A single cell transcriptional atlas of early synovial joint development.

Synovial joint development begins with the formation of the interzone, a region of condensed mesenchymal cells at the site of the prospective joint. Recently, lineage-tracing strategies have revealed that Gdf5-lineage cells native to and from outside the interzone contribute to most, if not all, of the major joint components. However, there is limited knowledge of the specific transcriptional and signaling programs that regulate interzone formation and fate diversification of synovial joint constituents. To address this, we have performed single cell RNA-Seq analysis of 7329 synovial joint progenitor cells from the developing murine knee joint from E12.5 to E15.5. By using a combination of computational analytics, in situ hybridization and in vitro characterization of prospectively isolated populations, we have identified the transcriptional profiles of the major developmental paths for joint progenitors. Our freely available single cell transcriptional atlas will serve as a resource for the community to uncover transcriptional programs and cell interactions that regulate synovial joint development.

High Dietary Fructose Intake on Cardiovascular Disease Related Parameters in Growing Rats.

The objective of this study was to determine the effects of a high-fructose diet on cardiovascular disease (CVD)-related parameters in growing rats. Three-week-old female Sprague Dawley rats were randomly assigned to four experimental groups; a regular diet group (RD: fed regular diet based on AIN-93G, n = 8), a high-fructose diet group (30Frc: fed regular diet with 30% fructose, n = 8), a high-fat diet group (45Fat: fed regular diet with 45 kcal% fat, n = 8) or a high fructose with high-fat diet group (30Frc + 45Fat, fed diet 30% fructose with 45 kcal% fat, n = 8). After an eight-week treatment period, the body weight, total-fat weight, serum glucose, insulin, lipid profiles and pro-inflammatory cytokines, abdominal aortic wall thickness, and expressions of eNOS and ET-1 mRNA were analyzed. The result showed that total-fat weight was higher in the 30Frc, 45Fat, and 30Frc + 45Fat groups compared to the RD group (p < 0.05). Serum triglyceride (TG) levels were highest in the 30Frc group than the other groups (p < 0.05). The abdominal aorta of 30Frc, 45Fat, and 30Frc + 45Fat groups had higher wall thickness than the RD group (p < 0.05). Abdominal aortic eNOS mRNA level was decreased in 30Frc, 45Fat, and 30Frc + 45Fat groups compared to the RD group (p < 0.05), and also 45Fat and 30Frc + 45Fat groups had decreased mRNA expression of eNOS compared to the 30Frc group (p < 0.05). ET-1 mRNA level was higher in 30Frc, 45Fat, and 30Frc + 45Fat groups than the RD group (p < 0.05). Both high fructose consumption and high fat consumption in growing rats had similar negative effects on CVD-related parameters.

Biochemical Responses of Anodized Titanium Implants with a Poly(lactide-co-glycolide)/Bone Morphogenic Protein-2 Submicron Particle Coating. Part 1: An In Vitro Study.

PURPOSE: This study was conducted to examine the effects of coating poly(D,L-lactide-co-glycolide) (PLGA)/recombinant human bone morphogenetic protein-2 (rhBMP-2) submicron particles by electrospray onto titanium (Ti) implants on the proliferation and differentiation capacity of mesenchymal stem cells. In addition, the duration of rhBMP-2 release was investigated. MATERIALS AND METHODS: Ti disks were fabricated and divided into four groups as follows. Group C (control) was anodized at 300 V, group P was anodized and then coated with 0.2% PLGA, group B1 was anodized and then coated with PLGA/rhBMP-2 submicron particles at 200 ng per disk, and group B2 was anodized and then coated with PLGA/rhBMP-2 submicron particles at 600 ng per disk. The topography of the PLGA/rhBMP-2-coated Ti surfaces was examined, and proliferation assays, alkaline phosphatase activity tests, and rhBMP-2 releasing tests were conducted in vitro. RESULTS: There was no difference in the roughness of control Ti disks and Ti disks coated with PLGA/rhBMP-2 submicron particles by electrospray. The proliferation of mesenchymal stem cells increased over time; at 7 days, cell proliferation on the Ti disks coated with rhBMP-2 was significantly higher than the other groups. Anodized Ti disks coated with PLGA/rhBMP-2 (groups B1 and B2) released rhBMP-2 for approximately 21 days. In accordance with the amount of rhBMP-2 released from the PLGA/rhBMP-2 construct, the differentiation capacity of mesenchymal stem cells on the PLGA/rhBMP-2-coated disks was increased significantly. CONCLUSION: Submicron PLGA/rhBMP-2 coating on Ti implants by electrospray facilitated cell proliferation and differentiation, which is important for early healing and integration of implants.

