Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development.

The levels of the mRNAs encoding sodium channels I, II and III in various regions of the developing rat central nervous system (from embryonal day 10 to postnatal day 90) have been examined by blot hybridization analysis with specific probes. The three sodium channel mRNAs exhibit different temporal and regional expression patterns. The expression of sodium channel I mRNA rises after a lag phase to adult levels during the second and third postnatal weeks with stronger increases in caudal regions of the brain and in spinal cord. Sodium channel II mRNA increases steadily until the first postnatal week, keeping high adult levels in rostral regions of the brain or reaching low adult levels after the second postnatal week in most caudal regions of the brain and in spinal cord; cerebellum shows low levels during the first two postnatal weeks but high adult levels. In all regions, sodium channel III mRNA attains maximum levels around birth and decreases during the first and second postnatal weeks to reach variable low adult levels. These results suggest that sodium channel III is expressed predominantly at fetal and early postnatal stages and sodium channel I predominantly at late postnatal stages, whereas sodium channel II is expressed throughout the developmental stages studied with greater regional variability.     

Expression of mRNAs Encoding Subunits of the NMDA Receptor in Developing Rat Brain

Abstract: Developmental changes in the levels of N -methyl- d -aspartate (NMDA) receptor subunit mRNAs were identified in rat brain using solution hybridization/RNase protection assays. Pronounced increases in the levels of mRNAs encoding NR1 and NR2A were seen in the cerebral cortex, hippocampus, and cerebellum between postnatal days 7 and 20. In cortex and hippocampus, the expression of NR2B mRNA was high in neonatal rats and remained relatively constant over time. In contrast, in cerebellum, the level of NR2B mRNA was highest at postnatal day 1 and declined to undetectable levels by postnatal day 28. NR2C mRNA was not detectable in cerebellum before postnatal day 11, after which it increased to reach adult levels by postnatal day 28. In cortex, the expression of NR2A and NR2B mRNAs corresponds to the previously described developmental profile of NMDA receptor subtypes having low and high affinities for ifenprodil, i.e., a delayed expression of NR2A correlating with the late expression of low-affinity ifenprodil sites. In cortex and hippocampus, the predominant splice variants of NR1 were those without the 5' insert and with or without both 3' inserts. In cerebellum, however, the major NR1 variants were those containing the 5' insert and lacking both 3' inserts. The results show that the expression of NR1 splice variants and NR2 subunits is differentially regulated in various brain regions during development. Changes in subunit expression are likely to underlie some of the changes in the functional and pharmacological properties of NMDA receptors that occur during development.     

Developmental Changes in Expression of a 60-kDa Somatostatin Receptor Immunoreactivity in the Rat Brain

Abstract: The neuropeptide somatostatin (SRIF) exerts several important physiological actions in the adult CNS through interactions with membrane-bound receptors. SRIF expression is developmentally regulated and this regulation is most apparent in the cerebellum, where SRIF immunoreactivity is expressed at early postnatal ages and then disappears toward adulthood. The transitory nature of SRIF expression at a time of major changes in cerebellum suggests that this peptide may have a role in cerebellar development. To further investigate the role of the SRIF transmitter system during development, we have examined the levels of expression of SRIF receptors in the developing rat brain by immunoblotting using antiserum selective for a 60-kDa brain SRIF receptor. In whole rat brain, SRIF receptor immunoreactivity first appears at embryonic day 13 (E13), is elevated at E16. increases at birth, peaks at early postnatal ages, and then gradually declines with age. No apparent changes in size of the receptor occur with age. No consistent changes in levels of SRIF receptor immunoreactivity are detected from early postnatal ages to adulthood in the hippocampus, cerebral cortex, and striatum, but levels gradually decline in the hypothalamus. In contrast, SRIF receptor immunoreactivity is expressed transiently in cerebellum. SRIF receptor immunoreactivity is detectable in cerebellum at E16, increases in levels at birth, is apparent from postnatal day 3 to postnatal day 8, and then disappears. The transitory nature of SRIF receptor expression in cerebellum is unique and parallels the expression of SRIF immunoreactivity in this brain region. These findings support the hypothesis that SRIF has a role in cerebellar development.     

Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development.

The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.     

Effect of prenatal and postnatal ethanol exposure on the developmental profile of mRNAs encoding NMDA receptor subunits in rat hippocampus

It has been proposed that assembly of the final NMDA receptor complex may be modified by prenatal ethanol exposure, resulting in long-term alterations of NMDA receptor pharmacology. We investigated the effect of prenatal and postnatal ethanol exposure on the developmental profile of mRNAs encoding NMDA receptor subunits in rat hippocampus. Female Sprague-Dawley rats were chronically intoxicated for 4 weeks with a 10% (v/v) ethanol solution administered throughout pregnancy and lactation. Hippocampus and cerebellum were isolated from pups (postnatal days 1-28) of the ethanol-exposed and ad libitum groups. Our results, using a semiquantitative RT-PCR technique, showed a selective effect of ethanol exposure on the various NMDA receptor subunits. Ethanol exposure significantly increased the levels of NR1(1XX), NR1(X11) and NR2(D) mRNAs on postnatal days 7 and 14 and decreased the level of NR2(C) on postnatal day 1. Immunoblot analyses demonstrated that NR2(D) protein levels were increased on postnatal day 7 after ethanol exposure. However, the developmental profile of mRNAs encoding for NR2(A-B), NR3(L/S), GBP and Gly/TCP-BP subunits were not affected. Moreover, no significant effects of ethanol exposure were observed on the developmental transition from expression of NR1(0XX) to NR(1XX) splice variants occurring in the cerebellum on postnatal day 19. Unexpectedly, [(3) H]MK-801 binding experiments showed that ethanol exposure increased the B (max) values of high-affinity sites on postnatal days 14 and 28, with no change of K (d) values. These findings indicate that prenatal and/or postnatal ethanol exposure alters the hippocampal levels of mRNAs encoding for certain subunits and the density of high-affinity [(3) H]MK-801 binding sites. As these subunits have been shown to modulate the functional properties of NMDA receptors, these results suggest that this altered expression could be involved in the neurodevelopmental disorders associated with fetal ethanol exposure.     

Developmental changes in lysophospholipid receptor expression in rodent heart from near-term fetus to adult

Lysophospholipids (LPs) are small signaling lipids that regulate diverse physiological and pathological processes through G protein-coupled receptors. To investigate the function of LP signaling in heart organogenesis and maturation, we measured the expression of 10 confirmed LP receptors (Lpar1-5 and S1pr1-5) in rat heart from embryonic day 19.5 (E19.5d) to postnatal week 12 (P12w). The expression of Lpar3 mRNA peaked at 37-fold higher than adult expression at P1d, while the expression levels of Lpar1 and Lpar4 increased markedly after P1d and peaked at 19- and 48-folds of adult expression on P7d. The expression levels of all three receptor mRNAs were significantly reduced by P21d and remained low thereafter. Expression of the corresponding receptor proteins also peaked during the early postnatal period but the subsequent decline was less dramatic from P14d to P12w compared to mRNA expression. In contrast, S1pr1 and S1pr3 exhibited more gradual developmental changes. Although early expression was higher than mature expression (3- to 6-fold), these receptors were still strongly expressed at P12w. The other isotypes examined, Lpar2, Lpar5, S1pr4, and S1pr5, were very weakly expressed at all developmental stages. Analysis of receptor distribution within the developing heart (P1d) revealed that Lpar1, Lpar3, and Lpar4 were expressed in the myocardium of all four chambers but not in valves, while Lpar3 was also uniquely expressed in the aorta and coronary vessels. Western blots revealed that the developmental changes in Lpar1, Lpar3, and Lpar4 protein expression mirrored changes in β-actin and β-tubulin expression. The increase in Lpar1 and Lpar4 receptors from P1d to P7d corresponds to the period of rapid myocardial growth and functional maturation. Moreover, the relatively high expression of Lpar1, Lpar3, and Lpar4 in the late prenatal rat heart suggests that these LPA receptors may also contribute to organogenesis. The increase in Lpar3 and Lpar4 expression concomitant with rising expression of cytoskeleton proteins further suggests a possible role for LPA signaling in cytoskeletal remodeling during cardiac development.     

