Interplay of Nkx3.2, Sox9 and Pax3 Regulates Chondrogenic Differentiation of Muscle Progenitor Cells

Muscle satellite cells make up a stem cell population that is capable of differentiating into myocytes and contributing to muscle regeneration upon injury. In this work we investigate the mechanism by which these muscle progenitor cells adopt an alternative cell fate, the cartilage fate. We show that chick muscle satellite cells that normally would undergo myogenesis can be converted to express cartilage matrix proteins in vitro when cultured in chondrogenic medium containing TGFß3 or BMP2. In the meantime, the myogenic program is repressed, suggesting that muscle satellite cells have undergone chondrogenic differentiation. Furthermore, ectopic expression of the myogenic factor Pax3 prevents chondrogenesis in these cells, while chondrogenic factors Nkx3.2 and Sox9 act downstream of TGFß or BMP2 to promote this cell fate transition. We found that Nkx3.2 and Sox9 repress the activity of the Pax3 promoter and that Nkx3.2 acts as a transcriptional repressor in this process. Importantly, a reverse function mutant of Nkx3.2 blocks the ability of Sox9 to both inhibit myogenesis and induce chondrogenesis, suggesting that Nkx3.2 is required for Sox9 to promote chondrogenic differentiation in satellite cells. Finally, we found that in an in vivo mouse model of fracture healing where muscle progenitor cells were lineage-traced, Nkx3.2 and Sox9 are significantly upregulated while Pax3 is significantly downregulated in the muscle progenitor cells that give rise to chondrocytes during fracture repair. Thus our in vitro and in vivo analyses suggest that the balance of Pax3, Nkx3.2 and Sox9 may act as a molecular switch during the chondrogenic differentiation of muscle progenitor cells, which may be important for fracture healing

A prospective hospital based study showing prevalence of eye manifestations in patients having systemic inflammatory autoimmune disease

Aim: To outline several common ocular complications and their prevalence in patients with systemic inflammatory autoimmune disease.Material and Methods:The study included 135 patients having systemic inflammatory autoimmune disease (rheumatoid arthritis) who attended ophthalmology out patient department in Govt. Medical College Jammu. The patients underwent detailed ocular examination, slit lamp biomicroscopy, ophthalmoscopy. The tear function of all the patients was assessed using Schirmer’s test, tear film breakup time and ocular surface staining.Results: Fifty three (39%) patients out of the 135 patients had ocular manifestations of rheumatoid arthritis. Dry eye was the most common manifestation which was seen in about 28 patients (53%). The ocular manifestations were more common in females (78%).The manifestations were bilateral in 45 patients(85%).Ten patients (19%) had features of scleritis, three (6%) with episcleritis, 10 patients having anterior uveitis (19%) and other manifestations peripheral ulcerative  keratitis (PUK) ,retinal vasculitis, keratitis contributed 1% each or less. Conclusion: Ocular manifestations contribute significantly to the extra articular manifestations of the Rheumatoid Arthritis. Dry eye is the commonest complication. There was a female preponderance in the study and also the manifestations were more frequently found bilaterally. Keywords: Ocular manifestations, Rheumatoid Arthritis, Episcleritis,Uveitis

Detection of Acid-Fast Bacilli in Postlysis Debris of Clinical Specimens Improves the Reliability of PCR

This article does not have an abstract

Orbital ectopic glial tissue in relation to medial rectus: a rare entity

Heterotopic brain tissue is a rare entity and it is rarer still in the orbit. There have been very few case reports of orbital ectopic glial tissue. The case is described herein of a 3-month-old baby presenting with an orbital glial hamartoma inseparable from the medial rectus muscle. The diagnosis was based on the histopathological features and a positive GFAP stain. The features of this case and the previously reported cases are discussed

Nkx3.2 and Sox9 inhibit muscle-specific gene expression in muscle satellite cells.

<p>Muscle satellite cells were infected with RCAS viruses encoding GFP, Nkx3.2HA, Sox9V5 or Nkx3.2HA+Sox9V5. Immunocytochemistry and qRT-PCR analyses were performed. Virus staining images were overlaid with Pax3, Pax7 and MHC images, and DAPI stains cell nuclei. For all qRT-PCR, GAPDH was used for normalization. (A) Pax3 immunostaining results. (B) Pax3 mRNA expression. (C) Pax7 immunostaining results. (D) Pax7 mRNA expression. (E) MHC immunostaining results. (F) MHC mRNA expression. “*” denotes statistically significant differences (p<0.05) relative to control samples.</p

Nkx3.2 is required for Sox9 to activate a cartilage program and to inhibit the muscle program in muscle satellite cells.

<p>For all qRT-PCR, GAPDH was used for normalization. (A) qRT-PCR analysis of Nkx3.2 expression in satellite cells infected with control RCAS-GFP virus or RCAS-Sox9V5 virus. (B) qRT-PCR analysis of Sox9 expression in satellite cells infected with control RCAS-GFP virus or RCAS-Nkx3.2HA virus. (C)–(G) Muscle satellite cells were co-infected with the following combination of viruses: RCAS-A-GFP+RCAS-B-AP (alkaline phosphatase); RCAS-A-AP+RCAS-B-Sox9V5; RCAS-A-Nkx3.2HA+RCAS-B-Sox9V5; RCAS-A-Nkx3.2-ΔC-VP16+RCAS-B-Sox9V5. These infected cells were subject to immunocytochemistry analysis and qRT-PCR. (C) Immunocytochemistry analysis of Collagen II protein expression in satellite cells. Virus staining images were overlaid with Collagen II images, and DAPI stains cell nuclei. (D) Collagen II mRNA expression. (E). Aggrecan mRNA expression. (F). Pax3 mRNA expression. (G). MHC mRNA expression. In A–B, “*” denotes p<0.05 in statistical analysis. In D–G, “*” denotes statistically significant differences (p<0.05) between RCAS-A-AP+RCAS-B-Sox9V5 and RCAS-A-Nkx3.2-ΔC-VP16+RCAS-B-Sox9V5 co-infected samples.</p

Nkx3.2 and Sox9 inhibit mouse Pax3 promoter activity.

<p>(A) Schematic diagram of the mouse Pax3 promoter luciferase construct. (B) Immunocytochemistry analysis showing equal infection efficiencies of all viruses (GFP, Sox9, Nkx3.2, Nkx3.2-ΔC-HA, Nkx3.2-ΔC-VP16). (C) Luciferase analysis on satellite cells infected with all viruses (GFP, Sox9, Nkx3.2, Nkx3.2-ΔC-HA, Nkx3.2-ΔC-VP16) and transfected with the Pax3 luciferase construct. A control luciferase vector was used for normalization. “**” denotes p<0.01 and “***” denotes p<0.001 in statistical analysis.</p

Viral infection with Pax3 in muscle satellite cells inhibits chondrogenesis while maintaining muscle gene expression.

<p>qRT-PCR analysis of satellite cells infected with RCAS-A-Pax3 or RCAS-A-AP (alkaline phosphatase, control virus) and cultured in 3D micromasses in chondrogenic media. (A) Pax3 mRNA, (B) Collagen II mRNA, (C) Aggrecan mRNA, and (D) MyoD, Myogenin, MHC mRNA expression. All PCR results were normalized to GAPDH. “*” denotes p<0.05 in statistical analysis.</p