piglets using in situ hybridization with two synthetic oligonucleotide probes followed by immunocytochemistry (ICC). CGRP mRNA was present in pulmonary neuroendocrine cells (PNEC) of both the solitary type and cluster type (neuroepithelial body; NEB) at all levels of the airway epithelium from bronchi to alveoli. The distribution of labelled cells was similar to that previously described with ICC. The 44-mer probe provided stronger hybridization signal than the 34-mer and the two combined increased labelling slightly. Formalin fixation reduced labelling and tended to increase background. Labelling for CGRP mRNA was evenly distributed over the cytoplasm, whereas CGRP-like immunoreactivity (LI) usually was of highest intensity toward the base of the PNEC, suggesting basal accumulation of synthesized peptide. CGRP-LI was also observed in occasional rat ganglia and in some, but not all, piglet ganglia. These local neurons may contribute to the CGRP fibers of airways and vasculature, and could theoretically bridge their dendrites and axons between NEB and the effector organ (e.g. artery or arteriole) thus accomplishing a function similar to the postulated axon reflex.
Animals↗
Distribution patterns of CCK and CCK mRNA in some neuronal and non-neuronal tissues.
Mutt and Jorpes (49) originally isolated cholecystokinin (CCK) from porcine intestine. Subsequently, it was recognized that CCK/gastrin-like material could be found in the rat brain (74), and it was later shown mainly to represent the C-terminal octapeptide (CCK-8) (2, 12-14, 48, 54, 55). These radioimmunoassay studies have been supplemented by numerous immunohistochemical investigations showing extensive CCK immunoreactive neuron systems in the brain and spinal cord (20, 26, 27, 31, 37, 40, 42, 44, 72, 75, 76). During recent years several groups have employed in situ hybridisation and radioactively labelled probes complementary to CCK mRNA and partly confirmed results from immunohistochemical studies but also revealed new interesting findings (3, 5, 6, 30, 41, 58, 64-66, 77). Several lines of evidence indicate that CCK-8 may act as a neurotransmitter or neuromodulator in many areas of the central nervous system. The development of new CCK antagonists has opened up new possibilities to understand the functional significance of CCK peptides in the neurons and other systems. The aim of the present article is to briefly review the distribution of some of the CCK systems and in this way define possible targets for these new types of drugs. Focus will be on cerebral cortex in view of the theme of the meeting, anxiety, on spinal cord as a basis for discussion of CCK and pain, and finally CCK/gastrin peptides in sperm will be discussed. The question of coexistence of CCK and dopamine in mesencephalic neurons is reviewed in a parallel article (28).
Animals↗
Neuropeptide Y and catecholamine synthesizing enzymes and their mRNAs in rat sympathetic neurons and adrenal glands: studies on expression, synthesis and axonal transport after pharmacological and experimental manipulations using hybridization techniques and radioimmunoassay.
The effects of reserpine treatment (10 mg/kg, i.p.) on the content of neuropeptide Y-like immunoreactivity and catecholamines were compared with the levels of mRNA coding for neuropeptide Y, tyrosine hydroxylase and phenylethanolamine N-methyltransferase in rat sympathetic neurons and adrenal gland. A reversible depletion of neuropeptide Y-like immunoreactivity was observed in the right atrium of the heart, kidney and masseter muscle, while the immunoreactive neuropeptide Y content in the stellate and lumbar sympathetic ganglia and its axonal transport in the sciatic nerve increased following reserpine. The increase in the stellate ganglion was maximal at 48 h and absent 9 days after reserpine treatment. The expression of neuropeptide Y mRNA and tyrosine hydroxylase mRNA in both the stellate and the superior cervical ganglion increased earlier than the neuropeptide Y content, with a clear cut two-fold elevation at 24 h after reserpine. The increase in both mRNAs in the superior cervical ganglion and the depletion of neuropeptide Y, but not of noradrenaline, in terminal areas was prevented after pretreatment both with a nicotinic receptor antagonist (chlorisondamine) and with surgical preganglionic denervation. A marked (75-90%) depletion of neuropeptide Y-like immunoreactivity and adrenaline in the adrenal gland, concomitant with 3-4-fold increases in neuropeptide