Temporal effects of Sprouty on lung morphogenesis

Paracrine signaling mediated by FGF-10 and the FGF-R2IIIb receptor is required for formation of the lung. To determine the temporal requirements for FGF signaling during pulmonary morphogenesis, Sprouty-4 (Spry-4), an intracellular FGF receptor antagonist, was expressed in epithelial cells of the fetal lung under control of a doxycycline-inducible system. Severe defects in lobulation and severe lung hypoplasia were observed when Spry-4 was expressed throughout fetal lung development (E6.5-E18.5) or from E6.5 until E13.5. Effects of Spry-4 on branching were substantially reversed by removal of doxycycline from the dam at E12.5, but not at E13.5. In contrast, when initiated late in development (E12.5 to birth), Spry-4 caused less severe pulmonary hypoplasia. Expression of Spry-4 from E16.5 to E18.5 reduced lung growth and resulted in perinatal death due to respiratory failure. Expression of Spry-4 during the saccular and alveolar stages, from E18.5 to postnatal day 21, caused mild emphysema. These findings demonstrate that the embryonic-pseudoglandular stage is a critical time period during which Spry-sensitive pathways are required for branching morphogenesis, lobulation, and formation of the peripheral lung parenchyma.     

Glucose and pyruvate metabolism during mouse gonadal sex differentiation

In the mouse, gonadal sex differentiation starts around E12 and meiosis begins in the ovary shortly after E13. In the search for metabolic changes that might be correlated to gonadal sex differentiation and/or possibly the onset of meiosis, we investigated the metabolism of glucose and pyruvate in the developing mouse ovary before (E11.5-E12.5), during (E14.5-16.5), and after meiosis (E18.5), and in fetal testes without meiosis. Gonads were cultured with 14C-labeled glucose (0.02 and 5.58 mM) and 14C-pyruvate (0.17 mM). The oxidation expressed as 14CO2 production and the organification expressed as retention of 14C in the tissues were measured and correlated to the protein content of the gonads. Using 0.02 mM glucose, a decline in oxidation and organification was found in ovaries as well as in testes, which is probably related to starvation. Using 5.58 mM glucose, a continuous decline in oxidation was seen only in the testis. Organification of 0.17 mM pyruvate increased at E12.5 and E14.5 in the ovary but not in the testis. This was in despite of an exponential increase of protein content in the testes compared to only a moderate increase in the ovary. The CO2 production from 5.58 mM glucose was equal to that from 0.17 mM pyruvate in gonads of both sexes. In conclusion, an increased metabolism of 5.58 mM glucose and 0.17 mM pyruvate in the ovaries as compared to the testes is related to sex differences during gonadal formation and onset of meiosis in the ovaries. J. Exp. Zool. 288:130-138, 2001.     

Role of Periostin in Adhesion and Migration of Bone Remodeling Cells

Periostin is an extracellular matrix protein highly expressed in collagen-rich tissues subjected to continuous mechanical stress. Functionally, periostin is involved in tissue remodeling and its altered function is associated to numerous pathological processes. In orthodontics, periostin plays key roles in the maintenance of dental tissues and it is mainly expressed in those areas where tension or pressing forces are taking place. In this regard, high expression of periostin is essential to promote migration and proliferation of periodontal ligament fibroblasts. However little is known about the participation of periostin in migration and adhesion processes of bone remodeling cells. In this work we employ the mouse pre-osteoblastic MC3T3-E1 and the macrophage-like RAW 264.7 cell lines to overexpress periostin and perform different cell-based assays to study changes in cell behavior. Our data indicate that periostin overexpression not only increases adhesion capacity of MC3T3-E1 cells to different matrix proteins but also hampers their migratory capacity. Changes on RNA expression profile of MC3T3-E1 cells upon periostin overexpression have been also analyzed, highlighting the alteration of genes implicated in processes such as cell migration, adhesion or bone metabolism but not in bone differentiation. Overall, our work provides new evidence on the impact of periostin in osteoblasts physiology.     

