Association of TIMP-2 with extracellular matrix exposed to mechanical stress and its co-distribution with periostin during mouse mandible development

Matrix remodeling is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Periostin, originally identified in a mouse osteoblastic library, plays a role in cell adhesion and migration and in mechanical stress-induced matrix remodeling. In this study, we analyzed and compared the distribution patterns of TIMP-2 and periostin during mouse mandible development. Immunohistochemical staining for TIMP-2 and periostin was carried out on serial cryosections obtained from mice at embryonic days 13–16, postnatal day 2 (P2), P35, and 12 weeks of age. TIMP-2 and periostin exhibited a strikingly similar protein distribution during mandible development. From bud to early bell stages of molars, TIMP-2 and periostin were highly expressed on the lingual and anterior sides of the basement membrane and on the adjacent jaw mesenchyme. In pre- and postnatal incisors, the basement membrane of the apical loop and dental follicle was immunostained for TIMP-2 and periostin. At postnatal stages, TIMP-2 and periostin were prominently confined to the extracellular matrix (ECM) of gingival tissues, periodontal ligaments, and tendons (all recipients of mechanical strain). However, periostin was solely detected in the lower portion of the inner root sheath of hair follicles. Gingiva of P2 cultured in anti-TIMP-2 antibody-conditioned medium showed markedly reduced staining of periostin. We suggest that TIMP-2 and periostin are co-distributed on ECM exposed to mechanical forces and coordinately function as ECM modulators. This work was supported by the Japanese Ministry of Education, Culture, Sports, Science, and Technology and by Niigata University Research Projects.     

金属蛋白酶-2(MMP-2)和金属蛋白酶抑制因子(TIMP-1,-3)mRNA在小鼠胎盘中的表达

本实验利用原位杂交对小鼠妊娠不同时期胎盘中MMP－2,TIMP－2,－3mRNA的表达进行了研究。结果表明;MMP－2主要在具有很强的侵润能力的海绵滋养层细胞中表达,到妊娠13．5天时,MMP－2的表达明显降低,说明此时的滋养层细胞基本上失去侵润能力。TMIP－1和TMIP－3在滋养层细胞和蜕膜细胞中都有表达,这两种抑制因子的协同表达,一方面能够调控滋养层细胞侵入子宫内膜的深度,另一方面,滋养层细胞自身既表达MMP－2又表达TIMPs,可能对其自身有保护作用,使得MMP的水解功能局限于子宫蜕膜的特定区域。在妊娠10．5天,滋养层巨细胞同时表达TIMP－1,－3mRNA,这可能与其功能的转换是一致的;因为此时小鼠滋养层巨细胞体积最大,且不再增殖,同时其功能屯从侵入型向内分泌型转换。所以,MMPs和TIMPs在小鼠滋养层细胞和子宫蜕膜中的协同表达表明其在着床过程中可能发挥重要作用。     

An in situ hybridization study of MMP-2, -9, -13, -14, TIMP-1, and -2 mRNA in fetal mouse mandibular condylar cartilage as compared with limb bud cartilage

The main purpose of this in situ hybridization study was to investigate MMPs and TIMPs mRNA expression in developing mandibular condylar cartilage and limb bud cartilage. At E14.0, MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the periosteum of mandibular bone, and in the condylar anlage. At E15.0 MMP-2, -14, TIMP-1 and -2 mRNAs were expressed in the perichondrium of newly formed condylar cartilage and the periosteum of developing bone collar, whereas, expression of MMP-14 and TIMP-1 mRNAs were restricted to the inner layer of the periosteum/perichondrium. This expression patterns continued until E18.0. Further, from E13.0 to 14.0, in the developing tibial cartilage, MMP-2, -14, and TIMP-2 mRNAs were expressed in the periosteum/perichondrium, but weak MMP-14 and no TIMP-1 mRNA expression was recognized in the perichondrium. These results confirmed that the perichondrium of condylar cartilage has characteristics of periosteum, and suggested that MMPs and/or TIMPs are more actively involved in the development of condylar (secondary) cartilage than tibial (primary) cartilage. MMP-9-positive cells were observed in the bone collar of both types of cartilage, and they were consistent with osteoclasts/chondroclasts. MMP-13 mRNA expression was restricted to the chondrocytes of the lower hypertrophic cell zone in tibial cartilage at E14.0, indicating MMP-13 can be used as a marker for lower hypertrophic cell zone. It was also expressed in chondrocytes of newly formed condylar cartilage at E15.0, and continuously expressed in the lower hypertrophic cell zone until E18.0. These results confirmed that progenitor cells of condylar cartilage are rapidly differentiated into hypertrophic chondrocytes, which is a unique structural feature of secondary cartilage different from that of primary cartilage.     