Y mRNA and tyrosine hydroxylase mRNA expression, was present at 24 h after reserpine treatment. Also in the adrenal gland, there was a reversal of the reserpine-induced increase in neuropeptide Y mRNA and tyrosine hydroxylase mRNA and depletion of neuropeptide Y and adrenaline following splanchnic denervation. Pharmacological, ganglionic blockade prevented the depletion of neuropeptide Y and the increased expression of neuropeptide Y mRNA, but not fully, the tyrosine hydroxylase mRNA elevation. In addition, a marked decrease in phenylethanolamine N-methyltransferase mRNA levels was noted after reserpine. This decrease was reversed by denervation and by ganglionic blockade. Denervation alone led to a small but significant decrease in all mRNAs examined both in the superior cervical ganglion and the adrenal medulla. The present data suggest that the depletion of neuropeptide Y-like immunoreactivity in sympathetic nerves and in the adrenal gland after reserpine is associated with a compensatory increase in neuropeptide Y synthesis and axonal transport, most likely due to increased nicotinic receptor stimulation. Whereas the reserpine depletion of neuropeptide Y in both sympathetic nerves and adrenal gland is related to neuronal activation, adrenal but not nerve terminal depletion of catecholamines can be prevented by the ganglionic blocker chlorisondamine.4+e difference in effect of pharmacological ganglionic
Adrenal Glands↗
A gene encoding a fibroblast growth factor receptor isolated from the Huntington disease gene region of human chromosome 4.
The gene responsible for Huntington disease (HD), an autosomal dominant neurodegenerative disorder, is located near the terminus of the short arm of chromosome 4. Detailed genetic linkage and physical mapping studies have defined a region of approximately 2.5 million basepairs where the disease gene is likely to be located. Efforts to identify the disease gene are now focused on the identification and characterization of expressed genes in this region. Nucleotide sequence analysis of a cDNA clone derived from the HD gene region has revealed that it encodes a member of the fibroblast growth factor subfamily of tyrosine kinase receptors, some members of which are known to be involved in the differentiation and survival of certain cell types within the central nervous system. Histochemical analysis using in situ hybridization revealed its expression in many areas of the brain, among them being the caudate and putamen. The nature of this gene, FGFR3, and its map location make it a possible candidate for the HD gene.
Amino Acid Sequence↗
Colocalization of neurotransmitters analyzed by in situ hybridization.
In situ hybridization and Northern blot analysis has been used to analyse in some detail the localization and regulation of the messenger molecules adrenaline, noradrenaline and neuropeptide tyrosine (NPY) within cells of the sympathetic nervous system and the adrenal medulla. In the rat adrenal gland, a novel NPY containing population of ganglion cells was found. Synthetic oligonucleotide probes complementary to mRNA coding for the catecholamine synthesizing enzymes phenylethanolamine N-methyltransferase (PNMT), tyrosine hydroxylase (TH) and NPY were used to analyse the regulation of these genes following administration of the catecholamine depleting drug reserpine. Twenty-four hours after a single dose of reserpine, a differential regulation of PNMT, TH and NPY was found. Thus, a dramatic decrease in PNMT mRNA was observed in the adrenal medulla. In contrast, mRNA for both TH and NPY exhibited an increase. Different regulatory mechanisms may thus operate for these three compounds coexisting in chromaffin cells of the adrenal medulla. The regulation of enzymes and peptides was also studied in human sympathetic ganglia. After brief electrical preganglionic stimulation of thoracic ganglia in humans, in situ hybridization was performed with synthetic oligonucleotide probes complementary to TH, dopamine beta-hydroxylase (DBH) and NPY mRNA respectively. A several fold increase in all three mRNAs was found in the principal ganglion cells. The results point to a very rapid regulation of genes involved in signal transmission in the sympathetic nervous system of humans. The results also suggest a novel way to define neuronal projections by visualizing increases in mRNA levels following electrical stimulation.
Adrenal Glands↗
Expression of the Wilms' tumor gene WT1 in the murine urogenital system.
