Expression of TMEFF1 mRNA in the mouse central nervous system: precise examination and comparative studies of TMEFF1 and TMEFF2

TMEFF1 and TMEFF2 are putative transmembrane proteins comprised of one epidermal growth factor (EGF)-like domain and two follistatin-like domains. Both TMEFF1 and TMEFF2 are predominantly expressed in the brain. We previously demonstrated that recombinant TMEFF2 protein can promote survival of neurons in primary culture and determined expression sites of TMEFF2 mRNA in the mouse central nervous system. To extend our understanding of TMEFF protein functions, we compared precise sites of expression of TMEFF1 and TMEFF2 mRNA using in situ hybridization analysis. Although both TMEFF genes are widely expressed in the brain, they exhibit different patterns of expression. TMEFF1 showed comparatively higher signals in the pyramidal cells of fifth layer of the cerebral neocortex, CA3, CA1 and subiculum regions of the hippocampus, locus coeruleus, and dentate cerebellar nucleus. In contrast, TMEFF2 is highly expressed in the medial habenular, CA2, CA3 and dentate gyrus region of the hippocampus, corpus callosum, cerebellar cortex and cranial nerve nuclei (III, IV, VII, X, XII). The results presented here indicate that expression of TMEFF1 and TMEFF2 are regulated differently and that they play region-specific roles in the central nervous system.     

Ontogeny of somatostatin mRNA-containing perikarya in the rat central nervous system.

The distribution of neuronal perikarya containing somatostatin mRNA in the developing rat brain was investigated with in situ hybridization histochemistry. This study describes the expression of somatostatin mRNA during selected perinatal stages and demonstrates regional changes in somatostatin mRNA expression at the single cell level. The mRNA expression closely parallels previously reported developmental localization of the peptide (Inagaki et al., 1982; Shiosaka et al., 1982). As early as embryonic day 13 (E13), somatostatin mRNA was observed in discrete spinal cord and brainstem regions. At birth, densely hybridized somata could be seen primarily in ventral and caudal brain areas with small scattered neurons in the hippocampus and dorsal neocortex. After birth, somatostatin mRNA increased in forebrain regions, such as the hippocampus, dorsal neocortex, and caudate. By postnatal day 14 (P14), the distribution in the telencephalic and diencephalic regions approached that of the adult brain. Several brain regions manifested large changes in the density of somatostatin mRNA hybridization during development. For example, the cerebellar vermis and brainstem contained somatostatin mRNA perikarya during early postnatal development but decreased in these regions in the adult. During perinatal development, increases in somatostatin mRNA content were the results of increases in both the number of neurons containing somatostatin mRNA as well as in the amount of this mRNA expressed in each cell. As the brain differentiates, the apparent numbers of somatostatin mRNA containing neurons in certain brain regions are reduced. These data provide evidence for transient somatostatinergic neurons during early development in discrete areas of the occipital cortex, pyriform cortex, cerebellum, and brainstem and suggest that this peptide may play a role in the development of these regions.     

Developmental expression of somatostatin in mouse brain. II. In situ hybridization

The distribution and the levels of expression of preprosomatostatin (PPSOM) mRNA were examined during pre- and postnatal development of the mouse brain using the in situ hybridization technique. The signal obtained by in situ hybridization of embryonic tissues at day 14 and day 17 of gestation was highest over the neurons of the pyriform cortex, amygdala, and entopeduncular nucleus. The signal was very low over cells of the neocortex and the developing hippocampal formation. The density of grains overlying the neurons of the amygdala and pyriform cortex continued to be high during early postnatal life, but decreased as the animals became adults. A progressive increase of PPSOM mRNA expression was observed in postnatal animals in the stratum oriens and dentate gyrus of the hippocampal formation. In the cerebral cortex and striatum, the number of these neurons became maximal between postnatal weeks 1 and 3. In the diencephalon, the highest densities of grains were found over neurons in the nucleus reticularis thalami and zona incerta at postnatal day 21; these levels declined slightly thereafter. The cells of the periventricular nucleus of the hypothalamus had high densities of grains as early as postnatal week 1 and continued to have high densities of grains in adult animals. These patterns of hybridization density parallelled the distribution of SOM-like immunoreactivity in the mouse brain. When PPSOM mRNA expression was examined in the cerebral cortices of mice that received lesions of the nucleus basalis of Meynert as neonates, a transient increase in the number of cells expressing PPSOM mRNA was observed in the frontoparietal cortex ipsilateral to the lesion at postnatal day 10, but not at postnatal day 30. Importantly, the density of grains over the individual cells was not altered in lesioned animals at these two ages.     