Chondromodulin-I and tenomodulin are differentially expressed in the avascular mesenchyme during mouse and chick development

Chondromodulin-I (ChM-I) and tenomodulin (TeM) are homologous angiogenesis inhibitors. We have analyzed the spatial relationships between capillary networks and the localization of these molecules during mouse and chick development. ChM-I and TeM proteins have been localized to the PECAM-1-negative avascular region: ChM-I is expressed in the avascular cartilage, whereas TeM is detectable in dense connective tissues, including tendons and ligaments. We have also examined the vasculature of chick embryos by injection with India ink and have performed in situ hybridization of the ChM-I and TeM genes. The onset of ChM-I expression is associated with chondrogenesis during mouse embryonic development. ChM-I expression is also detectable in precartilaginous or noncartilaginous avascular mesenchyme in chick embryos, including the somite, sclerotome, and heart. Hence, the expression domains of ChM-I and TeM during vertebrate development incorporate the typical avascular regions of the mesenchymal tissues. This study was partly supported by Grants-in-Aid from the Ministry of Education, Culture, Sport, Science, and Technology of Japan and by the Tanabe Medical Frontier Conference.     

Comparative analysis of ABCG2-expressing and label-retaining cells in mouse submandibular gland

The submandibular gland (SMG) is a tissue that can be regenerated in a tissue injury model and that has adult stem cells capable of self-renewal and differentiation into functional cells. We have analyzed the localization of label-retaining cells (LRCs), which are putative progenitor cells, by using the BrdU-labeling method. 5-Bromo-2′-deoxyuridine (BrdU) injection followed by a long chasing period permitted the identification of LRCs based on the slow-cycling characteristic. In order to confirm the accurate localization of LRCs, BrdU and SMG-specific markers, including aquaporin5, cytokeratin, and smooth muscle actin, were examined by double-immunofluoresence staining. We found that LRCs were distributed in the acinus, duct, myoepithelium, and connective tissue. Moreover, ABCG2 (a known stem cell marker) was used for the characterization of LRCs and the localization of cells as putative stem/progenitor cells. ABCG2-expressing cells were distributed in various regions of the SMG but did not co-localize with LRCs. We suggest that putative progenitor cells exist in various regions of the SMG and have diverse capacities to differentiate into specific cells. Yeun-Jung Kim and Hyuk-Jae Kwon contributed equally to this work. This work was supported by Korea Research Foundation Grant (KRF-2006–013-E00143).     

Influence of extended operation time and of occlusal force on determination of pulpal healing pattern in replanted mouse molars

The mechanism regulating the divergent healing processes following tooth replantation is unclear. This study clarifies the relationship between the healing pattern, the time taken for tooth replantation, and the influence of occlusal force. We investigated the pulpal healing process after tooth replantation by immunohistochemistry for 5-bromo-2′-deoxyuridine and nestin and by histochemistry for tartrate-resistant acid phosphatase. The upper right first molar of 3-week-old mice was extracted and repositioned in the original socket immediately or 30 min to 6 h after the operation. We divided the animals into a non-occluded group in which the lower right first molar was extracted and an occluded group without extraction of the counterpart tooth. In control teeth (upper left first molar), the periphery of the coronal dental pulp showed intense nestin-positive reaction. Tooth replantation weakened the nestin-positive reaction in the pulp tissue. On postoperative days 5–7, tubular dentin formation commenced next to preexisting dentin in which nestin-positive odontoblast-like cells were arranged in successful cases. In other cases, bone-like tissue formation occurred in the pulp chamber until day 14. The ratio of tertiary dentin formation was significantly higher in the non-occluded group. The intentionally prolonged time for the completion of tooth replantation induced bone-like tissue formation, expanded inflammatory reaction, or fibrous tissue formation in pulp tissue. Thus, the lack of a proper oxygenated medium is probably decisive for the survival of odontoblast-lineage cells, and occlusal force during and/or after operation worsens the fate of these cells. This work was supported in part by KAKENHI (B) from MEXT, Japan (no. 16390523 to H.O.) and by the Japan-Korea Joint Research Project from JSPS and KOSEF.     