Spatiotemporal expression of the selenoprotein P gene in postimplantational mouse embryos

Selenoprotein P (Sepp) is an extracellular glycoprotein which functions principally as a selenium (Se) transporter and antioxidant. In order to assess the spatiotemporal expression of the Sepp gene during mouse embryogenesis, quantitative RT-PCR and in situ hybridization analyses were conducted in embryos and extraembryonic tissues, including placenta. Sepp mRNA expression was detected in all embryos and extraembryonic tissues on embryonic days (E) 7.5 to 18.5. Sepp mRNA levels were high in extraembryonic tissues, as compared to embryos, on E 7.5-13.5. However, the levels were higher in embryos than in extraembryonic tissues on E 14.5-15.5, but were similar in both tissues during the subsequent periods prior to birth. According to the results of in situ hybridization, Sepp mRNA was expressed principally in the ectoplacental cone and neural ectoderm, including the neural tubes and neural folds. In whole embryos, Sepp mRNA was expressed abundantly in nervous tissues on E 9.5-12.5. Sepp mRNA was also expressed in forelimb and hindlimb buds on E 10.5-12.5. In the sectioned embryos, on E 13.5-18.5, Sepp mRNA was expressed persistently in the developing limbs, gastrointestinal tract, nervous tissue, lung, kidney and liver. On E 16.5-18.5, Sepp mRNA expression in the submandibular gland, whisker follicles, pancreas, urinary bladder and skin was apparent. In particular, Sepp mRNA was detected abundantly in blood cells during all the observed developmental periods. These results show that Sepp may function as a transporter of selenium, as well as an antioxidant, during embryogenesis.     

硫酸糖胺聚糖(Sulfated Glycosaminoglycans)在花背蟾蜍眼早期形态发生中的合成

本文用放射自显影追踪注射入胚胎的~(35)S-硫酸盐的方法,研究了花背蟾蜍早期形态发生时眼的各部分组织和细胞外基质中的硫酸糖胺聚糖(Sulfated Glycosaminoglycans简称:硫酸GAG)的合成,并分析了其在眼形态发生中的作用。结果表明:1.在眼早期形态发生时,合成的硫酸GAG主要是硫酸软骨素。2.眼各部分组织中在即将分化时硫酸GAG合成率增高,分化开始后逐渐下降到原基形成时的水平。3.在晶状体被诱导时,在视杯和晶状体相互贴近的组织及两者间的细胞外基质中硫酸GAG的合成率明显增加,提示这是晶状体诱导的重要因素。4.角膜上皮形成时即向角膜上皮下层和细胞外基质分泌硫酸GAG;角膜上皮透明时,合成更多的硫酸角质素。     

An in situ hybridization study of the insulin-like growth factor system in developing condylar cartilage of the fetal mouse mandible

The objective of this study was to investigate the involvement of the insulin-like growth factor (IGF) system in the developing mandibular condylar cartilage and temporomandibular joint (TMJ). Fetal mice at embryonic day (E) 13.0-18.5 were used for in situ hybridization studies using [35S]-labeled RNA probes for IGF-I, IGF-II, IGF-I receptor (-IR), and IGF binding proteins (-BPs). At E13.0, IGF-I and IGF-II mRNA were expressed in the mesenchyme around the mandibular bone, but IGF-IR mRNA was not expressed within the bone. At E14.0, IGF-I and IGF-II mRNA were expressed in the outer layer of the condylar anlage, and IGF-IR mRNA was first detected within the condylar anlage, suggesting that the presence of IGF-IR mRNA in an IGF-rich environment triggers the initial formation of the condylar cartilage. IGFBP-4 mRNA was expressed in the anlagen of the articular disc and lower joint cavity from E15.0 to 18.5. When the upper joint cavity was formed at E18.5, IGFBP-4 mRNA expression was reduced in the fibrous mesenchymal tissue facing the upper joint cavity. Enhanced IGFBP-2 mRNA expression was first recognized in the anlagen of both the articular disc and lower joint cavity at E16.0 and continued expression in these tissues as well as in the fibrous mesenchymal tissue facing the upper joint cavity was observed at E18.5. IGFBP-5 mRNA was continuously expressed in the outer layer of the perichondrium/fibrous cell layer in the developing mandibular condyle. These findings suggest that the IGF system is involved in the formation of the condylar cartilage as well as in the TMJ.     