Ontogenesis of the insulin receptor in the rabbit brain

We delineated the ontogeny of the brain insulin binding, insulin receptor number and affinity using plasma membranes isolated from the rabbit. Specific 125I-insulin binding and receptor number expressed per milligram of protein increased from the 20 day gestation fetus to the 1-day-old newborn, declining thereafter to attain adult values by day 6 of postnatal life. Specific 125I-insulin binding and the receptor number in the adult brain was less than the fetal and neonatal (1 day) brain receptors. Although a similar trend was observed specifically during fetal development, the changes in receptor number expressed per microgram DNA were not significant in the neonatal period. The adult brain insulin receptor number was higher than the 20- to 27-day fetus and similar to that of the 30-day fetus and the 1- to 5-day newborns. The total receptor number correlated linearly with the brain plasma membrane protein increment velocity. The affinity of the receptors increased during early fetal development (20-27 days) and remained constant thereafter in the postnatal period. We conclude that the ontogenic changes of the brain insulin receptors are similar to the ontogenic changes of brain plasma membrane protein. The developmental changes are more pronounced when the receptor number is expressed per milligram protein versus microgram DNA.     

Distinct, Developmentally Regulated Brain mRNAs Direct the Synthesis of Neurotransmitter Transporters

The Xenopus laevis oocyte expression system was utilized to define developmental and structural properties of neurotransmitter transporter mRNAs and the pharmacological characteristics of encoded carriers independent of the complexities of brain tissue preparations. Poly(A)+ RNA from dissected brain regions of neonatal and adult rats was microinjected into Xenopus oocytes and the expression of Na(+)-dependent neurotransmitter transporters determined 48 h later. Transport studies conducted with oocytes injected with RNAs derived from juvenile rat tissues indicate a region- and transporter-specific, postnatal increase in mRNA abundance as a major factor in the developmental changes observed for brain high-affinity amino acid uptake systems. Both L-glutamic acid (Glu) and gamma-aminobutyric acid (GABA) uptake systems were detectable by day 3 in postnatal forebrain mRNA and became progressively enriched during the next 2 weeks of forebrain development. In contrast, brainstem Glu and GABA transporter enrichment was 60-70% of adult values by day 3 and exceeded adult levels by day 10. Parallel determinations of L-glutamic acid decarboxylase mRNA abundance during development argue for distinct regulatory influences on mRNAs directing transmitter synthesis and reuptake. Glycine uptake could not be detected at any point of forebrain development and exhibited a gradual postnatal rise to adult levels over the first 3 postnatal weeks of brainstem development. Uptake studies conducted with well-characterized inhibitors of Glu, GABA, dopamine, and choline transport (D-aspartate, nipecotic acid, nomifensine, and hemicholinium-3, respectively) revealed that oocyte transporters encoded by adult rat brain mRNAs retained antagonist sensitivities exhibited by in vitro brain preparations. In addition, a differential regional sensitivity to the Glu transport antagonist dihydrokainate (1 mM) was observed, lending support to previous reports of region-specific Glu transporter subtypes. To determine the structural diversity present among brain transporter mRNAs, poly(A)+ RNA was size-fractionated on linear (10-31%) sucrose density gradients prior to oocyte injection. These experiments revealed two mRNA size classes (2.4-3.0 kb, 4.0-4.5 kb) independently capable of directing the synthesis of Glu, GABA, and glycine transporters. In regions other than the cerebellum, Glu and GABA transporter activities migrated as single, yet distinct, peaks of 4.0-4.5 kb. In contrast, both Glu and GABA transporters exhibited major peaks of activity at 2.5-3.0 kb with size-fractionated cerebellar mRNA. Brainstem glycine uptake exhibited a broad sedimentation profile, with peaks apparent at 2.4 and 4.0 kb. Taken together, these findings indicate previously unappreciated complexity in mRNA structure and regulation which underlies the expression of amino acid neurotransmitter uptake systems in the rodent CNS.     

