Wdr5 is required for chick skeletal development

Wdr5, a bone morphogenetic protein 2 (BMP‐2)–induced protein belonging to the family of the WD repeat proteins, is expressed in proliferating and hypertrophic chondrocytes of the growth plate and in osteoblasts. Although previous studies have provided insight into the mechanisms by which Wdr5 affects chondrocyte and osteoblast differentiation, whether Wdr5 is required in vivo for endochondral bone development has not been addressed. In this study, using an avian replication competent retrovirus (RCAS) system delivering Wdr5 short hairpin (sh) RNA to silence Wdr5 in the developing limb, we report that reduction of Wdr5 levels delays endochondral bone development and consequently results in shortening of the skeletal elements. Shortening of the skeletal elements was due to impaired chondrocyte maturation, evidenced by a significant reduction of Runx2, type X collagen, and osteopontin expression. A decrease in Runx2, type collagen I, and ostepontin expression in osteoblasts and a subsequent defect in mineralized bone was observed as well when Wdr5 levels were reduced. Most important, retroviral misexpression of Runx2 rescued the phenotype induced by Wdr5 shRNA. These findings suggest that during limb development, Wdr5 is required for endochondral bone formation and that Wdr5 influences this process, at least in part, by regulating Runx2 expression. © 2010 American Society for Bone and Mineral Research.     

Parathyroid hormone-related protein is required for normal intramembranous bone development.

It is well established that parathyroid hormone-related protein (PTHrP) regulates chondrocytic differentiation and endochondral bone formation. Besides its effect on cartilage, PTHrP and its major receptor (type I PTH/PTHrP receptor) have been found in osteoblasts, suggesting an important role of PTHrP during the process of intramembranous bone formation. To clarify this issue, we examined intramembranous ossification in homozygous PTHrP-knockout mice histologically. We also analyzed phenotypic markers of osteoblasts and osteoclasts in vitro and in vivo. A well-organized branching and anastomosing pattern was seen in the wild-type mice. In contrast, marked disorganization of the branching pattern of bone trabeculae and irregularly aligned osteoblasts were recognized in the mandible and in the bone collar of the femur of neonatal homozygous mutant mice. In situ hybridization showed that most of the osteoblasts along the bone surfaces of the wild-type mice and some of the irregularly aligned osteoblastic cells in the homozygous mice expressed osteocalcin. Alkaline phosphatase (ALP) activity and expression of osteopontin messenger RNA (mRNA) in primary osteoblastic cells did not show significant differences between cultures derived from the mixture of heterozygous mutant and wild-type mice (+/? mice) and those from homozygous mutant mice. However, both mRNA and protein levels of osteocalcin in the osteoblastic cells of homozygous mutant mice were lower than those of +/? mice, and exogenous PTHrP treatment corrected this suppression. Immunohistochemical localization of characteristic markers of osteoclasts and ruffled border formation did not differ between genotypes. Cocultures of calvarial osteoblastic cells and spleen cells of homozygous mutant mice generated an equivalent number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear and multinucleated cells and of pit formation to that of +/? mice, suggesting that osteoclast differentiation is not impaired in the homozygous mutant mice. These results suggest that PTHrP is required not only for the regulation of cartilage formation but also for the normal intramembranous bone development.     

Oxidant stress is increased within the glomerulus in experimental diabetic nephropathy

SUMMARY: The mechanisms by which the metabolic consequences of hyperglycaemia induce diabetic renal injury remain ill-defined. We hypothesized that oxidant stress, a consequence of hyperglycaemia, is increased in glomeruli from Lewis rats with streptozotocin (STZ)-induced diabetes prior to major structural and functional glomerular damage. After 12 weeks of diabetes, Lewis rats had not developed proteinuria and their glomeruli appeared normal by light microscopy. However, kidneys of diabetic animals had higher levels of lipid peroxides and malondialdehyde (MDA) than control rats. Immunohistochemistry demonstrated MDA–lysine adducts in glomeruli of diabetic rats, and that lipid peroxides and MDA were increased in glomerular lysates of diabetic rats. A possible mechanism for this finding was suggested by the observation that freshly isolated whole glomeruli from STZ rats showed a greater capacity than glomeruli from control rats to produce H₂O₂. Activity of the inducible form of superoxide dismutase (SOD) Mn-SOD was increased in glomerular lysates from STZ rats, consistent with its induction by oxidant stress. Immunostaining for Cu,Zn SOD showed increased protein in glomeruli, although compared with the increase in Mn-SOD activity, Cu,Zn-SOD activity was not substantially increased, potentially as a result of partial inactivation of this enzyme by glycation. The increased oxidant stress in untreated diabetic rats was a consequence of hyperglycaemia and not due to a direct nephrotoxic effect of STZ, as at least some of these changes were attenuated by insulin treatment of diabetic animals. Collectively, these results demonstrate that experimental diabetes mellitus is accompanied by increased oxidant stress within glomeruli.     

