Localization of retinol-binding protein messenger RNA in the rat kidney and in perinephric fat tissue

The cellular localization of retinol-binding protein (RBP) messenger RNA (mRNA) in the kidney, and the developmental pattern of the renal expression of the RBP gene, were studied in the Sprague-Dawley rat. In situ hybridization studies were conducted with single-stranded cRNA probes, using sections of adult and young rat kidneys. These studies revealed specific localization of RBP mRNA in the outer stripe of the medulla, specifically localized in the S3 segment of the proximal tubules. Northern blot analysis demonstrated that RBP mRNA was not detectable in the kidney before birth or during the first week postpartum, but was clearly detected by the end of the second week of age. No RBP mRNA was observed in the kidney by in situ hybridization at 12 days of age. At 26 days of age, however, RBP mRNA was clearly detected by the in situ hybridization technique, localized in the same anatomic region as that observed in the adult kidney. Transthyretin mRNA was not detected in the adult kidney. Previous studies have shown that immunoreactive RBP is localized in the convoluted segment of the proximal tubules of the rat kidney. The present results demonstrate that RBP mRNA in the kidney is localized in an anatomic region (the S3 segment of the proximal tubules) different from that of immunoreactive RBP. In addition, an intense RBP mRNA hybridization signal was detected in the perinephric fat tissue of 26- and 40-day-old and adult rats. Further analysis of RNA from epididymal fat showed a level of RBP mRNA approximately 20% of that of liver. The function of RBP synthesized in the kidney and adipose tissue remains to be determined. We have previously hypothesized that RBP synthesized in extrahepatic tissue may function in the recycling of retinol back to the liver or to other target tissues.     

Large-scale cellular-resolution gene profiling in human neocortex reveals species-specific molecular signatures

Although there have been major advances in elucidating the functional biology of the human brain, relatively little is known of its cellular and molecular organization. Here we report a large-scale characterization of the expression of ～1,000 genes important for neural functions by in?situ hybridization at?a cellular resolution in visual and temporal cortices of adult human brains. These data reveal diverse gene expression patterns and remarkable conservation of each individual gene's expression among individuals (95%), cortical areas (84%), and between human and mouse (79%). A small but substantial number of genes (21%) exhibited species-differential expression. Distinct molecular signatures, comprised of genes both common between species and unique to each, were identified for each major cortical cell type. The data suggest that gene expression profile changes may contribute to differential cortical function across species, and in particular, a shift from corticosubcortical to more predominant corticocortical communications in the human brain.     

Analysis of area-specific expression patterns of RORbeta, ER81 and Nurr1 mRNAs in rat neocortex by double in situ hybridization and cortical box method

Background

The mammalian neocortex is subdivided into many areas, each of which exhibits distinctive lamina architecture. To investigate such area differences in detail, we chose three genes for comparative analyses, namely, RORbeta, ER81 and Nurr1, mRNAs of which have been reported to be mainly expressed in layers 4, 5 and 6, respectively. To analyze their qualitative and quantitative coexpression profiles in the rat neocortex, we used double in situ hybridization (ISH) histochemistry and cortical box method which we previously developed to integrate the data of different staining and individuals in a standard three-dimensional space.

Principal Findings

Our new approach resulted in three main observations. First, the three genes showed unique area distribution patterns that are mostly complementary to one another. The patterns revealed by cortical box method matched well with the cytoarchitectonic areas defined by Nissl staining. Second, at single cell level, RORbeta and ER81 mRNAs were coexpressed in a subpopulation of layer 5 neurons, whereas Nurr1 and ER81 mRNAs were not colocalized. Third, principal component analysis showed that the order of hierarchical processing in the cortex correlates well with the expression profiles of these three genes. Based on this analysis, the dysgranular zone (DZ) in the somatosensory area was considered to exhibit a profile of a higher order area, which is consistent with previous proposal.

Conclusions/Significance

The tight relationship between the expression of the three layer specific genes and functional areas were revealed, demonstrating the usefulness of cortical box method in the study on the cerebral cortex. In particular, it allowed us to perform statistical evaluation and pattern matching, which would become important in interpreting the ever-increasing data of gene expression in the cortex.     

