Expression and localization of versican during postnatal development of rat temporomandibular joint disc

To analyze the growth-related changes in extracellular matrix components in temporomandibular joint (TMJ) discs, the expression and localization of the core protein of a large chondroitin sulphate proteoglycan, versican, in rat TMJ discs during postnatal development (2–32 weeks) were examined using Western blot analysis, real-time quantitative PCR and immunohistochemistry. Western blot analysis showed that rat TMJ discs predominantly expressed one isoform (V1) and the core protein sharply increased after birth, reached a peak at 8 weeks, and then gradually decreased up to 32 weeks. Real-time quantitative PCR with TaqMan probes indicated that mRNA expression of versican was highest at 2 weeks and gradually decreased with growth. An immunohistochemical study showed that staining for versican was weak and evenly distributed in TMJ discs at 2 weeks. Regional differences in staining for versican became prominent after 8 weeks; staining was intense in the anterior and posterior peripheral attachments, and weak in the central part of the discs. These results demonstrate that growth-related changes and regional differences exist in the expression of versican in the TMJ discs of growing rats, and these probably reflect the changes in the biomechanical environment caused by the development of orofacial functions.     

Expression of mitogen-activated protein kinase pathways during postnatal development of rat heart

The loss of ability to proliferate (terminal differentiation) and reduction in capability to resist ischemia are key phenomena observed during postnatal development of the heart. Mitogen-activated protein kinases (MAPKs) mediate signaling pathways for cell proliferation/differentiation and stress responses such as ischemia. In this study, the expression of these kinases and their associated kinases were investigated in rat heart ventricle. Extracts of 1-, 10-, 20-, 50-, and 365-day-old rat heart ventricles were probed with specific antibodies and their immunoreactivities were quantified by densitometry. Most of the mitogenic protein kinases including Raf1, RafB, Mek1, Erk2, and Rsk1 were significantly down-regulated, whereas the stress signaling kinases, such as Mlk3, Mekk1, Sek1, Mkk3, and Mapkapk2 were up-regulated in expression during postnatal development. Most MAP kinases including Erk1, JNKs, p38 Hog, as well as Rsk2, however, did not exhibit postnatal changes in expression. The proto-oncogene-encoded kinases Mos and Cot/Tpl 2 were up-regulated up to two- and four-fold, respectively, during development. Pak1, which may be involved in the regulation of cytoskeleton as well as in stress signaling, was downregulated with age, but the Pak2 isoform increased only after 50 days. All of these proteins, except RafB, were also detected in the isolated adult ventricular myocytes at comparable levels to those found in adult ventricle. Tissue distribution studies revealed that most of the protein kinases that were up-regulated during heart development tended to be preferentially expressed in heart, whereas the downregulated protein kinases were generally expressed in heart at relatively lesser amounts than in most of other tissues. J. Cell. Biochem. 71:286–301, 1998. © 1998 Wiley-Liss, Inc.     

Expression of TRPC6 in renal cortex and hippocampus of mouse during postnatal development

TRPC6, a member of the TRPC family, attracts much attention from the public because of its relationship with the disease. In both the brain and kidney, TRPC6 serves a variety of functions. The aim of the present study was to observe the expression and effects of TRPC6 in renal cortex and hippocampus during early?postnatal development of?the mouse. In the present study, immunohistochemistry and Western blotting were used to detect the expression of TRPC6 in the mouse kidney and hippocampus of postnatal day 1, 3, 5, 7, 14, 21, 28 and 49 (P1, P3, P5, P7, P14, P21, P28 and P49). Results showed that the expression of TRPC6 was increased in the mouse hippocampus, and there was a significant increase between P7 and P14 during the postnatal development. Meanwhile, the expression of TRPC6 was also detected in glomerulus and tubules, and a decreased expression was found during postnatal maturation of mouse renal cortex. From these in vivo experiments, we concluded that the expression of TRPC6 was active in the developing mouse kidney cortex, and followed a loss of expression with the development of kidney. Meanwhile, an increased expression was found in the hippocampus with the development. Together, these data suggested that the developmental changes in TRPC6 expression might be required for proper postnatal kidney cortex development, and played a critical role in the hippocampus during development, which formed the basis for understanding the nephrogenesis and neurogenesis in mice and provided a practically useful knowledge to the clinical and related research.     