Expression of low-molecular-weight neurofilament (NF-L) mRNA during postnatal development of the mouse brain

A regional Northern blot analysis demonstrated that the highest levels of NF-L mRNA in the adult mouse brain are present in brain stem followed by mid-brain, with lower levels found in neocortex, cerebellum, and hippocampus. The study was extended to the cellular level over the time course of postnatal development using in situ hybridization. This developmental analysis revealed that the expression of NF-L mRNA closely follows the differentiation pattern of many large neurons during postnatal neurogenesis. Neurons which differentiate early such as Purkinje, mitral, pyramidal, and large neurons of brain stem and thalamic nuclei, expressed high levels of NF-L mRNA at postnatal day 1. Early expression of NF-L mRNA may be required for the maintenance of the extensive neurofilament protein networks that are detected within the axons of larger neurons. Smaller neurons which differentiate later, such as dentate gyrus granule cells, small pyramidal and granule cells of the neocortex, and granule cells of the cerebellum, exhibit a delayed expression of NF-L mRNA.To whom to address reprint requests.     

Expression of Frizzleds and secreted frizzled-related proteins (Sfrps) during mammalian lens development

Recent studies indicate a role for Wnt signaling in regulating lens cell differentiation (Stump et al., 2003). Here we investigated expression patterns of Wnt receptors, the Frizzleds (Fzs) and the Wnt signaling regulators, the secreted frizzled-related proteins (Sfrps), during rodent lens development. RT-PCR showed that Fz receptors, Fz1-Fz8 are expressed in lens. In situ hybridization showed that all the Fz genes examined have similar expression patterns. Fzs are expressed throughout the early lens primordium. At embryonic day 14.5 (E14.5), Fz gene expression is predominantly localized to the epithelium and elongating cells at the lens equator. Fz expression is absent from lens fibers. This pattern of Fz gene expression continues throughout early postnatal development. Immunolocalization studies showed that Fz protein distribution closely follows that of the mRNAs. In addition, epithelial cells in FGF-treated explants show strongest Fz reactivity in cellular protrusions as they migrate and elongate. Sfrp1- Sfrp5 are expressed and all, except Sfrp2, have similar patterns of expression to each other and to the Fzs during lens development. Sfrp2 is strongly expressed in all lens pit cells but becomes restricted to the presumptive epithelial cells of the lens vesicle. By E14.5, Sfrp2 is only present in a few cells above the lens equator. Sfrp2 is not detected in the lens at E18.5 or at later stages. This study shows that multiple Fz and Sfrp genes are expressed during lens morphogenesis and differentiation. This is consistent with a role for Wnt-Fz signaling during both embryonic and postnatal lens development.     

Analysis by mRNA levels of the expression of six G protein alpha-subunit genes in mammalian cells and tissues.

The distribution and levels of expression of Gs alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Go alpha, and Gx alpha mRNAs were compared by Northern blot analysis using several rat tissues and selected human and rat cell lines. Gi1 alpha, Go alpha, and Gx alpha, were detected in a limited number of tissue and cells whereas Gi2 alpha, Gi3 alpha, and Gs alpha, were expressed in all the tissues and cells tested albeit in varying amounts. The expression of these six genes appears to be differentially regulated during postnatal development of the rat brain. High expression levels particularly of Go alpha, in young rat brain may be related to the formation of neurites during differentiation of nerve cells.