基质金属蛋白酶-2、-9及其组织抑制因子(TIMPs)在动情周期大鼠子宫中的表达(英文)

基质金属蛋白酶（ＭＭＰｓ）家族的作用是降解所有细胞外基质,其活性受其特异性组织抑制因子（ＴＩＭＰｓ）的抑制。细胞外基质成分的降解与重组在动物生殖生长过程中起重要作用,其变化可以通过ＭＭＰｓ和ＴＩＭＰｓ两者表达水平的变化进行监测。大鼠虽然没有月经形成,但是在其子宫内膜也出现类似灵长类的生殖生物学变化。本文从ＭＭＰｓ和ＴＩＭＰｓ两者的表达水平,对大鼠子宫内膜的这些变化进行了研究。于大鼠动情周期的不同时期,将其处死、取子宫制备酶粗提液和组织切片,采用酶谱法（ｚｙｍｏｙｒａｎｈｎ）和原位杂交方法研究动情周期大鼠子宫中ＭＭＰ－２和－９的活性变化以及ＭＭＰ－２、－９和ＴＩＭＰ－１、－２、－３ｍＲＮＡ的表达。并通过光密度扫描方法对酶谱结果进行半定量分析。所用杂交探针见Ｔａｂｌｅ１。酶谱结果显示：在动情周期大鼠子宫中只检测到６７ｋＤａ的ＭＭＰ－２活性,而没有检测到ＭＭＰ－９的活性（Ｆｉｇ．１）。ＭＭＰ－２的活性在动情前期最高,动情期和动情后期次之,间情期最低（Ｆｉｇ．２）。原位杂交结果显示：ＭＭＰ－２、－９、ＴＩＭＰ－１、－２、－３ｍＲＮＡ主要在子宫内膜基底部的基质细胞中表达。ＭＭＰ－２和－９ｍＲＮＡ在动情前期、动情期和动     

Differential expression of 11beta-hydroxysteroid dehydrogenase types 1 and 2 mRNA and glucocorticoid receptor protein during mouse embryonic development

Accumulating evidence suggests that the actions of glucocorticoids in target tissues are critically determined by the expression of not only the glucocorticoid receptor (GR) but also the glucocorticoid-metabolizing enzymes, known as 11β-hydroxysteroid dehydrogenase types 1 and 2 (11β-HSD1 and 11β-HSD2). To gain insight into the role of glucocorticoids in fetal development, the expression patterns of the two distinct 11β-HSD isozymes and GR were studied in the mouse embryo from embryonic day 12.5 (E12.5, TERM = E19) to postnatal day 0.5 (P0.5) by in situ hybridization and immunohistochemistry, respectively. 11β-HSD1 mRNA was detected in the heart as early as E12.5 and maintained thereafter. In the lung and liver, 11β-HSD1 mRNA was first detected between E14.5 and E16.5, increased to high levels towards term and maintained after birth. Relatively low levels of 11β-HSD1 mRNA were also detected in the kidney, adrenal glands and gastrointestinal tract at E18.5. However, the mRNA for 11β-HSD1 was undetectable in all other embryonic tissues including the brain. In contrast, kidney was the only organ that expressed appreciable levels of 11β-HSD2 mRNA during embryonic life. The level of 11β-HSD2 mRNA in the kidney increased dramatically in the newborn, which coincided with expression of 11β-HSD2 mRNA in the whisker follicle, tooth and salivary gland. Distinct from the profiles of 11β-HSD1 and 11β-HSD2 mRNA, GR protein was detectable in all tissues at all ages studied except for the thymus, salivary gland, and bone. Taken together, the present study demonstrates that tissue- and developmentally-stage specific expression of 11β-HSD1 and 11β-HSD2 as well as GR occurs in the developing mouse embryo, thus highlighting the importance of these two enzymes and GR in regulating glucocorticoid-mediated maturational events in specific tissues during murine embryonic development.     