Expression of galectin 4 in the rat small intestine during postnatal development

We determined the expression of an endogenous lectin, galectin 4, in the rat small intestine during postnatal development. The mRNA levels of galectin 4 did not change significantly between birth and adulthood. In contrast, the protein was present at higher levels after than before weaning, and the potential ligands for galectin 4 were more highly represented in the enterocyte microvilli of weaned than of suckling rats. These results suggest possible differences either in galectin 4 mRNA stability, in its translation regulation or in the protein stability.     

Expression of P2X receptors on immune cells in the rat liver during postnatal development

Single and double-labeling immunofluorescence and RT-PCR expression of P2X receptor proteins and mRNAs were used in a study of the liver of postnatal rats. OX62 and ED1 were used as markers for dendritic and macrophage (Kupffer) cells respectively. The results showed that the P2X₆ receptor subunit was up-regulated by 15-fold on hepatic sinusoid cells during postnatal days P1 to P60. Subpopulations of Kupffer cells co-expressed P2X₄ and P2X₆ receptor subunits and dendritic cells co-expressed P2X₄ and P2X₇ receptor subunits. Lipopolysaccharide (endotoxin) injected into the peritoneal cavity led to increased expression of the P2X₆ receptor on Kupffer cells, suggesting that the P2X₆ receptor subunit may be up-regulated by endotoxin. This study presents the first evidence that P2X receptors are widely distributed in the rat liver immune system and that activation of Kupffer and dendritic cells in the rat liver might be regulated by extracellular ATP.     

Expression of GABA transporters,GAT-1 and GAT-3, in the cerebral cortex and thalamus of the rat during postnatal development

The cellular and subcellular localization of two GABA transporters, GAT-1 and GAT-3, was investigated using immunocytochemical methods in the rat cerebral cortex and thalamus during postnatal development. The distribution of the transporters is compared with that of the neuronal marker GABA, and with that of vimentin and of glial fibrillary acidic protein, which identify immature and mature astrocytes, respectively. Our observations show that the two transporters are already expressed at birth in both brain areas with the same cellular localization as in adult rats, as GAT-1 is present in growth cones and terminals only in the cortex, whereas both transporters are expressed in astrocytes in the cortex and thalamus. The distribution of GAT-1 and GAT-3 undergoes postnatal changes reflecting in general the neurogenetic events of the neocortex and thalamus and, more specifically, the maturation of GABAergic innervation. The adult-like pattern of expression is achieved in the third postnatal week in the cortex and in the second postnatal week in the thalamus. The early expression of GAT-1 in GABAergic terminals confirms previous studies showing the existence of neuronal mechanisms of GABA uptake from the embryonic stages. As for the glial localization, the precocious existence of two astrocytic GABA transporters suggests that they operate through different functional mechanisms from birth, whereas their exclusively glial expression in the thalamus indicates that the astroglia plays a major role in the transport, recycling and metabolism of thalamic GABA.     

Characterization of Na+/H+ Exchanger Isoform (NHE1, NHE2 and NHE3) Expression in Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. Na⁺/H⁺ exchange (NHE) is one of the major Na⁺ absorptive pathways in gallbladder. In this study, we measured gallbladder Na⁺/H⁺ exchange and characterized the NHE isoforms expressed in prairie dogs. Na⁺/H⁺ exchange activity was assessed by measuring amiloride-inhibitable transepithelial Na⁺ flux and apical ²²Na⁺ uptake using dimethylamiloride (DMA). HOE-694 was used to determine NHE2 and NHE3 contributions. Basal J ^Na _ms was higher than J ^Na _sm with J ^Na _net absorption. Mucosal DMA inhibited transepithelial Na⁺ flux in a dose-dependent fashion, causing J ^Na _ms equal to J ^Na _sm and blocking J ^Na _net absorption at 100 μm. Basal ²²Na⁺ uptake rate was 10.9 ± 1.0 μmol · cm⁻²· hr⁻¹ which was inhibited by ∼43% by mucosal DMA and ∼30% by mucosal HOE-694 at 100 μm. RT-PCR and Northern blot analysis demonstrated expression of mRNAs encoding NHE1, NHE2 and NHE3 in the gallbladder. Expression of NHE1, NHE2 and NHE3 polypeptides was confirmed using isoform-specific anti-NHE antibodies. These data suggest that Na⁺/H⁺ exchange accounts for a substantial fraction of gallbladder apical Na⁺ entry and most of net Na⁺ absorption in prairie dogs. The NHE2 and NHE3 isoforms, but not NHE1, are involved in gallbladder apical Na⁺ uptake and transepithelial Na⁺ absorption. Received: 9 February 2001/Revised: 11 April 2001     