Rax-CreERT2 knock-in mice: a tool for selective and conditional gene deletion in progenitor cells and radial glia of the retina and hypothalamus.

To study gene function in neural progenitors and radial glia of the retina and hypothalamus, we developed a Rax-CreERT2 mouse line in which a tamoxifen-inducible Cre recombinase is inserted into the endogenous Rax locus. By crossing Rax-CreER(T2) with the Cre-dependent Ai9 reporter line, we demonstrate that tamoxifen-induced Cre activity recapitulates the endogenous Rax mRNA expression pattern. During embryonic development, Cre recombinase activity in Rax-CreER(T2) is confined to retinal and hypothalamic progenitor cells, as well as progenitor cells of the posterior pituitary. At postnatal time points, selective Cre recombinase activity is seen in radial glial-like cell types in these organs--specifically Müller glia and tanycytes--as well as pituicytes. We anticipate that this line will prove useful for cell lineage analysis and investigation of gene function in the developing and mature retina, hypothalamus and pituitary.

A gene trap knockout of the Tiam-1 protein results in malformation of the early embryonic brain.

Tiam-1 has been implicated in the development of the central nervous system. However, the in vivo function of Tiam-1 has not been fully determined in the developing mouse brain. In this study, we generated Tiam-1 knockout mice using a Tiam-1 gene-trapped embryonic stem cell line. Insertion of a gene trap vector into a genomic site downstream of exon 5 resulted in a mutant allele encoding a truncated protein fused with the ß-geo LacZ gene. Primary mouse embryonic fibroblasts lacking Tiam-1 revealed a significant decrease in Rac activity and cell proliferation. In addition, whole-mount embryonic LacZ expression analysis demonstrated that Tiam-1 is specifically expressed in regions of the developing brain, such as the caudal telencephalon and rostral diencephalon. More importantly, mouse embryos deficient in Tiam-1 gene expression displayed a severe defect in embryonic brain development, including neural tube closure defects or a dramatic decrease in brain size. These findings suggest that embryonic Tiam-1 expression plays a critical role during early brain development in mice.

EphA8-ephrinA5 signaling and clathrin-mediated endocytosis is regulated by Tiam-1, a Rac-specific guanine nucleotide exchange factor.

Recent studies indicate that endocytosis of Eph-ephrin complexes may be one of the mechanisms by which a high affinity cell-cell adhesion is converted to a repulsive interaction. In this study, we show that EphA8 undergoes clathrin-mediated endocytosis upon treatment with ephrin-A5, and that EphA8 is associated tightly with Tiam-1, a Rac-specific guanine nucleotide exchange factor. Analysis of EphA8 deletion mutants revealed that a juxtamembrane region in EphA8 is critically involved in endocytosis of EphA8-ephrinA5 complexes. An EphA8 mutant lacking this juxtamembrane portion was defective for endocytosis with ephrinA5, and also displayed a weak association with Tiam-1. Expression of an endocytosis-defective version of EphA8 resulted in a low level of Rac activity in response to ephrin-A5 stimulation. More importantly, down-regulation of Tiam-1 resulted in inefficient endocytosis of EphA8-ephrinA5 complexes. These results suggest that Tiam-1 plays a role in clathrin-dependent endocytosis of EphA8-ephrinA5 complexes.

The SAM domains of Anks family proteins are critically involved in modulating the degradation of EphA receptors.

We recently reported that the phosphotyrosine-binding (PTB) domain of Anks family proteins binds to EphA8, thereby positively regulating EphA8-mediated signaling pathways. In the current study, we identified a potential role for the SAM domains of Anks family proteins in EphA signaling. We found that SAM domains of Anks family proteins directly bind to ubiquitin, suggesting that Anks proteins regulate the degradation of ubiquitinated EphA receptors. Consistent with the role of Cbl ubiquitin ligases in the degradation of Eph receptors, our results revealed that the ubiquitin ligase c-Cbl induced the ubiquitination and degradation of EphA8 upon ligand binding. Ubiquitinated EphA8 also bound to the SAM domains of Odin, a member of the Anks family proteins. More importantly, the overexpression of wild-type Odin protected EphA8 and EphA2 from undergoing degradation following ligand stimulation and promoted EphA-mediated inhibition of cell migration. In contrast, a SAM domain deletion mutant of Odin strongly impaired the function of endogenous Odin, suggesting that the mutant functions in a dominant-negative manner. An analysis of Odin-deficient primary embryonic fibroblasts indicated that Odin levels play a critical role in regulating the stability of EphA2 in response to ligand stimulation. Taken together, our studies suggest that the SAM domains of Anks family proteins play a pivotal role in enhancing the stability of EphA receptors by modulating the ubiquitination process.