EphB3 receptor and ligand expression in the adult rat brain

Summary Eph receptors and ligands are two families of proteins that control axonal guidance during development. Their expression was originally thought to be developmentally regulated but recent work has shown that several EphA receptors are expressed postnatally. The EphB3 receptors are expressed during embryonic development in multiple regions of the central nervous system but their potential expression and functional role in the adult brain is unknown. We used in situ hybridization, immunohistochemistry, and receptor affinity probe in situ staining to investigate EphB3 receptors mRNA, protein, and ligand (ephrin-B) expression, respectively, in the adult rat brain. Our results indicate that EphB3 receptor mRNA and protein are constitutively expressed in discrete regions of the adult rat brain including the cerebellum, raphe pallidus, hippocampus, entorhinal cortex, and both motor and sensory cortices. The spatial profile of EphB3 receptors was co-localized to regions of the brain that had a high level of EphB3 receptor binding ligands. Its expression pattern suggests that EphB3 may play a role in the maintenance of mature neuronal connections or re-arrangement of synaptic connections during late stages of development.     

Expression of the endothelial lipase gene in murine embryos and reproductive organs

Endothelial lipase (EL) is a recently discovered member of the triglyceride-lipase family that is involved in plasma HDL metabolism. In this study, we investigated the putative role of EL in mouse reproduction by studying EL gene expression in mouse embryos and adult reproductive organs. PCR analysis revealed that EL mRNA is expressed in mouse embryos on embryonic day 8.5 (E8.5) to E11.5, but not later in development. In situ hybridization studies on E10.5 whole embryos and embryonic sections showed expression of EL mRNA in multiple tissues, although of varying intensity. High expression was found in the neuroepithelium of the brain and the neural tube, the mesenchymal cells between organs, the optic lens and cup, and the otocyst. In adult mice, EL mRNA expression was high in ovaries from pregnant mice but low in ovaries from nonpregnant mice. EL mRNA was also highly expressed in placenta and testes. In situ hybridization studies demonstrated intense EL mRNA staining of lutein cells in corpora lutei in ovaries, of spermatocytes in the late pachytene and diplotene stages in testes, and of principal cells in epididymis. These results suggest that EL, in addition to its effects on plasma lipoprotein metabolism, plays a role in murine reproduction.     

Developmental regulation of rat Ubc13 and Uev1B genes in the nervous system

Ubiquitin is a highly conserved protein in eukaryotes, and regulates diverse cellular processes. Lys-63-linked poly-ubiquitination has been recently identified to be involved in non-proteolytic processes such as DNA repair and cytokine-mediated signal transduction. Although, the heterodimeric enzymes Ubc13 and Uev are required for ubiquitination, their expressional regulation is not known. We have analyzed changes in their expression during brain development. Northern blot analysis revealed that the expression levels of the two genes were very similar. Expression of both genes decreased gradually during the embryonic stages, then increased in the late postnatal period and was moderate in the adult. In situ hybridization analyses revealed that the expression patterns of the two genes were similar. Expression was observed in various regions in the embryonic brain but became restricted to specific regions after birth. In the adult, their expression was similar in regions such as the cerebral cortex, hippocampus, and substantia nigra, but different in the cerebellum. These results suggest that Ubc13 may be closely associated with Uev1B.     

Neural-specific expression of miR-344-3p during mouse embryonic development

MicroRNAs are small noncoding RNAs involved in various biological processes. We characterized the expression of miR-344-3p during mouse embryonic development. At E9.5–E10.5 and E15.5, in situ hybridization detected strong miR-344-3p signal in the central nervous system, including the cerebral cortex, hindbrain, cerebellum, thalamus, hindbrain, medulla oblongata, spinal cord, and dorsal root ganglia. Further, qRT-PCR analysis identified miR-344-3p expression at E15.5, with expression stably maintained in the brain from E12.5 to E18.5 before decreasing to relatively low levels postnatally. We also analyzed miR-344-3p expression using immunofluorescence in situ hybridization at E18.5 and within the adult brain. miR-344-3p signal was mainly detected in cortical regions surrounding the ventricular system, choroid plexus, glomerular layer of the olfactory bulb, and granular cell layer of the cerebellar cortex. Altogether, our results indicate miR-344-3p may play an important role in morphogenesis, nervous system development in the brain.     