Kidney development and disease in the zebrafish

Unraveling the molecular pathogenesis of human disease presents many experimental challenges, not the least of which is that experiments on humans are generally frowned upon. Model organisms, including the zebrafish, allow for experimental analysis of gene function and the detailed characterization of disease processes. Zebrafish have matured as a vertebrate model organism now that genetic tools for targeted "knockdowns" and unbiased mutagenesis approaches are in hand. The fish larval pronephros is a relevant kidney in which to pursue many aspects of human kidney development and disease. This short review outlines recent progress in applying the zebrafish pronephros to issues of human health and development.     

c-myc is required for osteoclast differentiation.

The role of the receptor activator of nuclear factor kappaB (NF-kappaB) ligand (RANKL)-a tumor necrosis factor (TNF)-related cytokine-in osteoclast formation has been established clearly. However, the downstream signaling pathways activated by this cytokine remain largely unknown. To identify genes that play a role in osteoclastogenesis, we used RAW 264.7 mouse monocytes as a model system for the differentiation of multinucleated osteoclasts from mononucleated precursors. RAW 264.7 cells were induced with RANKL to form multinucleated giant osteoclast-like cells (OCLs) that expressed a number of osteoclast-specific markers and were able to form resorption pits on both calcium phosphate films and bone slices. This system was used to identify genes that are regulated by RANKL and may play a role in osteoclast differentiation. The proto-oncogene c-myc was strongly up-regulated in RANKL-induced OCLs but was absent in undifferentiated cells. Expression of Myc partners Max and Mad, on the other hand, was constant during OCL differentiation. We expressed a dominant negative Myc in RAW 264.7 cells and were able to block RANKL-induced OCL formation. Northern Blot analysis revealed a delay and a significant reduction in the level of messenger RNA (mRNA) for tartrate-resistant acid phosphatase (TRAP) and cathepsin K. We conclude that c-myc is a downstream target of RANKL and its expression is required for RANKL-induced osteoclastogenesis.     

The cellular history of the glomerulus

Knowledge about the structure and functions of the cells of the glomeruli has accumulated slowly over the past 350 years. Marcello Malpighi originated the work, but it failed to progress far until Schleiden and Schwann developed their cellular theory in 1839. William Bowman linked the glomeruli to the tubules, described the parietal epithelial cells, the basement membranes, and (with Robert Todd) apparently first identified endothelial cells. Electron microscopy contributed especially to an understanding of epithelial and endothelial cell structure. Axel Key first described mesangial cells, but acceptance of these fell into abeyance for many years until Yamada incontrovertibly demonstrated their existence. Techniques such as tissue culture and molecular biological investigations have, more recently, provided much information about glomerular cell function. Progress has, throughout, depended upon the discovery of ever more powerful methods of microscopy, the development of ancillary experimental methods, the formulation of persuasive explanations for observations, and the suggestion of succinct terminology to describe the features observed.     

Expression of the nonmuscle myosin heavy chain IIA in the human kidney and screening for MYH9 mutations in Epstein and Fechtner syndromes.

Mutations in the MYH9 gene, which encodes the nonmuscle myosin heavy chain IIA, have been recently reported in three syndromes that share the association of macrothrombocytopenia (MTCP) and leukocyte inclusions: the May-Hegglin anomaly and Sebastian and Fechtner syndromes. Epstein syndrome, which associates inherited sensorineural deafness, glomerular nephritis, and MTCP without leukocyte inclusions, was shown to be genetically linked to the same locus at 22q12.3 to 13. The expression of MYH9 in the fetal and mature human kidney was studied, and the 40 coding exons of the gene were screened by single-strand conformation polymorphism in 12 families presenting with the association of MTCP and nephropathy. MYH9 is expressed in both fetal and mature kidney. During renal development, it is expressed in the late S-shaped body, mostly in its lower part, in the endothelial and the epithelial cell layers. Later, as well as in mature renal tissue, MYH9 is widely expressed in the kidney, mainly in the glomerulus and peritubular vessels. Within the glomerulus, MYH9 mRNA and protein are mostly expressed in the epithelial visceral cells. Four missense heterozygous mutations that are thought to be pathogenic were found in five families, including two families with Epstein syndrome. Three mutations were located in the coiled-coil rod domain of the protein, and one was in the motor domain. Two mutations (E1841K and D1424N) have been reported elsewhere in families with May-Hegglin anomaly. The two others (R1165L and S96L) are new mutations, although one of them affects a codon (R1165), found elsewhere to be mutated in Sebastian syndrome.     

咪唑安定对斑马鱼胚胎血管发育的影响

目的 观察眯唑安定对斑马鱼胚胎血管发育的影响.方法 根据培养液成分将特异性标记血管内皮细胞的转基因斑马鱼胚胎随机分为四组.每组120个.A组咪唑安定浓度1 mg/L、B组10 nag/L、C组20 mg/L、D组不加咪唑安定作为对照.咪唑安定处理时间为胚胎受精后4.3～30 h.随后用共聚焦显微镜观察斑马鱼胚胎血管发育情况.结果 与D组比较,A组斑马鱼胚胎无明显血管发育改变;C组胚胎发育停滞在原肠胚时期;B组胚胎体节间血管和尾部血管发育均受到明显的抑制(P<0.01).结论 10 mg/L 的咪唑安定干预可抑制斑马鱼胚胎血管发育.