Identification and characterization of zebrafish ocular formation genes.

To study genes that are specifically expressed in the eyes, we employed microarray and in situ hybridization analyses to identify and characterize differentially expressed ocular genes in eyeless masterblind (mbl-/-) zebrafish (Danio rerio). Among 70 differentially expressed genes in the mbl-/- mutant identified by microarray analysis, 8 down-regulated genes were characterized, including 4 eye-specific genes, opsin 1 short-wave-sensitive 1 (opn1sw1), crystallinbetaa1b (cryba1b), crystallinbetaa2b (cryba2b), and crystallingamma M2d3 (crygm2d3); 2 eye and brain genes, ATPase, H+ transporting, lysosomal, V0 subunit c (atp6v0c) and basic leucine zipper and W2 domains 1a (bzw1a); and 2 constitutive genes, heat shock protein 8 (hspa8) and ribosomal protein L7a (rpl7a). In situ hybridization experiments confirmed down-regulation of these 8 ocular formation genes in mbl-/- zebrafish and showed their ocular and dynamic temporal expression patterns during zebrafish early development. Further, an automated literature analysis of the 70 differentially expressed genes identified a sub-network of genes with known associations, either with each other or with ocular structures or development, and shows how this study contributes to the current body of knowledge.     

Discovery of sonic hedgehog expression in postnatal growth plate chondrocytes: differential regulation of sonic and Indian hedgehog by retinoic acid

Sonic hedgehog (Shh) is a key signal protein in early embryological patterning of limb bud development. Its analog, Indian hedgehog (Ihh), primarily expressed during early cartilage development in prehypertrophic chondrocytes, regulates proliferation and suppresses terminal differentiation of postnatal growth plate (GP) chondrocytes. We report here for the first time that both Shh and Ihh mRNA are expressed in the GP of rapidly growing 6-week-old broiler-strain chickens. They are also expressed in other tissues such as articular chondrocytes, kidney, and bone. In situ hybridization and RT-PCR analyses reveal Shh in all zones of the GP, with peak expression in late hypertrophy. Using primary cultures of GP chondrocytes in serum-containing medium, we followed the patterns of Shh and Ihh mRNA expression as the cultures matured and mineralized. We find a cyclical expression of both hedgehog genes during the early period of culture development between day 10 and 14; when one is elevated, the other tended to be suppressed, suggesting that the two hedgehogs may play complementary roles during GP development. Retinoic acid (RA), a powerful modulator of gene expression in cell differentiation, stimulates GP chondrocytes toward terminal differentiation, enhancing mineral formation. We find that RA strongly suppresses Ihh, but enhances expression of Shh in this system. While Ihh suppresses maturation of GP chondrocytes to hypertrophy, we hypothesize that Shh acts to push these cells toward hypertrophy.     

Nedd4-mediated AMPA receptor ubiquitination regulates receptor turnover and trafficking

α-Amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptors (AMPARs) are the primary mediators of excitatory synaptic transmission in the brain. Alterations in AMPAR localization and turnover have been considered critical mechanisms underpinning synaptic plasticity and higher brain functions, but the molecular processes that control AMPAR trafficking and stability are still not fully understood. Here, we report that mammalian AMPARs are subject to ubiquitination in neurons and in transfected heterologous cells. Ubiquitination facilitates AMPAR endocytosis, leading to a reduction in AMPAR cell-surface localization and total receptor abundance. Mutation of lysine residues to arginine residues at the glutamate receptor subunit 1 (GluA1) C-terminus dramatically reduces GluA1 ubiquitination and abolishes ubiquitin-dependent GluA1 internalization and degradation, indicating that the lysine residues, particularly K868, are sites of ubiquitination. We also find that the E3 ligase neural precursor cell expressed, developmentally down-regulated 4 (Nedd4) is enriched in synaptosomes and co-localizes and associates with AMPARs in neurons. Nedd4 expression leads to AMPAR ubiquitination, leading to reduced AMPAR surface expression and suppressed excitatory synaptic transmission. Conversely, knockdown of Nedd4 by specific siRNAs abolishes AMPAR ubiquitination. These data indicate that Nedd4 is the E3 ubiquitin ligase responsible for AMPAR ubiquitination, a modification that regulates multiple aspects of AMPAR molecular biology including trafficking, localization and stability.     