Expression of cyclin E in postmitotic neurons during development and in the adult mouse brain

Cyclin E, a member of the G1 cyclins, is essential for the G1/S transition of the cell cycle in cultured cells, but its roles in vivo are not fully defined. The present study characterized the spatiotemporal expression profile of cyclin E in two representative brain regions in the mouse, the cerebral and cerebellar cortices. Western blotting showed that the levels of cyclin E increased towards adulthood. In situ hybridization and immunohistochemistry showed the distributions of cyclin E mRNA and protein were comparable in the cerebral cortex and the cerebellum. Immunohistochemistry for the proliferating cell marker, proliferating cell nuclear antigen (PCNA) revealed that cyclin E was expressed by both proliferating and non-proliferating cells in the cerebral cortex at embryonic day 12.5 (E12.5) and in the cerebellum at postnatal day 1 (P1). Subcellular localization in neurons was examined using immunofluorescence and western blotting. Cyclin E expression was nuclear in proliferating neuronal precursor cells but cytoplasmic in postmitotic neurons during embryonic development. Nuclear cyclin E expression in neurons remained faint in newborns, increased during postnatal development and was markedly decreased in adults. In various adult brain regions, cyclin E staining was more intense in the cytoplasm than in the nucleus in most neurons. These data suggest a role for cyclin E in the development and function of the mammalian central nervous system and that its subcellular localization in neurons is important. Our report presents the first detailed analysis of cyclin E expression in postmitotic neurons during development and in the adult mouse brain.     

Expression of osteopontin mRNA in developing rat brainstem and cerebellum

This study was undertaken to investigate the developmental expression of osteopontin (OPN) in the rat brainstem and cerebellum by Northern blotting and in situ hybridization. The expression of OPN was noted in the mesencephalic Vth nucleus initially at embryonic day 16 (E16). At E20, the labeling extended into other brainstem nuclei including the cochlear, vestibular, facial motor, and hypoglossal nuclei. During the first week of postnatal life, the OPN signal in the brainstem increased markedly, and by P14, OPN expression was found in functionally diverse areas including motor-related areas, sensory relay nuclei, and the reticular formation. The adult labeling pattern was established in central neurons at this time. These results corresponded well with those from Northern blot analysis. On the basis of morphological and distribution criteria, the OPN signal in several nuclei appeared to be contained exclusively within neuronal soma. OPN expression in neurons occurred during the period of neuronal differentiation and increased with maturation. Our results therefore suggest that OPN contributes to developmental processes, including the differentiation and maturation of specific neuronal populations, in the rat brain.     

In situ study of c-myc protein expression during avian development

The distribution of the c-myc protein was studied in the developing embryo from the two-somite stage to embryonic day 17 (E17). A triple labelling method was used, with a polyclonal serum recognizing the human and avian c-myc proteins as the first marker followed by Hoechst 33258 for nuclear staining and the monoclonal antibody 13F4 which reveals the avian myogenic lineage. In situ hybridization was carried out at three selected stages (E3, E6 and E8), in order to compare the distribution of myc mRNA and myc protein. The c-myc protein signal was barely detectable in blastodisc nuclei during the period of somite formation, after which it became ubiquitous in the embryonic body until E4. Myotomal cell nuclei displayed a strong signal until their organization into premuscular masses. On day 4, the level of c-myc protein decreased in all embryonic tissues. By doubling the antibody titre and amplifying the signal by means of the streptavidin-biotin method, c-myc could still be detected in nuclei of defined groups of cells. Such was the case in some mesenchyme-derived tissues at critical periods of organogenesis, for instance in prechondrogenic condensations or hemopoietic cell foci at E6, the latter becoming negative at E9. The heart ventricle displayed a patch-work of positive and negative nuclei from E6 to E10. A myc signal restricted to the quail species was found in the wall of the carotid arteries. Cell nuclei in the nervous system displayed a detectable signal which became restricted to postmitotic neurones. In the ectoderm, the c-myc protein was generally not present after E4, except in presumptive feather buds at the time of epitheliomesenchymal interactions. Endodermal cells (such as hepatocytes, oesophageal and tracheal epithelia) did not express detectable levels of c-myc at any time. Our results reveal a time- and tissue-specific expression of c-myc during avian development. It is noteworthy that the expression of the c-myc protein often appears dissociated from cell proliferation as shown by the absence of the signal in endodermal cells at E3-E13 as well as its presence in postmitotic neurones. Finally, although RNA and protein are simultaneously detected in some structures such as presumptive feather buds, their expression is dissociated in endodermal tissues, notably hepatocytes, where in situ hybridization detects a large number of RNA copies with no detectable protein signal.     