Impaired Abdominal Wall Development and Deficient Wound Healing in Mice Lacking Aortic Carboxypeptidase-Like Protein

Aortic carboxypeptidase-like protein (ACLP) is a member of a diverse group of proteins that contain a domain with similarity to that of the Dictyostelium discoideum protein discoidin I. The discoidin domain has been identified in mammalian milk fat globule membrane proteins, blood coagulation factors, and receptor tyrosine kinases, where it may facilitate cell aggregation, adhesion, or cell-cell recognition. Here we show that ACLP is a secreted protein that associates with the extracellular matrix (ECM). During mouse embryogenesis, ACLP is abundantly expressed in the ECM of collagen-rich tissues, including the vasculature, dermis, and the developing skeleton. We deleted the ACLP gene in mice by homologous recombination. The majority of ACLP(-/-) mice die perinatally due to gastroschisis, a severe disruption of the anterior abdominal wall and herniation of the abdominal organs. ACLP(-/-) mice that survived to adulthood developed nonhealing skin wounds. Following injury by a dermal punch biopsy, ACLP(-/-) mice exhibited deficient wound healing compared with controls. In addition, dermal fibroblasts isolated from ACLP(-/-) 18.5-day-postconception embryos exhibited a reduced proliferative capacity compared with wild-type cells. These results indicate that ACLP is an ECM protein that is essential for embryonic development and dermal wound healing processes.     

Developmental expression of the murine Prl-1 protein tyrosine phosphatase gene

The expression of the murine Prl-1 protein tyrosine phosphatase gene was examined in normal embryos from E10.5 through E18.5. Prl-1 mRNA was detected in the brain, neural tube, and dorsal root ganglia, and in several non-neuronal tissues, including the skeletal system. Heart and skeletal muscle were consistently negative. At E13.5, Prl-1 was expressed in the condensing prechondrogenic cells of the vertebrae, whereas at E18.5, Prl-1 mRNA was localized to the hypertrophic chondrocytes. The dynamic expression of Prl-1 during cartilage differentiation may suggest a functional role in skeletal development.     

Collagen XXVII is developmentally regulated and forms thin fibrillar structures distinct from those of classical vertebrate fibrillar collagens

We have generated an antiserum to the variable domain of mouse collagen XXVII, a recently discovered novel member of the fibrillar collagen family. Collagen XXVII protein was first detectable in the mouse at embryonic day 12.5 (E12.5). By E14.5, the protein localized to cartilage, developing dermis, cornea, the inner limiting membrane of the retina, and major arteries of the heart. However, at E18.5, collagen XXVII protein was no longer apparent in most tissues and appeared restricted mainly to cartilage where expression continued into adulthood. Type XXVII collagen immunolocalized to 10-nm-thick nonstriated fibrils that were distinct from fibrils formed by the classical fibrillar collagens. The transient nature of its expression and unusual fibrillar structure suggest that collagen XXVII plays a developmental role distinct from those of the classical fibrillar collagens.     

sec24d encoding a component of COPII is essential for vertebra formation, revealed by the analysis of the medaka mutant, vbi

We characterized a medaka mutant, vertebra imperfecta (vbi), that displays skeletal defects such as craniofacial malformation and delay of vertebra formation. Positional cloning analysis revealed a nonsense mutation in sec24d encoding a component of the COPII coat that plays a role in anterograde protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus. Immunofluorescence analysis revealed the accumulation of type II collagen in the cytoplasm of craniofacial chondrocytes, notochord cells, and the cells on the myoseptal boundary in vbi mutants. Electron microscopy analysis revealed dilation of the ER and defective secretion of ECM components from cells in both the craniofacial cartilage and notochord in vbi. The higher vertebrates have at least 4 sec24 paralogs; however, the function of each paralog in development remains unknown. sec24d is highly expressed in the tissues that are rich in extracellular matrix and is essential for the secretion of ECM component molecules leading to the formation of craniofacial cartilage and vertebra.