Expression of second messenger- and cyclin- dependent protein kinases during postnatal development of rat heart

During early postnatal development, cardiomyocytes, which comprise about 80% of ventricular mass and volume, become phenotypically developed to facilitate their contractile functions and terminally differentiated to grow only in size but not in cell number. These changes are due to the expression of contractile proteins as well as the regulation of intracellular signal transduction proteins. In this study, the expression patterns of several protein kinases involved in various cardiac functions and cell-cycle control were analyzed by Western blotting of ventricular extracts from 1-, 10-, 20-, 50-, and 365-day-old rats. The expression level of cAMP-dependent protein kinase was slightly decreased (20%) over the first year, whereas no change was detected in cGMP-dependent protein kinase I. Calmodulin-dependent protein kinase II, which is involved in Ca²⁺ uptake into the sarcoplasmic reticulum, was increased as much as ten-fold. To the contrary, the expressions of protein kinase C-α and ι declined 77% with age. Cyclin-dependent protein kinases (CDKs) such as CDK1, CDK2, CDK4, and CDK5, which are required for cell-cycle progression, abruptly declined to almost undetectable levels after 10–20 days of age. In contrast, other CDK-related kinases, such as CDK8 or Kkialre, did not change significantly or increased up to 50% with age, respectively. Protein kinases implicated in CDK regulation such as CDK7 and Wee1 were either slightly increased in expression or did not change significantly. All of the proteins that were detected in ventricular extracts were also identified in isolated cardiac myocytes in equivalent amounts and analyzed for their relative expression in ten other adult rat tissues. J. Cell. Biochem. 69:506–521, 1998. © 1998 Wiley-Liss, Inc.     

Expression of GFRalpha-1, receptor for GDNF, in rat brain capillary during postnatal development of the BBB

It is well known thatthe blood-brain barrier (BBB) matures at ~2 wk after birth in therat. Recently, we showed that glial cell line-derived neurotrophicfactor (GDNF) enhances the barrier function of porcine endothelialcells forming the BBB in culture. In the present study, we examined therelation between permeability of the BBB, using Evans blue as a tracer,and expression of the GDNF family receptor (GFR-1) during postnataldevelopment of the BBB. Morphometric analysis showed that exudation ofEvans blue from capillaries of the cerebral cortex progressivelydecreased until postnatal day 21. Inversely,immunohistochemical examinations showed expression of GFR-1 in thecapillaries at postnatal day 3 and expression that reachedthe same levels as observed in adult rats by postnatal day10. However, c-ret, which is thought to mediate asignal evoked by binding of GDNF to GFR-1, was not expressed in thecapillaries of the brain cortex in 3-mo-old rats. On the other hand,the tight junction proteins occludin and ZO-1 appeared to be fullyexpressed at birth. The reciprocal relation between GFR-1 expressionand the permeability of the BBB strongly suggests active participationof GDNF in postnatal development of the BBB, although the mechanism(s)involved is still veiled.