Fibromodulin is expressed by both chondrocytes and osteoblasts during fetal bone development

Fibromodulin, a keratan-sulfate proteoglycan, was first isolated in articular cartilage and tendons. We have identified fibromodulin as a gene regulated during BMP-2-induced differentiation of a mouse prechondroblastic cell line. Because expression of fibromodulin during endochondral bone formation has not been studied, we examined whether selected cells of the chondrocytic and osteoblastic lineage expressed fibromodulin. Fibromodulin mRNA was detected in conditionally immortalized murine bone marrow stromal cells, osteoblasts, and growth plate chondrocytes, as well as in primary murine calvarial osteoblasts. We, therefore, investigated the temporo-spatial expression of fibromodulin in vivo during endochondral bone formation by in situ hybridization. Fibromodulin was first detected at 15.5 days post coitus (dpc) in the perichondrium and proliferating chondrocytes. Fibromodulin mRNA was also detected at 15.5 dpc in the bone collar and periosteum. At later time points fibromodulin was expressed in the primary spongiosa and the endosteum. To determine whether fibromodulin was expressed during intramembranous bone formation as well, in situ hybridization was performed on calvariae. Fibromodulin mRNA was present in calvarial osteoblasts from 15.5 dpc. These results demonstrate that fibromodulin is developmentally expressed in cartilage and bone cells during endochondral and intramembranous ossification. These findings suggest that this extracellular matrix protein plays a role in both endochondral and intramembranous bone formation.     

Knockdown of the intraflagellar transport protein IFT46 stimulates selective gene expression in mouse chondrocytes and affects early development in zebrafish

Bone morphogenetic proteins (BMPs) act as multifunctional regulators in morphogenesis during development. In particular they play a determinant role in the formation of cartilage molds and their replacement by bone during endochondral ossification. In cell culture, BMP-2 favors chondrogenic expression and promotes hypertrophic maturation of chondrocytes. In mouse chondrocytes we have identified a BMP-2-sensitive gene encoding a protein of 301 amino acids. This protein, named mIFT46, is the mouse ortholog of recently identified Caenorhabditis elegans and Chlamydomonas reinhardtii intraflagellar transport (IFT) proteins. After generation of a polyclonal antibody against mIFT46, we showed for the first time that the endogenous protein is located in the primary cilium of chondrocytes. We also found that mIFT46 is preferentially expressed in early hypertrophic chondrocytes located in the growth plate. Additionally, mIFT46 knockdown by small interfering RNA oligonucleotides in cultured chondrocytes specifically stimulated the expression of several genes related to skeletogenesis. Furthermore, Northern blotting analysis indicated that mIFT46 is also expressed before chondrogenesis in embryonic mouse development, suggesting that the role of mIFT46 might not be restricted to cartilage. To explore the role of IFT46 during early development, we injected antisense morpholino oligonucleotides in Danio rerio embryos to reduce zebrafish IFT46 protein (zIFT46) synthesis. Dramatic defects in embryonic development such as a dorsalization and a tail duplication were observed. Thus our results taken together indicate that the ciliary protein IFT46 has a specific function in chondrocytes and is also essential for normal development of vertebrates.     

Identification of developmentally expressed proteins that functionally interact with Nedd4 ubiquitin ligase.

Nedd4 is a HECT domain-containing ubiquitin ligase that mediates ubiquitylation and proteasome degradation of target proteins. The molecular basis for the interaction of Nedd4 with substrates lies in its WW domains, which can bind proline-rich (PY) domains in target proteins. Nedd4 is a developmentally expressed protein and may have a fundamental role to play in embryonic processes. However, whether Nedd4 has such a function is currently unknown, in part because few developmentally regulated ubiquitylation substrates have been identified or characterized. We have carried out a yeast two-hybrid screen and identified four proteins expressed in the mid-gestation embryo that are able to interact with Nedd4. Characterization of their functional interaction with Nedd4 in vitro and in vivo demonstrated that three of the four are bona fide Nedd4 binding partners, and two have the capacity to be ubiquitylation substrates. One of these is the first identified nonviral substrate for Nedd4-mediated monoubiquitylation. Interestingly, neither of these two ubiquitylated proteins interacts with Nedd4 through PY-mediated mechanisms. For one of the three Nedd4 binding partners, there was no discernable evidence of ubiquitylation. However, this protein clearly associates with Nedd4 through its PY domains and can alter the location of Nedd4 in cells, suggesting a role other than as a ubiquitylation substrate.     