The Wilms' tumor gene WT1 is a recessive oncogene that encodes a putative transcription factor implicated in nephrogenesis during kidney development. In this report we analyze expression of WT1 in the murine urogenital system. WT1 is expressed in non-germ-cell components of the testis and ovaries in both young and adult mice. In situ mRNA hybridization studies demonstrate that WT1 is expressed in the granulosa and epithelial cells of ovaries, the Sertoli cells of the testis, and in the uterine wall. In addition to the 3.1-kb WT1 transcript detected by Northern blotting of RNA from kidney, uterus, and gonads, there is an approximately 2.5-kb WT1-related mRNA species in testis. The levels of WT1 mRNA in the gonads are among the highest observed, surpassing amounts detected in the embryonic kidney. During development, these levels are differentially regulated, depending on the sexual differentiation of the gonad. Expression of WT1 mRNA in the female reproductive system does not fluctuate significantly from days 4 to 40 postpartum. In contrast, WT1 mRNA levels in the tesis increase steadily after birth, reaching their highest expression levels at day 8 postpartum and decreasing slightly as the animal matures. Expression of WT1 in the gonads is detectable as early as 12.5 days postcoitum (p.c.). As an initial step toward exploring the tissue-specific expression of WT1, DNA elements upstream of WT1 were cloned and sequenced. Three putative transcription initiation sites, utilized in testis, ovaries, and uterus, were mapped by S1 nuclease protection assays. The sequences surrounding these sites have a high G + C content, and typical upstream CCAAT and TATAA boxes are not present. These studies allowed us to identify the translation initiation site for WT1 protein synthesis. We have also used an epitope-tagging protocol to demonstrate that WT1 is a nuclear protein, consistent with its role as a transcription factor. Our results demonstrate regulation of WT1 expression during development of the gonads, implicate WT1 in genitourinary development, and provide a molecular framework toward understanding genitourinary defects observed among hereditary cases of Wilms' tumor.
Amino Acid Sequence↗
Substance P is present in cholinergic paravertebral sympathetic neurons innervating exocrine sweat glands.
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Animals↗
Changes in levels of mRNA coding for catecholamine synthesizing enzymes and neuropeptide Y in the adrenal medulla of the newborn rat.
We have measured levels of mRNA coding for the catecholamine synthesizing enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (D beta H), phenylethanolamine N-methyltransferase (PNMT) and for neuropeptide Y (NPY) in rat adrenal medulla by using in situ hybridization histochemistry. Ages of one day before birth (E21), 12 h, 24 h, 2 days and 4 days after birth and in adults were studied. TH, D beta H and NPY mRNA levels increased markedly postnatally. Twelve hours after birth the levels of mRNA for TH, D beta H and NPY were, respectively, 512 +/- 18%, 370 +/- 24% and 253 +/- 21% of E21 levels. At 24 h of age NPY mRNA level was 437 +/- 73% of fetal value. In contrast, the levels of mRNA coding for PNMT increased more slowly and reached 196 +/- 9% of E21 level on postnatal day four and was further increased in adult rats.
Adrenal Medulla↗
Localization of neuropeptide receptor mRNA in rat brain: initial observations using probes for neurotensin and substance P receptors.
The expression of receptors for neurotensin and substance P was examined in rat brain and spinal cord using in situ hybridization with synthetic oligonucleotide probes. Strong hybridization signals for neurotensin receptor mRNA were observed over neurons i.a. in the diagonal band, medial septal nucleus, nucleus basalis magnocellularis, suprachiasmatic nucleus, supramammillary area, substantia nigra and ventral tegmental area. Strong hybridization signals for substance P receptor mRNA were observed over scattered, large neurons in the striatum, and in the spinal cord over neurons in the dorsal horn, the area around the central canal and preganglionic autonomic neurons. Thus, discrete neurons in several brain regions express a G-protein-coupled receptor with which endogenous neurotensin and substance P may interact.
Animals↗
Evidence for coexistence between calcitonin gene-related peptide and serotonin in the bulbospinal pathway in the monkey.