     

5alpha-reductase isoenzymes 1 and 2 in the rat testis during postnatal development

The pubertal initiation of spermatogenesis is reliant on androgens, and during this time, 5alpha-reduced androgens such as dihydrotestosterone (DHT) are the predominant androgens in the testis. Two 5alpha-reductase (5alphaR) isoenzymes (5alphaR1 and 5alphaR2) have been identified, which catalyze the conversion of testosterone to the more potent androgen DHT. The present study aimed to investigate the developmental pattern of 5alphaR isoenzymes and their relationship to the production of 5alpha-reduced androgens in the postnatal rat testis. Both 5alphaR1 and 5alphaR2 isoenzyme mRNAs were measured by real-time polymerase chain reaction, isoenzyme activity levels by specific assays, and testicular androgens by radioimmunoassay after high-performance liquid chromatographic separation. Both 5alphaR1 and 5alphaR2 mRNAs and activity levels were low in the 10-day-old (prepubertal) testis, peaked between Days 20 and 40 during puberty, and then declined to low levels at 60-160 days of age. The developmental pattern of both 5alphaR isoenzyme activity levels was mirrored by the testicular production of 5alpha-reduced metabolites. Although 5alphaR1 was greater than 5alphaR2 at all ages, it is likely, given the substrate preferences of the two, that both isoenzymes contribute to the pubertal peak of 5alpha-reduced androgen biosynthesis. The peak in 5alphaR isoenzymes and 5alpha-reduced metabolite production coincided with the first wave of spermatogenesis in the rat, suggesting a role for 5alpha-reduced metabolites in the initiation of spermatogenesis. This was explored by acute administration of a 5alphaR inhibitor (L685,273) to immature rats. The L685,273 markedly suppressed testicular 5alphaR activity during puberty by 75%-86%. However, a marked increase was observed in testicular testosterone levels (in the absence of changes in LH), and no decrease was observed in the absolute levels of 5alpha-reduced metabolites. Therefore, whether the formation of DHT in the presence of low testosterone levels in the pubertal testis is required for the initiation of spermatogenesis cannot be tested using 5alphaR inhibitors. We conclude that both 5alphaR1 and 5alphaR2 isoenzymes are involved in the peak of 5alpha-reduced androgen biosynthesis in the testis during the pubertal initiation of spermatogenesis.     

Expression of p97/VCP and ubiquitin during postnatal development of the degenerating rat retina

In this study, we aimed to investigate the distribution pattern of ubiquitin and p97/VCP in the rat retina during postnatal development. Eyeballs from 1-, 4-, 10-, 36- and 72-week-old rats were examined by immunohistochemistry, and protein colocalization was determined by immunofluorescence microscopy. In the 1-week-old rat retina, p97/VCP was strongly expressed in the neuroblast layer, however no ubiquitin immunoreactivity was observed. p97/VCP immunoreactivity was present in the ganglion cell layer (GCL), inner nuclear layer (INL), outer nuclear layer (ONL), inner segment (IS) of the photoreceptor layer, and retinal pigment epithelium in the 4- and 10-week-old rat retinas. p97/VCP immunoreactivity increased significantly in the 10-week-old rat retinas. Ubiquitin was barely seen in the 4-week-old rat retinas, and ubiquitin expression was weak in the GCL and the IPL of the 10-week-old rat retinas. In the 36- and 72-week-old rats, the presence of ubiquitin was remarkable in the IS, INL, IPL and GCL, however, p97/VCP immunoreactivity was significantly decreased. Colocalization of ubiquitin and p97/VCP was also observed in the INL, IS, GCL and ONL of 36- and 72-week-old rat retinas. Our results indicate that p97/VCP immunoreactivity in the retina significantly decreases after rats reach 10 weeks of age, whereas ubiquitin immunoreactivity increases with aging. These results suggest that an altered expression pattern of p97/VCP and ubiquitin in the developing rat retina may associate with age-related retinal degeneration.     

Expression of TLR2 and TLR4 in murine small intestine during postnatal development

The important role played by the gut microbiota in host immunity is mediated, in part, through toll-like receptors (TLRs). We evaluated the postnatal changes in expression of TLR2 and TLR4 in the murine small intestine and assessed how expression is influenced by gut microbiota. The expression of TLR2 and TLR4 in the murine small intestine was highly dynamic during development. The changes were especially profound during the suckling period, with the maximal mRNA levels detected in the mid-suckling period. Immunohistochemical and flow-cytometric analyses indicated that the changes in TLR2 and TLR4 expression involve primarily epithelial cells. The germ-free mice showed minor changes in TLR2/TLR4 mRNA and TLR2 protein during the suckling period. This study demonstrated that the postnatal expression of TLR2 and TLR4 in small intestinal epithelial cells is dynamic and depends on the presence of commensal intestinal microbiota.     