Heart-type fatty acid binding proteins are upregulated during terminal differentiation of mouse cardiomyocytes,as revealed by proteomic analysis

At birth, the cardiomyocytes in the mouse neonatal heart still retain their ability to proliferate. However, this lasts only a few days and then the cardiomyocytes irreversibly lose their potential to divide. It is still not fully understood what factors are involved in the cessation of cardiomyocyte proliferation. Using proliferating cell nuclear antigen (PCNA) antibodies, we established that cardiomyocytes could divide extensively in 2-day-old mouse neonatal hearts and to a lesser extent in 6-day-old hearts. By 13 days, the cardiomyocytes have mostly stopped dividing. Comparative two-dimensional gel electrophoresis (2-DE) was performed on total proteins extracted from the 2-day- and 13-day-old hearts, in order to identify peptides that might be involved in the inhibition of cardiomyocyte proliferation. Using matrix-assisted laser desorption ionization mass spectroscopy (MALDI-TOF), we identified two protein spots that have the same molecular weight (approximately 14 kDa) but different pIs (5.9 and 6.1). Mass spectra analysis determined the proteins to be isoforms of the heart-type fatty acid binding protein (H-FABP). The pI 6.1 H-FABP is also known as mammary-derived growth inhibitor (MDGI; Specht et al. 1996). MGDI is a breast tumour growth suppressor gene capable of inhibiting tumour cell proliferation (Huynh et al. 1995). Both H-FABP isoforms were expressed in 2-day-old hearts but became strongly upregulated in 13-day-old hearts. We examined whether H-FABPs and PCNA were coexpressed in 2-, 6- and 13-day-old heart histological sections, using MDGI antibodies. The antibody could detect both forms of H-FABPs. It was established that there was a correlation between an increase in H-FABP expression and a decrease in PCNA expression. Hence, we tentatively propose that H-FABP isoforms are involved in regulating cardiomyocyte growth and differentiation in mouse neonatal hearts.This project was supported by a grant from the National Natural Science Foundation of China (Project No. 30340038).     

Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development

Frem1 belongs to a family of structurally related extracellular matrix proteins of which Fras1 is the founding member. Mutations in Fras1 and Frem1 have been identified in mouse models for Fraser syndrome, which display a strikingly similar embryonic skin blistering phenotype due to impaired dermal-epidermal adhesion. Here we show that Frem1 originates from both epithelial and mesenchymal cells, in contrast to Fras1 that is exclusively derived from epithelia. However, both proteins are localized in an absolutely overlapping fashion in diverse epithelial basement membranes. At the ultrastructural level, Frem1 exhibits a clustered arrangement in the sublamina densa coinciding with fibrillar structures reminiscent of anchoring fibrils. Furthermore, in addition to its extracellular deposition, around E16, Frem1 displays an intracellular distribution in distinct epidermal cell types such as the periderm layer and basal keratinocytes. Since periderm cells are known to participate in temporary epithelial fusions like embryonic eyelid closure, defective function of Frem1 in these cells could provide a molecular explanation for the "eyes open at birth" phenotype, a feature unique for Frem1 deficient mouse mutants. Finally, we demonstrate loss of Frem1 localization in the basement membrane but not in periderm cells in the skin of Fras1(-/-) embryos. Taken together, our findings indicate that besides a cooperative function with Fras1 in embryonic basement membranes, Frem1 can also act independently in processes related to epidermal differentiation.     

Identification and characterization of mouse PSF1-binding protein, SLD5

Although most somatic cells cannot proliferate, immature cells proliferate continuously to produce mature cells. Recently, we cloned mouse PSF1 from a hematopoietic stem cell specific cDNA library and reported that PSF1 is indispensable for the proliferation of immature cells. To identify the PSF1-binding protein, we used the yeast two-hybrid system with PSF1 as bait, and identified and cloned SLD5. SLD5 interacted with a central region of PSF1. Tissue distribution of SLD5 was quite similar to that of PSF1. When overexpressed, SLD5 protein was co-localized with PSF1. These data suggest that PSF1 and SLD5 may cooperate in the proliferation of immature cell populations.     

Immunohistochemical localization of the calcium/calmodulin-dependent protein phosphatase,calcineurin, in the mouse testis: its unique accumulation in spermatid nuclei

Immunohistochemical localization of a calmodulin-dependent protein phosphatase, calcineurin, was studied in the mouse testis in relation to previous observations showing that calmodulin is unusually rich in spermatogenic stages from mid-pachytene spermatocytes to elongating spermatids. The antibodies raised against calcineurin from scallop testis reacted with subunit B, but not subunit A, of calcineurin isoforms from mouse brain and testis. Indirect immunofluorescence using these antibodies on the mouse testis revealed positive reactions only in the nuclei of round or elongating spermatids: calcineurin started to accumulate in nuclei from the acrosomal cap phase, peaked at the initial stage of nuclear elongation, and decreased thereafter. There was almost no signal in the cytoplasm; spermatogenic cells at other stages, including spermatogonia, spermatocytes, mature sperm, and other somatic cells in the seminiferous tubules were totally negative. Immuno-electron microscopy gave the same result, on the basis of measuring the density of immunogold particles. These results suggest a role for calcineurin in remodeling of the nuclear chromatin in metamorphosing spermatids.     