Seven protein tyrosine phosphatases are differentially expressed in the developing rat brain

Regulation of protein function through tyrosine phosphorylation is critical in the control of many developmental processes, such as cellular proliferation and differentiation. Growing evidence suggests that tyrosine phosphorylation also regulates key events in neural development. Although a large body of data has demonstrated that protein tyrosine kinases play an important role in neural development, much less is known about their counterparts, protein tyrosine phosphatases (PTPases). Using polymerase chain reaction (PCR) with degenerate primers and a neonatal rat cortex cDNA library, we have identified seven PTPases expressed in the developing rat brain. Four of these are transmembrane PTPases: LAR, LRP, RPTP_γ, and CPTP1. Three are nonreceptor PTPases: PTP-1, P19-PTP, and SHP. Northern hybridization analysis demonstrates that only CPTP1 is preferentially expressed in neural tissues, whereas the others are found abundantly in nonneural tissues as well as in the brain. Within the embryonic and early postnatal brain, the seven PTPases have overlapping, yet unique, distributions. For example, LAR mRNA is highly expressed by both proliferating and postmitotic cells in the cerebral cortex at embryonic day 17 and in all layers of the cortex at postnatal day 4. In contrast, RPTP_γ mRNA is expressed by postmitotic neurons in the embryo and predominantly by neurons in the superficial layers of the postnatal cortex. Several of the PTPases examined here are expressed at very high levels in the embryonic cortical plate and postnatal neocortex, including the subplate and subventricular zone. The spatial and temporal regulation of PTPase agene expression suggests that these PTPases have important roles in signal transduction during early neuronal differentiation and neural development. © 1995 Willy-Liss, Inc.     

Postnatal distribution of cpp32/caspase 3 mRNA in the mouse central nervous system: an in situ hybridization study.

Apoptotic cell death is a major feature of the developing nervous system and of certain neurodegenerative diseases. Various gene effectors and repressors of this type of cell death have been identified. Among them, bcl-xl and bax, which encode for antiapoptotic and proapoptotic proteins, respectively, play major roles during development. The gene cpp32 encodes for the caspase 3 cysteine protease and is a critical mediator of cell death during embryonic development in the mammalian brain. To gain insight into the possible implications of these cell death genes during the postnatal development, we investigated the expression of bax, bcl-xl, and cpp32 mRNAs by in situ hybridization in the mouse brain from birth to adulthood. Whereas bax and bcl-xl mRNAs were expressed widely in neonates and adult mice, our results showed that cpp32 mRNA levels were decreased strongly from 12 postnatal days. From 1 postnatal day to 12 postnatal days, cpp32 mRNA was expressed ubiquitously in all brain nuclei, including areas where neurogenesis occurred. A positive correlation between areas displaying high levels of mRNA and apoptotic nuclei also was shown. In the adult, cpp32 mRNA was restricted to the piriform and entorhinal cortices, the neocortex, and to areas where neurogenesis is observed (e.g., olfactory bulb and dentate gyrus). The same pattern of expression was observed in adult mice over-expressing the antiapoptotic protein Bcl-2. These results demonstrate that the expression of cpp32 mRNA is highly regulated during the mouse postnatal period, leading to a specific distribution in the adult central nervous system. Moreover, the prevention of cell death by Bcl-2 likely is not linked to the regulation of caspase mRNA levels.     

Developmental expression of neuronal calmodulin mRNA species in the rat brain analyzed by in situ hybridization.