Expression pattern of the maternally imprinted gene Gtl2 in the forebrain during embryonic development and adulthood

Recent work has uncovered a large number of imprinted genes, many of which are thought to play a role in neurodevelopment and behavior. In order to begin to understand the role of specific genes in these processes, their expression patterns will be key. In this study we used in situ hybridization to study the developmental expression of Gtl2 in the forebrain from E12.5 to adulthood, since preliminary data from a microarray study indicated differential expression between the ventral and dorsal telencephalon of the mouse at a critical time point in the generation and migration of cortical neuronal populations. Strong expression was observed in the diencephalon, ventral telencephalon, post mitotic cell layers of the neocortex and pyramidal cell layer of the hippocampus. Additionally, heavily labeled subpopulations of laminar restricted cells were seen in the latter two areas.     

Area and layer patterning in the developing cerebral cortex

Two anatomical patterns characterize the neocortex, and both are essential for normal cortical function. First, neocortex is divided into anatomically distinct and functionally specialized areas that form a species-specific map. Second, neocortex is composed of layers that organize cortical connectivity. Recent studies of layer and area development have used time-lapse microscopy to follow cortical cell division and migration, gene arrays to find layer- or area- specific regulatory genes, time- and region- specific manipulations of candidate genes, and optical imaging to compare area maps in wild type with genetically altered mice. New observations clarify the molecular and cellular mechanisms that generate each pattern, and stress the links between layer and area formation.     

An in situ hybridization protocol for planarian embryos: monitoring myosin heavy chain gene expression

The monitoring of gene expression is fundamental for understanding developmental biology. Here we report a successful experimental protocol for in situ hybridization in both whole-mount and sectioned planarian embryos. Conventional in situ hybridization techniques in developmental biology are used on whole-mount preparations. However, given that the inherent lack of external morphological markers in planarian embryos hinders the proper interpretation of gene expression data in whole-mount preparations, here we used sectioned material. We discuss the advantages of sectioned versus whole-mount preparations, namely, better probe penetration, improved tissue preservation, and the possibility to interpret gene expression in relation to internal morphological markers such as the epidermis, the embryonic and definitive pharynges, and the gastrodermis. Optimal fixatives and embedding methods for sectioning are also discussed. A. Cardona and J. Fernández have contributed equally to this work.     

The Drosophila homolog of the immunoglobulin recombination signal-binding protein regulates peripheral nervous system development.

The J kappa RBP binds to the immunoglobulin recombination signal sequence flanking the kappa-type J segment. We previously isolated the highly conserved homolog of the J kappa RBP gene from D. melanogaster, which is not thought to have immunoglobulin molecules. Using many deficiency mutants and in situ hybridization, we mapped the Drosophila J kappa RBP gene in a region containing two recessive lethal mutations, i.e., br26 and br7, which shows the dominant Suppressor of Hairless (Su(H)) phenotype in heterozygotes. All six Su(H) alleles analyzed at the DNA level contained mutations in the Drosophila J kappa RBP gene. Since the Su(H) mutation affects peripheral nervous system development, the Drosophila J kappa RBP gene product is involved in gene regulation of peripheral nervous system development. The results also imply that the immunoglobulin recombination signal sequence and the target sequence of the Drosophila J kappa RBP protein might have a common evolutionary origin.     

Expression of alpha2A adrenoceptors during rat neocortical development

Norepinephrine has been suggested to play a neurotrophic role during development and is present in the brain as early as embryonic day (E) 12. We have recently demonstrated that the alpha2A adrenoceptor subtype is widely expressed during times of neuronal migration and differentiation throughout the developing brain. Here, we report the temporal and spatial expression pattern of alpha2A adrenoceptors in neocortex during late embryonic and early postnatal development using in situ hybridization and receptor autoradiography. Functional alpha2 receptors in embryonic rat cortex were also detected using agonist stimulated [35S]GTPgammaS autoradiography. Both alpha2A mRNA and protein expression were strongly increased by E19 and E20, respectively. The increased expression was in the cortical plate and intermediate and subventricular zones, corresponding to tiers of migrating and differentiating neurons. This transient up-regulation of alpha2A adrenoceptors was restricted to the lateral neocortex. At E20, functional alpha2 adrenoceptors were also detected in deep layers of lateral neocortex. During the first week of postnatal development, the expression of alpha2A mRNA and protein changed markedly, giving rise to a more mature pattern of anatomical distribution. The temporal and spatial distribution of alpha2A adrenoceptors in developing neocortex is consistent with expression of functional proteins on migrating and differentiating layer IV to II neurons. These findings suggest that alpha2A receptors may mediate a neurotrophic effect of norepinephrine during fetal cortical development. The early delineation of the lateral neocortex, which will develop into somatosensory and auditory cortices, suggests an intrinsic regulation of alpha2A mRNA expression.     