Expression profile of Xenopus banded hedgehog, a homolog of mouse Indian hedgehog, is related to the late development of endochondral ossification in Xenopus laevis

Late development of endochondral ossification occurs at the boundary between the growth cartilage and bone marrow during the formation of long bones in Xenopus laevis. Since the Indian hedgehog (Ihh) is involved in endochondral ossification in mouse, we investigated the expression of Xenopus banded hedgehog (X-bhh), which is a homolog of mouse Ihh. RT-PCR analysis demonstrated that the X-bhh mRNA was detected from an early stage of limb formation to formation of femurs in mature frogs, and it was associated with the expression of Xenopus-ptc1 (X-ptc1), Xenopus-gli1 (X-gli1), Xenopus-type II collagen (X-col II), Xenopus-runx2 (X-runx2), and Xenopus-osteocalcin (X-ocn) mRNAs. In situ hybridization revealed that chondrogenic cells observed at early limb development expressed X-bhh and X-gli1. At later stages of limb development, chondrocytes, located slightly away from the boundary between the cartilage and bone marrow, expressed the X-bhh, X-ptc1, and X-gli1 mRNAs; however, the mesenchymal cells at the boundary failed to express these mRNAs. The X-bhh, X-ptc1, and X-gli1 mRNAs as well as those of X-runx2 and X-ocn were expressed by the mesenchymal cells in the periosteal region at the tip of the cortical bone, indicating an intimate relationship between X-bhh expression and bone formation in this region. Considered collectively, the present study suggests that X-bhh evolutionally acquired the function to induce osteogenesis; however, the expression profile of X-bhh in epiphysis is closely related to the late development of endochondral ossification in X. laevis.     

Dlx5 regulates chondrocyte differentiation at multiple stages

Endochondral ossification, in which cartilaginous templates are progressively replaced by marrow and bone, represents the dominant mode of development of the axial and appendicular skeleton of vertebrates. Chondrocyte differentiation within the cartilaginous core of these skeletal elements is tightly regulated, both spatially and temporally. Here, we describe the expression of Dlx5 in the cartilaginous core of limb skeletal elements in chicken and mouse embryos. We find that Dlx5 is one of the earliest genes expressed in condensing limb mesenchyme that will give rise to the limb skeleton. Later, when proliferating and differentiating chondrocytes are found in spatially distinct regions of the cartilaginous model, Dlx5 is expressed in the zone of hypertrophy and in proliferating chondrocytes that are poised to differentiate. Consistent with this pattern of expression, we show that forced expression of Dlx5 potentiates early and late chondrocyte differentiation and inhibits proliferation in cultured cells. Examination of the limbs of mutant Dlx5 mouse embryos revealed that they displayed a delay in chondrocyte maturation compared with wild type littermates. Taken together, our data reveal a positive role for Dlx5 during multiple stages of chondrocyte differentiation and, along with previous studies of Dlx5 and osteogenesis, identify Dlx5 as a general regulator of differentiation in the mouse skeleton.     

MAPK信号通路基因在小鼠肝再生中的表达谱变化

为了分析丝裂原活化蛋白激酶(Mitogen-Activated Protein Kinases,MAPK)信号通路基因在肝再生中的表达图谱,以及探讨MAPK信号通路在肝再生中的作用,文章利用四氯化碳(Carbon Tetrachloride,CCl4)诱导的小鼠肝损伤再生模型对MAPK信号通路基因的表达进行检测.首先,采用CCl4腹腔注射的方法建立小鼠肝损伤再生模型,通过肝脏切片HE染色和测定血清中谷丙转氨酶活性确认模型的质量,然后,在注射CCl4后的第0、0.5、1.5、4.5、7 d分别采集小鼠肝脏样本,应用Affymetrix公司的小鼠基因表达芯片,检测MAPK信号通路中93个基因的差异表达图谱,并用荧光实时定量PCR法验证芯片检测的结果.结果表明,在芯片检测到的93个MAPK信号通路基因中,有31个在肝再生中有不同程度差异表达,且经荧光实时定量RT-PCR检测的结果与基因芯片的结果相符合.基因表达谱芯片技术可以筛选出肝再生中差异表达的基因,在小鼠肝再生中的第0.5和1.5 d,MAPK信号通路中表达水平上调的基因增多,而在第4.5和7 d,则表达水平下调的基因明显增多.这一结果表明MAPK信号通路对肝再生不同阶段的双重调控作用.     

Differential expression of two msh-related homeobox genes Chox-7 and Chox-8 during chick limb development.

We have isolated two closely related cDNAs, Chox-7 and Chox-8, encoding homeodomain-containing proteins homologous to Drosophila msh. The Chox-7 and Chox-8 genes are chicken cognates of mouse Hox-7.1 and Hox-8.1, respectively. In situ hybridization using 3' regions of the cDNAs as probes revealed that the Chox-7 gene is highly expressed in the mesenchyme subjacent to the apical ectodermal ridge whereas Chox-8 expression is localized in the anterodistal mesenchymal region at early stages of limb formation, suggesting different roles during limb development. At later stages, both genes are expressed in the anterior and posterior mesenchymes and in the interdigital mesenchyme where programmed cell death occurs.