The temporal and spatial distribution of calmodulin mRNAs which are preferentially expressed in neurons was determined during postnatal development of rat central nervous system. Expression of these mRNAs was strongly detected in the developing neocortex, hippocampus, and cerebellum. Differences in the pattern of expression of a 1.8 and 4.0 kb neuronal calmodulin mRNA species were identified in the developing cerebellum. Expression of the smaller mRNA appeared to correlate with proliferating and developing cerebellar granule neurons while the larger mRNA was present in the mature granule neuron population. A transient elevation in the neuronal calmodulin mRNA species was observed in the superior and inferior colliculus and in the thalamus at postnatal days 5 and 10.     

Viral regulation of aquaporin 4, connexin 43, microcephalin and nucleolin

The current study investigated whether human influenza viral infection in midpregnancy leads to alterations in proteins involved in brain development. Human influenza viral infection was administered to E9 pregnant Balb/c mice. Brains of control and virally-exposed littermates were subjected to microarray analysis, SDS-PAGE and western blotting at three postnatal stages. Microarray analysis of virally-exposed mouse brains showed significant, two-fold change in expression of multiple genes in both neocortex and cerebellum when compared to sham-infected controls. Levels of mRNA and protein levels of four selected genes were examined in brains of exposed mice. Nucleolin mRNA was significantly decreased in day 0 and day 35 neocortex and significantly increased in day 35 cerebellum. Protein levels were significantly upregulated at days 35 and 56 in neocortex and at day 56 in cerebellum. Connexin 43 protein levels were significantly decreased at day 56 in neocortex. Aquaporin 4 mRNA was significantly decreased in day 0 neocortex. Aquaporin 4 protein levels decreased in neocortex significantly at day 35. Finally, microcephalin mRNA was significantly decreased in day 56 neocortex and protein levels were significantly decreased at 56 cerebellum. These data suggest that influenza viral infection in midpregnancy in mice leads to long-term changes in brain markers for enhanced ribosome genesis (nucleolin), increased production of immature neurons (microcephalin), and abnormal glial-neuronal communication and neuron migration (connexin 43 and aquaporin 4).     

Characterization of a novel mRNA expressed by neurons in mature brain.

In previous studies, differential hybridization screening of an activated murine T-lymphocyte cDNA library identified an interleukin 2-responsive mRNA, designated F5, expressed in lymphoid tissues and brain only. We now report characterization of a full-length clone isolated from an adult mouse brain cDNA library. Neither the nucleic acid nor amino acid sequences demonstrated similarity to reported sequences. On Southern blotting, the protein coding sequence hybridized to genomic DNA from a variety of species. On Northern blotting, F5 mRNA was expressed in adult mouse brain, spinal cord, eye, and dorsal root ganglia but not in peripheral nerve. In situ hybridization studies demonstrated prominent expression by neurons in brain. F5 mRNA expression was undetectable in embryonic rat cerebral hemisphere and low until postnatal day 21. F5 is a novel mRNA selectively expressed by proliferating lymphocytes and mature neurons.     

Isolation of a mouse brain cDNA expressed in developing neuroblasts and mature neurons

A previously uncharacterized 4.5-kb mouse cDNA clone, designated mc7, was isolated and found to be predominantly expressed in brain. This cDNA predicts a 1035-bp open reading frame that encodes for a 345-amino acid polypeptide especially rich in glutamic acid residues located in the region from residues 80 to 174. Computational analysis revealed among other features, putative zinc-finger motifs and coiled-coil regions. The corresponding mc7 gene is detected in mouse, rat, pig and human genomes. In mouse the mc7 mRNA is expressed predominantly in brain and to a much lesser extent in kidney, lung and spleen. In brain it is detectable as early as embryonic day 14 while it is retained in the adult. In situ hybridization studies revealed that mc7 mRNA is widely, albeit unevenly, expressed in neurons throughout the adult brain. Developmental in situ hybridization studies in the cerebellar cortex demonstrated that at postnatal day 5 mc7 mRNA is mainly expressed in neuroblasts of the external granular layer and in developing neurons of the internal granular layer. Some staining is also present in purkinje cells becoming particularly pronounced at postnatal day 10, the time of arborarization of their dendritic tree. In the adult cerebellar cortex expression is mainly confined in purkinje cells and to a lesser extent in granule neurons. The early expression of mc7 in neuroblasts and developing neurons as well as its retention in a wide variety of mature neurons suggest that it may play a role in the process of differentiation and maturation of these cells in the brain.     