The Expression Pattern of Classical MHC Class I Molecules in the Development of Mouse Central Nervous System

Classical major histocompatibility complex (MHC) class I, first identified in the immune system, is also expressed in the developing and adult central nervous system (CNS). Although the MHC class I molecules have been found to be expressed in the CNS of different species, a necessary step to elucidate the temporal and spatial expression patterns of MHC class I molecules in the brain development has never been taken. Frozen sections were made from the brains of embryonic and postnatal C57BL/6 J mice, and the expression of H-2D^b mRNA was examined by in situ hybridization. Immunofluorescence was also performed to define the cell types that express H2-D^b in P15 mice. At E10.5, the earliest stage we examined, H2-D^b was expressed in neuroepithelium of the brain vesicles. From E12.5 to P0, H2-D^b expression was mainly located at cerebral cortex, neuroepithelium of the lateral ventricle, neuroepithelium of aquaeductus and developing cerebellum. From P4 to adult, H2-D^b mRNA was detected at olfactory bulb, hippocampus, cerebellum and some nerve nuclei. The major cell types expressing H-2D^b in P15 hippocampus, cerebral cortex and olfactory bulb were neuron. H2-K^b signal paralleled that of H2-D^b and the expression levels of the two molecules were comparable throughout the brain. The investigation of the expression pattern of H-2D^b at both embryonic and postnatal stages is important for further understanding the physiological and pathological roles of H2-D^b in the developing CNS.     

The TRH neuronal phenotype forms embryonic cell clusters that go on to establish a regionalized cell fate in forebrain

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.     

Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster, Homarus americanus

Neuropeptides in the stomatogastric ganglion (STG) and the brain of adult and late embryonic Homarus americanus were compared using a multi-faceted mass spectral strategy. Overall, 29 neuropeptides from 10 families were identified in the brain and/or the STG of the lobster. Many of these neuropeptides are reported for the first time in the embryonic lobster. Neuropeptide extraction followed by liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry enabled confident identification of 24 previously characterized peptides in the adult brain and 13 peptides in the embryonic brain. Two novel peptides (QDLDHVFLRFa and GPPSLRLRFa) were de novo sequenced. In addition, a comparison of adult to embryonic brains revealed the presence of an incompletely processed form of Cancer borealis tachykinin-related peptide 1a (CabTRP 1a, APSGFLGMRG) only in the embryonic brain. A comparison of adult to embryonic STGs revealed that QDLDHVFLRFa was present in the embryonic STG but absent in the adult STG, and CabTRP 1a exhibited the opposite trend. Relative quantification of neuropeptides in the STG revealed that three orcokinin family peptides (NFDEIDRSGFGF, NFDEIDRSGFGFV, and NFDEIDRSGFGFN), a B-type allatostatin (STNWSSLRSAWa), and an orcomyotropin-related peptide (FDAFTTGFGHS) exhibited higher signal intensities in the adult relative to the embryonic STG. RFamide (Arg-Phe-amide) family peptide (DTSTPALRLRFa), [Val¹]SIFamide (VYRKPPFNGSIFa), and orcokinin-related peptide (VYGPRDIANLY) were more intense in the embryonic STG spectra than in the adult STG spectra. Collectively, this study expands our current knowledge of the H. americanus neuropeptidome and highlights some intriguing expression differences that occur during development.     

Polysialic Acid Synthase (ST8Sia II/STX) mRNA Expression in the Developing Mouse Central Nervous System

Abstract: A comparative study was undertaken to correlate the immunohistochemical localization of polysialic acid (PSA) and the in situ localization of ST8Sia II mRNA. In situ hybridization of postnatal day 3 mouse brain showed high levels of ST8Sia II mRNA expression in the cerebral neocortex, striatum, hippocampus, subiculum, medial habenular nucleus, thalamus, pontine nuclei, and inferior colliculus; intermediate-level expression in the olfactory bulb, hypothalamus, superior colliculus, and cerebellum; and low-level expression in other regions. The distribution of ST8Sia II mRNA in the neocortex and cerebellum coincided with the immunohistochemical localization of PSA. During brain development, ST8Sia II mRNA started decreasing and had almost disappeared by postnatal day 14. Comparison between ST8Sia II and IV mRNA expression was also undertaken by northern blot analysis and competitive PCR analysis. During the late embryonic to early postnatal stages of the mouse CNS, the ST8Sia II mRNA showed abundant mRNA expression compared with the ST8Sia IV mRNA. Competitive PCR analysis of the adult mouse CNS showed weak expression of the two genes in the olfactory bulb, thalamus, hippocampus, and eyes. The regional and transient expression of ST8Sia II mRNA coincides with that of PSA, suggesting that ST8Sia II is closely involved in the biosynthesis and expression of PSA in the developing mouse CNS.     