Expression of gap junction genes during postnatal neural development

The timing of appearance of mRNAs encoding gap junction proteins was examined during development of the rat and mouse brain. Complementary DNAs (cDNAs) specific for the mRNA for the liver-type gap junction protein, connexin32, and the heart-type gap junction protein, connexin43, were used to probe Northern blots of total RNA isolated from the forebrain and hindbrain of mice and rats at various times before and after birth. Prior to postnatal day 10, connexin32 mRNA is detectable only at low levels. By postnatal days 10 to 16, a sharp increase occurs in the level of this mRNA. This increase is detectable first in the hindbrain, and subsequently in the forebrain. In contrast, connexin43 mRNA is readily detectable at birth, and the level of this mRNA also increases during subsequent development. The developmental appearance of the gap junction proteins, connexin32 and connexin43, was similar to that of their respective mRNAs. These results indicate that the genes encoding connexin32 and connexin43 are differentially expressed during neural development.     

The septal organ of the rat during postnatal development

The septal organ of Masera (SO) is a small, isolated patch of olfactory epithelium, located in the ventral part of the nasal septum. We investigated in this systematic study the postnatal development of the SO in histological sections of rats at various ages from the day of birth (P1) to P666. The SO-area increases to a maximum at P66-P105, just as the animals reach sexual maturity, and decreases thereafter, significantly however only in males, indicating a limited neurogenetic capacity for regeneration. In contrast, the main olfactory epithelium area continues to expand beyond P300. The modified respiratory epithelium ('zwischen epithelium') separating the SO and the main olfactory epithelium contains a few olfactory neurons up to age P66. Its length increases postnatally so that the SO becomes more ventral to the OE. Although the position of the SO relative to other anatomical landmarks changes with development it is consistently located just posterior to the opening of the nasopalatine duct (NPAL). Thus, a possible function of the SO is in sensing chemicals in fluids entering the mouth by licking and then delivered to the nasal cavity via the NPAL; therefore the SO may be involved in social/sexual behavior as is the vomeronasal organ (VNO). We suggest that the SO is a separate accessory olfactory organ with properties somewhat different from both OE and VNO and may exist only in species where the NPAL does not open into the VNO.     

Expression of α2A 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 α_2A 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 α_2A adrenoceptors in neocortex during late embryonic and early postnatal development using in situ hybridization and receptor autoradiography. Functional α₂ receptors in embryonic rat cortex were also detected using agonist stimulated [³⁵S]GTPγS autoradiography. Both α_2A 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 α_2A adrenoceptors was restricted to the lateral neocortex. At E20, functional α₂ adrenoceptors were also detected in deep layers of lateral neocortex. During the first week of postnatal development, the expression of α_2A mRNA and protein changed markedly, giving rise to a more mature pattern of anatomical distribution. The temporal and spatial distribution of α_2A adrenoceptors in developing neocortex is consistent with expression of functional proteins on migrating and differentiating layer IV to II neurons. These findings suggest that α_2A 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 α_2A mRNA expression. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 259–269, 1999     

Growth patterns of rat hepatocytes during postnatal development.

During postnatal growth in the liver of the rat, a characteristic shift towards binuclear cells and cells of higher ploidy class occurs. When the protein content of individual isolated hepatocytes of different ploidy classes is analysed cytophotometrically using the specific protein stain Naphthol Yellow S, it appears that the growth in mass in the period 30-99 days is due mainly to increase of protein content of binuclear diploid (BD) and mononuclear tetraploid (MT) cells. The mononuclear diploid (MD) cells play a quickly diminishing role in the parenchymal population after the initial growth phase and cells of highest ploidy degree remain unimportant quantitatively. The quickly growing BD and MT cells only reach a Naphthol Yellow S protein value twice that of MD cells after a certain period of growth, whereas changes in protein content are slight or absent from 99 days onwards in all cell types investigated.