Ephrin-B2/EphA4 forward signaling is required for regulation of radial migration of cortical neurons in the mouse

Postmitotic neurons in the neocortex migrate to appropriate positions and form layered structures of nascent cortex during brain development. The migration of these neurons requires precise control and coordination of a large number of molecules such as axon guidance cues. The Eph-ephrin signaling pathway plays important roles in the development of the nervous system in a wide variety of ways, including cell segregation, axon pathfinding, and neuron migration. However, the role of ephrin-B2/EphA4 signaling in cortical neuron migration remains elusive. Here we demonstrated that ephrin-B2 and its receptor EphA4 were expressed in complementary and overlapping patterns in the developing neocortex. Deletion of the EphA4 gene in the embryonic cerebral cortex resulted in faster migration of cortical neurons, whereas knockdown or overexpression of ephrin-B2 did not alter the normal process of migration. These results suggest that ephrin-B2 forward signaling through EphA4 is required for the precise control of cortical neuron migration.     

Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans

Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain.     

Northern blot and in situ hybridization analyses for the neurogranin mRNA in the developing monkey cerebral cortex

Neurogranin is a postsynaptic substrate for protein kinase C, and its expression is related to dendritic spine development and postsynaptic plasticity. Using both Northern blot analysis and in situ hybridization techniques, we investigated the developmental changes of neurogranin expression in the monkey cerebral cortex. In each of four neocortical areas examined, i.e., the prefrontal area (area FD of von Bonin and Bailey), the temporal association area (TE), the primary somatosensory area (PB), and the primary visual area (OC), the Northern blot analysis showed that the amount of neurogranin mRNA was low during the prenatal and perinatal periods until postnatal day 8. It increased during postnatal development and reached its peak value at postnatal day 70 (in area OC) or postnatal month 6 (in area FD, TE, and PB). After that, the amount of neurogranin mRNA in the cerebral neocortex decreased gradually until postnatal years 2-3. The in situ hybridization experiments also showed a transient increase of neurogranin mRNA in the neocortex during postnatal day 70 to postnatal month 6. The transient increase was prominent in layers II and III of areas FD and TE; deep in layer III of area PB; and in layers II, III, and IV of area OC. In the hippocampus, in contrast to the results in the neocortex, the expression of neurogranin mRNA was decreased almost continuously during the postnatal period. The transiently increased expression of neurogranin in the postnatal neocortex may be a molecular basis for the postsynaptic modification of afferent inputs possibly from subcortical structures.     

Developmental expression of the basic fibroblast growth factor gene in rat brain.

Basic fibroblast growth factor (bFGF) is a trophic factor for a variety of neuronal/glial cell populations. The RNase protection assay, with a cRNA complementary to the coding region of bFGF mRNA, was used to investigate the brain distribution and developmental regulation of bFGF mRNA expression. In adult rats bFGF mRNA is distributed throughout the brain, the highest levels being observed in cerebral cortex, hippocampus and spinal cord. The levels of bFGF mRNA in all the brain structures are low in newborn rats, increase thereafter to reach a peak of expression around postnatal day 21. bFGF mRNA levels are significantly different between various brain structures during the first and second postnatal week. Adult and aged rats (Fisher 344) express the same levels of bFGF mRNA in the various brain regions. The onset of bFGF mRNA expression suggests that this growth factor is important for the maturation as well as for the maintenance of different cell populations of the central nervous system.     

