Seita Hagino

Slit and glypican-1 mRNAs are coexpressed in the reactive astrocytes of the injured adult brain

The slit family serves as a repellent for growing axons toward correct targets during neural development. A recent report describes slit mRNAs expressed in various brain regions in adult rats. However, their functions in the adult nervous system remain unknown. In the present study, we investigated whether slit mRNAs were expressed in the cryo‐injured brain, using in situ hybridization. All slit family members were expressed at the lesion. Slit2 mRNA was the most intensely expressed in the cells surrounding the necrotic tissue. A double‐labeling study showed that slit2 mRNA was expressed in the glial fibrillary acidic protein (GFAP)‐positive reactive astrocytes. In addition, glypican‐1, a heparan sulfate proteoglycan that serves as a high‐affinity receptor for Slit protein, was coexpressed with slit2 mRNA in the reactive astrocytes. These findings suggested that slit2 might prevent regenerating axons from entering into the lesion in concert with glypican‐1. GLIA 42:130–138, 2003.

The LIM homeobox gene, L3/Lhx8, is necessary for proper development of basal forebrain cholinergic neurons.

Basal forebrain cholinergic neurons (BFCNs) are involved in cognitive functions such as learning and memory, and are affected in several neurodegenerative diseases (e.g. Alzheimers disease). Despite their importance, the molecular mechanisms of their development are not fully elucidated. A recent report demonstrated that some BFCNs in adult rat are positive for L3/Lhx8, a LIM homeobox transcription factor. To examine the function of L3/Lhx8 in the development of BFCNs, we generated L3/Lhx8 gene‐disrupted mice. In these mice, cells expressing cholinergic neuron markers, such as choline acetyltransferase, vesicular acetylcholine transporter and p75 low‐affinity NGF receptor, were markedly reduced in the basal forebrain, whereas other cholinergic neurons including brain stem and spinal motor neurons expressed the markers. Neurotransmitter phenotypes other than cholinergic in the basal forebrain appeared intact. From these results, we suggested that L3/Lhx8 has a pivotal and specific role in the development and/or maintenance of BFCNs.

Increased syndecan expression by pleiotrophin and FGF receptor-expressing astrocytes in injured brain tissue.

Syndecan‐1, ‐2, ‐3, and ‐4 are heparan sulfate proteoglycans that are differentially expressed during development and wound repair. To determine whether syndecans are also involved in brain injury, we examined the expression of syndecan core proteins genes in cryo‐injured mouse brain, using in situ hybridization. All syndecan mRNA transcripts were similarly expressed in the region surrounding the necrotic tissue, exhibiting peak levels at day 7 after injury. Comparison with cellular markers showed that reactive astrocytes were the primary source of syndecans. Syndecans serve as co‐receptors for fibroblast growth factor (FGF) and as a reservoir for another heparin‐binding growth factor, pleiotrophin (PTN, or heparin‐binding growth‐associated molecule. In our model, FGF receptor1 (FGFR1) and PTN mRNA levels were upregulated in reactive astrocytes. The distribution patterns of FGFR1 and PTN overlapped considerably with those of syndecan‐1 and ‐3 mRNAs, respectively. These results suggest that syndecans are expressed primarily in reactive astrocytes, and may provide a supportive environment for regenerating axons in concert with heparin‐binding growth factors (e.g., FGF and PTN) in the injured brain. GLIA 39:1–9, 2002.

Comparison of expression patterns between CREB family transcription factor OASIS and proteoglycan core protein genes during murine tooth development.

The transcription factor OASIS gene, which encodes for a CREB/ATF family member, is specifically expressed in the salivary gland, the cartilage and the tooth germs of the mouse embryo. In the present study, the expression patterns were compared between OASIS mRNA and major vertebrate proteoglycans, which might be the downstream genes of OASIS in the tooth germs of mouse first mandibular molars, through in situ hybridization histochemistry. OASIS mRNA expression was observed in the inner enamel epithelium during the cap and bell stages (E14.5-E18.5) in the preodontoblasts during differentiation stage (E18.5-P0) and in the differentiating odontoblasts during the early secretory stage (P2.5-P4.5). Proteoglycans (versican, decorin, biglycan, glypican, syndecan-1, and syndecan-3) were expressed in the tooth germs in various patterns. Decorin, biglycan, syndecan-1 and syndecan-3 showed gene expressions overlapping with OASIS. Especially the expression pattern of decorin and syndecan-3 coincided temporally and spatially exactly with that of OASIS. These results suggest that the OASIS gene might be related to proteoglycan expression and may play an important role in the differentiation of the odontoblast and cells in inner enamel epithelium.

Expression pattern of glypican-1 mRNA after brain injury in mice.

Glypican-1, a heparan sulfate proteoglycan, is expressed in various tissues including developing and postnatal central nervous system. It serves as a receptor for heparin-binding molecules such as fibroblast growth factors (FGFs). We investigated whether glypican-1 was expressed after brain injury in adult mice. In situ hybridization study showed that glypican-1 mRNA was expressed in the region surrounding necrotic tissue, and that the signal intensity peaked 7 days after the cryo-injury. In addition, both FGF-2 and amyloid precursor protein (APP) were concurrently upregulated and colocalized with glypican-1 mRNA. Since FGF-2 and APP can bind to glypican-1 in vitro, the present study suggested that their autocrine/paracrine interactions with glypican-1 may be involved in neuronal regeneration and/or neurite-outgrowth inhibition after brain injury.