The spatial and temporal expression of delta-like protein 1 in the rat pituitary gland during development

An analysis of secreted proteins by the signal sequence trap method using a cDNA library of the rat pituitary anlage at embryonic days (E) 13.5 revealed the abundant expression of delta-like protein 1 (Dlk1) in the pituitary gland. Dlk1, an epidermal growth factor-like repeat protein in preadipocytes, functions in maintaining the preadipose state. Expression of Dlk1 mRNA in the pituitary at E13.5 and in the adult pituitary was confirmed by in situ hybridization. The expression pattern of Dlk1 during pituitary development was also studied by immunohistochemistry. Dlk1 protein first appeared in Rathke’s pouch and the infundibulum at E11.5; as development proceeded, expression became restricted to the pars distalis and pars tuberalis (PT). Dlk1 was expressed in most ACTH cells during the embryonic stages, but its expression was limited to only a few ACTH cells in the adult pituitary. It was also expressed in a small population of TSH, GTH, and PRL cells throughout development, whereas it was present in the cytoplasm of most GH cells at all developmental stages. Similarly, Dlk1 was localized in the cytoplasm of PT cells during development. These findings provide new insights into the mechanism of Dlk1 regarding its regulation of pituitary hormone-secreting cells during development.     

Expression of Toll-like receptors in the developing brain

Toll-like receptors (TLR) are key players of the innate and adaptive immune response in vertebrates. The original protein Toll in Drosophila melanogaster regulates both host defense and morphogenesis during development. Making use of real-time PCR, in situ hybridization, and immunohistochemistry we systematically examined the expression of TLR1-9 and the intracellular adaptor molecules MyD88 and TRIF during development of the mouse brain. Expression of TLR7 and TLR9 in the brain was strongly regulated during different embryonic, postnatal, and adult stages. In contrast, expression of TLR1-6, TLR8, MyD88, and TRIF mRNA displayed no significant changes in the different phases of brain development. Neurons of various brain regions including the neocortex and the hippocampus were identified as the main cell type expressing both TLR7 and TLR9 in the developing brain. Taken together, our data reveal specific expression patterns of distinct TLRs in the developing mouse brain and lay the foundation for further investigation of the pathophysiological significance of these receptors for developmental processes in the central nervous system of vertebrates.     

Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain

The canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that are activated by increases in intracellular Ca(2+) and G(q)/phospholipase C-coupled receptors. We used quantitative real-time PCR, in situ hybridization, immunoblots and patch-clamp recording from several brain regions to examine the expression of the predominant TRPC channels in the rodent brain. Quantitative real-time PCR of the seven TRPC channels in the rodent brain revealed that TRPC4 and TRPC5 channels were the predominant TRPC subtypes in the adult rat brain. In situ hybridization histochemistry and immunoblotting further resolved a dense corticolimbic expression of the TRPC4 and TRPC5 channels. Total protein expression of HIP TRPC4 and 5 proteins increased throughout development and peaked late in adulthood (6-9 weeks). In adults, TRPC4 expression was high throughout the frontal cortex, lateral septum (LS), pyramidal cell layer of the hippocampus (HIP), dentate gyrus (DG), and ventral subiculum (vSUB). TRPC5 was highly expressed in the frontal cortex, pyramidal cell layer of the HIP, DG, and hypothalamus. Detailed examination of frontal cortical layer mRNA expression indicated TRPC4 mRNA is distributed throughout layers 2-6 of the prefrontal cortex (PFC), motor cortex (MCx), and somatosensory cortex (SCx). TRPC5 mRNA expression was concentrated specifically in the deep layers 5/6 and superficial layers 2/3 of the PFC and anterior cingulate. Patch-clamp recording indicated a strong metabotropic glutamate-activated cation current-mediated depolarization that was dependent on intracellular Ca(2+)and inhibited by protein kinase C in brain regions associated with dense TRPC4 or 5 expression and absent in regions lacking TRPC4 and 5 expression. Overall, the dense corticolimbic expression pattern suggests that these Gq/PLC coupled nonselective cation channels may be involved in learning, memory, and goal-directed behaviors.