By the use of the indirect immunofluorescence and in situ hybridization techniques, the distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity (LI) and CGRP mRNA was studied in the spinal cord as well as in the midline raphe nuclei and the hypoglossal nucleus in the medulla oblongata of the monkey (Macaca fascicularis). In the spinal cord only a few large neurons in the motor nucleus contained CGRP-LI, while a majority of the neurons in the hypoglossal nucleus contained CGRP-LI. A relatively dense innervation by CGRP-immunoreactive (IR) fibers was also seen close to cell bodies and proximal dendrites of large neurons in the motor nucleus, especially in its ventral part. 5-Hydroxytryptamine (5-HT)-, substance P- and thyrotropin-releasing hormone (TRH)-IR varicosities were also observed in a similar position around large neurons in the motor nucleus. Double labeling disclosed that the majority of CGRP-IR axon terminals also contained 5-HT-LI. Expression of CGRP mRNA was found in neurons in the medullary midline raphe nuclei and in large neurons in the motor nucleus at the cervical spinal cord level. In adjacent sections of the medulla oblongata, CGRP-labeled neurons in the midline raphe nuclei also expressed preprotachykinin mRNA. The present results show that CGRP- and 5-HT-LI coexist in fibers within the motor nucleus of the monkey spinal cord and that this coexistence is probably due to the presence of CGRP in the descending bulbospinal, serotonergic pathway.
Animals↗
Comparison of gene expression of the dopamine D-2 receptor and DARPP-32 in rat brain, pituitary and adrenal gland.
In situ hybridization histochemistry was used to compare the distribution of dopamine D-2 receptor mRNA and DARPP-32 mRNA in rat brain, pituitary and adrenal gland. In many regions such as the caudate nucleus, nucleus accumbens and olfactory tubercle, there was an excellent correlation with dopamine receptor binding studies, whereas in some discrete brain regions mismatches were observed.
Adenylyl Cyclases↗
Differential effects of intracerebroventricular colchicine administration on the expression of mRNAs for neuropeptides and neurotransmitter enzymes, with special emphasis on galanin: an in situ hybridization study.
The axonal transport blocker colchicine has been extensively used in immunohistochemical studies to induce accumulation of neuroactive compounds, especially neuropeptides, in neuronal somata and thus improve their visualization. To assess whether colchicine might, in addition, influence the synthesis of such compounds, we have now used in situ hybridization to examine the levels of mRNAs encoding for several neuropeptides (galanin [GAL], cholecystokinin [CCK], somatostatin [SOM], neuropeptide Y [NPY]) and neurotransmitter-synthesizing enzymes (choline acetyltransferase [ChAT], tyrosine hydroxylase [TH], amino acid decarboxylase [AADC], and glutamic acid decarboxylase [GAD]) after intraventricular administration of the drug. The results show that colchicine differentially modifies the levels of several mRNA species in different brain areas. Thus GAL mRNA levels increase in virtually all regions examined, including the basal forebrain, hypothalamus, dorsal raphe nucleus, locus coeruleus, and nucleus tractus solitarii. In addition, after colchicine treatment, GAL mRNA appears to be induced in the ipsilateral hemisphere in regions such as the cortex, hippocampus, striatum, lateral septum, and some nuclei of the thalamus as well as within white matter, where it cannot be detected in control animals. Although GAL mRNA in the vast majority of cases is neuronal, some findings indicate a possible glial localization. In parallel, colchicine depletes ChAT mRNA and increases GAD mRNA in the basal forebrain and striatum and decreases AADC mRNA in the dorsal raphe nucleus and locus coeruleus. In the latter nucleus, NPY and TH mRNA levels are increased by colchicine. In contrast, TH mRNA and also CCK mRNA levels decrease in the substantia nigra. In the cortex, hippocampus, and thalamus ipsilateral to colchicine injection CCK mRNA levels are markedly decreased, whereas SOM mRNA is decreased and NPY mRNA increased in the hippocampus but unchanged in the cortex. The results are discussed with reference to the possible artifacts that the use of colchicine might induce in immunohistochemical mapping studies and in relation to possible neurotoxic actions of colchicine, in some cases perhaps related to impaired retrograde transport of growth factor(s).
Animals↗
Peptides and transmitter enzymes in hypothalamic magnocellular neurons after administration of hyperosmotic stimuli: comparison between messenger RNA and peptide/protein levels.