Expression of the neuregulin receptor ErbB4 in the brain of the rhesus monkey (Macaca mulatta)

We demonstrated recently that frontal cortical expression of the Neuregulin (NRG) receptor ErbB4 is restricted to interneurons in rodents, macaques, and humans. However, little is known about protein expression patterns in other areas of the brain. In situ hybridization studies have shown high ErbB4 mRNA levels in various subcortical areas, suggesting that ErbB4 is also expressed in cell types other than cortical interneurons. Here, using highly-specific monoclonal antibodies, we provide the first extensive report of ErbB4 protein expression throughout the cerebrum of primates. We show that ErbB4 immunoreactivity is high in association cortices, intermediate in sensory cortices, and relatively low in motor cortices. The overall immunoreactivity in the hippocampal formation is intermediate, but is high in a subset of interneurons. We detected the highest overall immunoreactivity in distinct locations of the ventral hypothalamus, medial habenula, intercalated nuclei of the amygdala and structures of the ventral forebrain, such as the islands of Calleja, olfactory tubercle and ventral pallidum, and medium expression in the reticular thalamic nucleus. While this pattern is generally consistent with ErbB4 mRNA expression data, further investigations are needed to identify the exact cellular and subcellular sources of mRNA and protein expression in these areas. In contrast to in situ hybridization in rodents, we detected only low levels of ErbB4-immunoreactivity in mesencephalic dopaminergic nuclei but a diffuse pattern of immunofluorescence that was medium in the dorsal striatum and high in the ventral forebrain, suggesting that most ErbB4 protein in dopaminergic neurons could be transported to axons. We conclude that the NRG-ErbB4 signaling pathway can potentially influence many functional systems throughout the brain of primates, and suggest that major sites of action are areas of the "corticolimbic" network. This interpretation is functionally consistent with the genetic association of NRG1 and ERBB4 with schizophrenia.     

Chromosomal localization of the retinol binding protein gene and its elimination as a candidate gene for the repeated epilation (Er) mutation in mice.

The repeated epilation (Er) mutation is an autosomal defect that blocks differentiation in stratified epithelia and appendages in mice. Plasma retinol binding protein (RBP) was tested as a possible candidate gene for the Er defect because of the importance of retinol as a modulator of epithelial morphogenesis and differentiation. Two approaches were used: (1) cloning and sequencing of the RBP cDNA from normal and mutant mice, and (2) the chromosomal localization of the mouse RBP gene. The mouse RBP sequence differs slightly from that of the rat RBP, but mutant and normal mouse RBP have identical sequences. The mouse RBP gene was localized by in situ hybridization to the distal portion of chromosome 19. This physical mapping confirms the recent assignment of the gene to chromosome 19 by linkage analysis. These results eliminate the RBP gene as a candidate gene for the defect in the Er mutation that maps to chromosome 4.     

A family with Huntington disease and reciprocal translocation 4;5.

We report the clinical and cytogenetic findings in a family in which a balanced reciprocal translocation between the long arm of chromosome 4 and the short arm of chromosome 5 is segregating together with Huntington disease in 2 generations. In situ hybridization studies revealed that the linked human DNA marker is located on the short arm of the normal and translocated chromosome 4 in the region 4p16. The association between Huntington disease and the translocation in this family may represent a chance occurrence. However, it is also possible that there is an undetected rearrangement of DNA on chromosome 4 involving the gene for Huntington disease but not affecting the site of the linked marker. Finally, the likelihood that this represents heterogeneity cannot be excluded.     

Directed cosmid isolation of bovine markers for physical assignment by fish

Abstract

A directed PCR‐based iterative screening protocol was developed to isolate cosmids containing microsatellite markers linked to chromosomal regions of interest, such as those near the ends of linkage groups or quantitative trait loci. This method was optimized for large‐scale screening of total genomic libraries and used to purify bovine cosmids that anchor the ends of the bovine linkage group 28 (BTA28). Cosmids containing ms markers RBP3 and BMS2060 were purified for fluorescence in situ hybridization and assigned to 28ql8‐ql9 and 28ql2, respectively. These assignments indicated that approximately 73% of BTA28 (90% excluding the centromere) is covered by the current linkage map. Since this method is applicable to any target gene sequence suitable for PCR amplification, it may be extended to comparative mapping of genes.