Synergistic interaction of the two paralogous Arabidopsis genes LRX1 and LRX2 in cell wall formation during root hair development

LRR-extensins (LRX) form a family of structural cell wall proteins containing a receptor-like domain. The functional analysis of Arabidopsis LRX1 has shown that it is involved in cell morphogenesis of root hairs. In this work, we have studied LRX2, a paralog of LRX1. LRX2 expression is mainly found in roots and is responsive to factors promoting or repressing root hair formation. The function of LRX1 and LRX2 was tested by the expression of a truncated LRX2 and different LRX1/LRX2 chimaeric proteins. Using complementation of the lrx1 phenotype as the parameter for protein function, our experiments indicate that LRX1 and LRX2 are functionally similar but show differences in their activity. Genetic analysis revealed that single lrx2 mutants do not show any defect in root hair morphogenesis, but synergistically interact with the lrx1 mutation. lrx1/lrx2 double mutants have a significantly enhanced lrx1 phenotype, resulting in frequent rupture of the root hairs soon after their initiation. Analysis of the root hair cell wall ultrastructure by transmission electron microscopy (TEM) revealed the formation of osmophilic aggregates within the wall, as well as local disintegration of the wall structure in the double mutant, but not in wild-type plants. Our results indicate that LRX1 and LRX2 have overlapping functions in root hair formation, and that they likely regulate cell morphogenesis by promoting proper development of the cell wall.     

Selective inhibition of ventral temporal but not dorsal nasal neurites from mouse retinal explants during contact with chondroitin sulphate

We have determined whether chondroitin sulphate (CS) glycosaminoglycans are sufficient to direct a selective inhibition of neurite growth from ventral temporal (VT) but not from dorsal nasal (DN) retina in mouse embryos; this may underlie the formation of axon divergence in the optic chiasm. Explants from the retinal region of embryonic day-14 mouse were grown on a laminin–polylysine substrate near to a circular spot coated with CS. In control cultures, in which no CS was added to the spot, both VT and DN retinal neurites grew extensively into the coated territory. When presented with spots coated with 10 mg/ml CS, neurite growth from the VT retina into the CS territory was dramatically reduced but that from the DN retina was not significantly affected. The selective inhibition to VT neurites was completely abolished by treatment with chondroitinase ABC, indicating a specific contribution of CS glycosaminoglycan in this regionally specific behaviour. This differential behaviour was not observed in explants presented with a lower or higher concentration of CS or in explants grown on substrate coated with a different laminin concentration. Thus, a critical ratio of CS to laminin seems to be essential to induce this differential behaviour in retinal neurites towards contact with CS. Furthermore, this behavior was not observed in explants cultured directly on a CS-rich substrate, suggesting that contact with growth-promoting molecules is necessary for the selective responses of retinal neurites during subsequent contact with CS. We concluded that CS glycosaminoglycan is sufficient to drive selective inhibition of VT but not DN neurites and that, together with a critical combination of growth-promoting factors, it may control the axon divergence process at the mouse optic chiasm.This project is partially supported by a grant from the Research Grant Council of the Hong Kong Special Administrative Region (project no. CUHK 4417/03M) and a Direct Grant from the Chinese University of HK (project no. 2004.1.051).     

Dkk1, -2, and -3 expression in mouse craniofacial development

Summary The Dickkopf family is important for embryogenesis and postnatal development and growth. Dkk1 is a strong head inducer and knockout of this gene leads to absence of anterior head structures, which are predominantly formed through neural crest migration. During early craniofacial development, Dkk1 to Dkk3 show developmentally regulated expression in a number of elements. However, their expression and roles in late times of craniofacial development are largely unknown. This study focuses on the expression profile of Dkk1-3 on late embryonic and early postnatal stages. It was found that Dkks were involved in a variety of craniofacial developmental processes, including facial outgrowth, myogenesis, osteogenesis, palatogenesis, olfactory epithelium and tooth development; and the expression persisted to postnatal stage in the muscles and bones. Their expression patterns suggest important roles in these processes; further study is warranted to elucidate these roles.