In situ hybridization histochemistry and indirect immunofluorescence histochemistry were used to study changes in the expression of vasopressin (VP), oxytocin (OXY), tyrosine hydroxylase (TH), galanin (GAL), dynorphin (DYN) and cholecystokinin (CCK) in hypothalamic magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nuclei of rats. After prolonged administration of 2% sodium chloride as drinking water (salt-loading), the treatment increased the levels of VP, OXY, TH, GAL, DYN and CCK mRNA in the PVN and SON. The increase in CCK mRNA was, however, proportionally higher in the PVN than in the SON. Within cell bodies of the PVN and SON of salt-loaded rats, a depletion of VP- and OXY-like immunoreactivity (LI) and an increase in TH-LI were seen. In salt-loaded/colchicine-treated rats, a marked decrease in GAL- and DYN-LI, but no specific changes in CCK-LI were observed. Within nerve fibers of the posterior pituitary of salt-loaded rats, a marked depletion of VP-, GAL- and DYN-LI was found. Less pronounced depletion was observed in OXY- and CCK-LI, and no specific changes in TH-LI were seen. The results show that high plasma osmolality induces increased mRNA levels for VP, OXY, TH, GAL, DYN and CCK, presumably indicating increased synthesis, an increased export from cell somata of VP, OXY, GAL and DYN, and a decrease in levels of these peptides in the posterior pituitary, suggesting increased release. The catecholamine-synthesizing enzyme TH, however, which has a cytoplasmic localization and is not released from nerve endings, remains high in the cell bodies and nerve endings during this state of increased activity.
Administration, Oral↗
Distribution and cellular localization of DARPP-32 mRNA in rat brain.
In situ hybridization histochemistry has been used to determine the regional distribution and cellular localization of DARPP-32 mRNA in the rat brain. Results support the concept that DARPP-32 is present primarily in cells expressing the dopamine D1 subtype receptor, and that DARPP-32 is not synthesized in dopamine-containing cells. Strongly labelled neuronal cell bodies were found in the caudate nucleus, nucleus accumbens, olfactory tubercle, parts of the bed nucleus of the stria terminalis, and the amygdaloid complex. In addition large amounts of DARPP-32 mRNA were visualized in the medial habenula and around the third ventricle, in ependymal cells and tanycytes, and in the cerebellar Purkinje cells. A less pronounced activity was seen in layers II-III and VI throughout the cerebral cortex. The present studies together with previous biochemical and immunocytochemical studies demonstrate that DARPP-32 gene expression is greatest primarily in D1 dopaminoceptive cells, although there are exceptions. In situ hybridization may thus be used to quantitate regulation of DARPP-32 mRNA in discrete brain regions.
Animals↗
Neuropeptide gene expression in hypothalamic magnocellular neurons of normal and hypophysectomized rats: a combined immunohistochemical and in situ hybridization study.
Hypothalamic magnocellular neurons of the paraventricular and supraoptic nuclei contain several peptides and non-peptide putative neurotransmitters co-existing with vasopressin and oxytocin. However, the functional role of these substances is still unknown. In the present paper the temporal course of changes in the expression of vasopressin, oxytocin, galanin, cholecystokinin, dynorphin and tyrosine hydroxylase in magnocellular hypothalamic neurons of rats subjected to hypophysectomy was examined. Following different survival times the animals were processed either for immunohistochemistry with antibodies against the above mentioned peptides or for in situ hybridization with synthetic oligonucleotide probes complementary to the mRNAs encoding for the peptides. The results obtained showed a marked rise in vasopressin mRNA levels at two days followed by a decrease up to 36 days of survival. Oxytocin mRNA responded to the lesion with a transient decrease, with its lowest values between five and seven days. This was followed by a recovery which almost reached normal values at 36 days of survival. The results also showed a marked, transient activation of the synthetic pathway for galanin and cholecystokinin. The numbers of cells expressing these peptides were maximal between five and seven days, and the respective mRNA levels were significantly increased at these survival times. This was followed by a decrease in the amount of galanin- and cholecystokinin-like immunoreactivity as well as in the levels of their respective mRNAs. Dynorphin-like immunoreactivity showed a course similar to that of galanin and cholecystokinin in operated animals. However, the amounts of dynorphin mRNA were significantly increased at two days, but were followed by a reduction at five days and remained low throughout the different survival times tested. The experiments performed with the tyrosine hydroxylase antibodies and probe showed undetectable levels of the enzyme and its mRNA in normal and hypophysectomized animals. These results demonstrate that, in magnocellular hypothalamic neurons, expression of several peptides occur in differential ways after hypophysectomy. The possibility is discussed that these changes represent part of the mechanisms underlying the process of degeneration and regeneration known to occur in magnocellular hypothalamic neurons after hypophysectomy.
Animals↗