Expression of OASIS, a CREB/ATF family transcription factor, in CNS lesion and its transcriptional activity.

We reported the expression patterns of a novel member of the CREB/ATF family, OASIS, in central nervous system (CNS) lesions and its transcriptional activity. OASIS gene expression was upregulated in the stab-injured spinal cord. Double labeling experiments revealed that the distribution of OASIS mRNA-positive cells overlapped with a population of GFAP-immunoreactive cells. This finding suggested that OASIS might regulate expression of important downstream molecules in certain subset of the reactive astrocytes (e.g. inhibitory substances in injured brain). In gel shift assays, OASIS was able to specifically bind to CRE as CREB family members were. We then examined transcriptional activity of full-length OASIS with GAL4-UAS-luciferase reporter assay in COS7 cells. OASIS protein activated transcription, but did not inhibit basal transcription driven by AdML promoter. To determine critical portion(s) of the OASIS protein in transcriptional activation, we examined the activity of various deletion constructs of OASIS gene. The assay revealed that a strong transcriptional activation domain lay in the N-terminal region where acidic amino acids clustered and a possible repression domain, which had not been reported for other CREB/ATF family members, lay in the more C-terminal region. We therefore proposed that OASIS protein positively regulated gene transcription in a subset of reactive astrocytes, and thereby influenced the reaction of injured CNS tissues.

Uninephrectomy induces progressive glomerulosclerosis and apoptosis in anti-Thy1 glomerulonephritis

Administration of the anti‐Thy1 antibody in rats induces reversible glomerulonephritis resembling human mesangiolytic and mesangioproliferative diseases. The purpose of the present study was to design a model of irreversible glomerulosclerosis, using the anti‐Thy1 antibody injection after uninephrectomy, and examine it, focusing on apoptosis in the process of progressive sclerotic changes. Wistar rats were divided into three groups: one‐kidney groups (group I and III) and a two‐kidney group (group II). All groups were injected with the anti‐Thy1 antibody (OX‐7) at day 0, and group I and III were uninephrectomized at day −6. Only group III rats were given a half dose of OX‐7 as compared with group I and II. Rats were killed for histological examinations at days 7, 14 and 30. In group I, progressive glomerular lesions, such as glomerular adhesion to Bowmans capsule, crescent formation, and collapse of capillary tufts were observed at days 14 and 30. No significant differences were observed in the pathological findings between group I and III. There was a significantly higher number of glomerular terminal deoxynucleotidyltransferase‐mediated dUTP nick end labeling‐positive cells in group I as compared to group II at days 7 and 14. Moreover, the glomerular expression of transforming growth factor‐β, heparan sulfate proteoglycan and chondroitin sulfate proteoglycan significantly increased in group I as compared to group II at days 7 and 14. Progressive glomerulosclerosis can be induced in the rat by a single injection of the anti‐Thy1 antibody after unilateral nephrectomy. It is suggested that apoptosis and extracellular matrix accumulation play an important role in the development of glomerulosclerosis.

Differential expression of decorin and biglycan genes during palatogenesis in normal and retinoic acid-treated mice

Proteoglycans are involved in secondary palate formation. In the present study, we focused on two small leucine‐rich proteoglycans, decorin and biglycan, because they assembled extracellular matrix molecules such as collagens and modulated signaling pathway of transforming growth factor‐β. To investigate the functions of decorin and biglycan in palatogenesis, we compared their mRNA expression patterns between normal palate and retinoic acid‐induced cleft palate in mice by using in situ hybridization analysis during the period of embryonic day 13.5 (E13.5) to E15.5. On E13.5, decorin mRNA was expressed in the epithelia and mesenchyme on the nasal side of the developing secondary palate. During the period the palate shelves were fusing (E14.5), decorin mRNA was strongly expressed in the mesenchyme but its expression pattern was asymmetric; decorin mRNA expression area in the nasal side was broader than that in the oral side. The expression of decorin mRNA was hardly detected in the mesenchyme on either side of the medial edge epithelium. After fusion (E15.5), its expression converged to the mesenchyme just around the palatine bone. Biglycan mRNA was ubiquitously distributed throughout the palatal mesenchyme for the mid‐gestation period. Its expression area became limited to the ossification area within the palate after the late gestation period. In the retinoic acid–treated mice, the area of the decorin gene expression expanded to the core region of the palate primordium where little signal was observed in control mice. On the other hand, biglycan in the retinoic acid–treated mice did not show remarkable change in its distribution patterns compared with that in the control mice. These findings suggest that decorin and biglycan play distinct roles in palatogenesis, and decorin was more actively involved in the process of secondary palate formation than biglycan. Up‐regulation of decorin gene expression in the retinoic acid–treated mice might influence the pathogenesis of cleft palate. Developmental Dynamics 226:618–626, 2003.