Identification of the novel developmentally regulated gene, Bdm2, which is highly expressed in fetal rat brain

Most of the neurogenesis take place during the embryonic stage; the genes expressed predominantly in this stage may play important roles in the control of development of the central nervous system. Using a differential display method, we identified the novel rat gene, brain development-related molecule 2 (Bdm2), that is expressed more abundantly in the embryonic brain than in the adult brain. Full-length Bdm2 cDNA consists of 1842 base pairs (bp) and contains an open reading frame of 1260 bp. Northern blot analysis demonstrated that Bdm2 was strongly expressed in the late embryonic brain and was still detected at lower levels in an early postnatal period; in adults, Bdm2 mRNA was decreased to an undetectable level in brain, though the expression of this mRNA was revealed in other tissues. Level of Bdm2 mRNA was maintained during neuronal differentiation of mouse embryonal carcinoma cell P19, but decreased during the differentiation to glial and unidentified non-neuronal cells. In situ hybridization study demonstrated the wide distribution of Bdm2 mRNA in the embryonic brain; in the adult brain, the hybridization signals became more restricted to the hippocampus, olfactory bulb, cerebellum, and neocortex, almost coinciding with the regions where nascent and immature neurons are present. Thus, it appears likely that Bdm2 encodes a protein that is involved in both the regulation of growth of undifferentiated neural cells and the terminal differentiation of neuronal cells.     

Initiation of protein synthesis and heat-shock protein-72 expression in the rat brain following severe insulin-induced hypoglycemia

Summary Following stress such as heat shock or transient cerebral ischemia, global brain protein synthesis initiation is depressed through modulation of eucaryotic initiation factor (eIF) activities, and modification of ribosomal subunits. Concomitantly, expression of a certain class of mRNA, heat-shock protein (HSP) mRNA, is induced. Here we report that the activity of eucaryotic initiation factor-2 (eIF-2), a protein that participates in the regulation of a rate-limiting initiation step of protein synthesis, transiently decreases following insulin-induced severe hypoglycemia in the rat brain neocortex. Expression of HSP 72, a 72-kDa HSP, in surviving neurons was seen at 1–7 days of recovery following 30 min of hypoglycemic coma, but not at 1 h and 6 h of recovery. In the neocortex, HSP 72 was first seen in layer IV, and later also in surviving neurons in layer II. In the CA1 region and in the crest of dentate gyrus, HSP 72 expression was evident in cells adjacent to irreversibly damaged neurons. In the CA3 region and the hilus of dentate gyrus, HSP 72 was expressed in a few scattered neurons. In septal nucleus, HSP 72 was expressed in a lateral to medial fashion over a period of 1–3 days of recovery. We conclude that severe insulin-induced hypoglycemia induces a stress response in neurons in the recovery phase, including inhibition of protein synthesis initiation, depression of eIF-2 activity, and a delayed and prolonged expression of HSP 72 in surviving neurons. The HSP 72 expression may be a protective response to injurious stress.     

Patterns of Jagged1, Jagged2, Delta-like 1 and Delta-like 3 expression during late embryonic and postnatal brain development suggest multiple functional roles in progenitors and differentiated cells

The Notch-DSL signaling system, consisting of multiple receptors and ligands, inhibits neurogenesis and promotes gliogenesis during embryonic development, but the specific function of the various ligands and receptors at later developmental stages are unknown. Here, we examined the expression pattern of four Delta, Serrate and Lag-2 (DSL) ligands, Jagged1, Jagged2, Delta-like1 (Dl1) and Delta-like 3 (Dl3), in late embryonic and postnatal rat brain by in situ hybridization. In late embryos, Jagged1, Dl1 and Dl3 mRNAs were present in the periventricular germinal epithelia, but this expression diminished during postnatal ages. Jagged1 mRNA was also expressed in the inner aspect of the dentate gyrus at early postnatal times. Dl3 was detectable in the external granule cell layer (EGL) of the cerebellum, another site of postnatal neurogenesis. Jagged2 mRNA was expressed in virtually all postnatal neurons. Jagged1 mRNA was highly expressed in several brain nuclei during postnatal development, with lower levels of expression in other grey matter regions. In white matter, Dl1 and Dl3 mRNAs were expressed during the first week of postnatal development but only the expression of Dl1 mRNA persisted through the second week. Dl1 mRNA was present at lower levels throughout grey matter during the first few weeks of development. Jagged1 mRNA was expressed in blood vessels, choroid plexus, and menninges throughout development and in the adult. Jagged2 mRNA was transiently expressed in cerebral blood vessels and choroid plexus during the first postnatal week. Taken together, these results support multiple and differing roles for the various ligands during and after central nervous system (CNS) development.