Laser Capture Microdissection of Mouse Growth Plate Cartilage.

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Abstract

[Indexed for MEDLINE] 17. Bone. 2015 Jul;76:97-106. doi: 10.1016/j.bone.2015.02.031. Epub 2015 Apr 4. Role of c-Myb in chondrogenesis. Oralová V(1), Matalová E(2), Janečková E(3), Drobná Krejčí E(4), Knopfová L(5), Šnajdr P(4), Tucker AS(6), Veselá I(7), Šmarda J(5), Buchtová M(3). Author information: (1)Institute of Animal Physiology and Genetics CAS, v.v.i., Brno, Czech Republic; Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic. Electronic address: 211812@mail.muni.cz. (2)Institute of Animal Physiology and Genetics CAS, v.v.i., Brno, Czech Republic; Department of Physiology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic. (3)Institute of Animal Physiology and Genetics CAS, v.v.i., Brno, Czech Republic; Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic. (4)Institute of Anatomy, Charles University, Prague, Czech Republic. (5)Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic. (6)Department of Craniofacial Development and Stem Cell Biology, King's College London, London, UK. (7)Department of Anatomy, Histology and Embryology, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic. The Myb locus encodes the c-Myb transcription factor involved in controlling a broad variety of cellular processes. Recently, it has been shown that c-Myb may play a specific role in hard tissue formation; however, all of these results were gathered from an analysis of intramembranous ossification. To investigate a possible role of c-Myb in endochondral ossification, we carried out our study on the long bones of mouse limbs during embryonic development. Firstly, the c-myb expression pattern was analyzed by in situ hybridization during endochondral ossification of long bones. c-myb positive areas were found in proliferating as well as hypertrophic zones of the growth plate. At early embryonic stages, localized expression was also observed in the perichondrium and interdigital areas. The c-Myb protein was found in proliferating chondrocytes and in the perichondrium of the forelimb bones (E14.5-E17.5). Furthermore, protein was detected in pre-hypertrophic as well as hypertrophic chondrocytes. Gain-of-function and loss-of-function approaches were used to test the effect of altered c-myb expression on chondrogenesis in micromass cultures established from forelimb buds of mouse embryos. A loss-of-function approach using c-myb specific siRNA decreased nodule formation, as well as downregulated the level of Sox9 expression, a major marker of chondrogenesis. Transient c-myb overexpression markedly increased the formation of cartilage nodules and the production of extracellular matrix as detected by intense staining with Alcian blue. Moreover, the expression of early chondrogenic genes such as Sox9, Col2a1 and activity of a Col2-LUC reporter were increased in the cells overexpressing c-myb while late chondrogenic markers such as Col10a1 and Mmp13 were not significantly changed or were downregulated. Taken together, the results of this study demonstrate that the c-Myb transcription factor is involved in the regulation and promotion of endochondral bone formation. Copyright © 2015 Elsevier Inc. All rights reserved. DOI: 10.1016/j.bone.2015.02.031