Investigating the molecular regulation of tissue segregation and distinction in knee joint formation

Abstract

Synovial-lined articular joints are complex structures that facilitate friction-free movement between skeletal elements. Disorders of joint formation and maintenance are a common cause of pain and disability. Joint formation begins with establishment of an interzone at sites within the cartilaginous anlagen of the future skeleton and is followed by differentiation and segregation of tissues in the joint region. Chondrocytes abutting the interzone remain throughout adulthood and form articular cartilage, whereas the more distal epiphyseal chondrocytes are consumed by endochondral ossification. Previous studies have identified that the transcription factor Erg may have a role in regulating the articular/epiphyseal chondrocyte distinction. Members of the cadherin family of adhesion molecules have been implicated in tissue segregation during joint formation. This study therefore aimed to: i) Evaluate model systems for the study of tissue distinction, including limb bud culture and knockout mouse models; ii) Examine the role of Erg in the specification of articular cartilage; and, iii) Identify which cadherin(s) are expressed during joint formation and explore their roles. This study found that the articular/epiphyseal chondrocyte distinction was maintained in limb bud culture indicating that absence of movement in limb bud culture does not cause reversal of this distinction. This model system was therefore not suitable for examining the role of movement in the maintenance of the articular/epiphyseal distinction.In situ hybridisation studies found that Erg was expressed by cells that are not destined to be consumed by endochondral ossification at E13.5 in the mouse. Its expression surrounded that of Matn1 and spanned the interzone, with the exception the intermediate zone (expressing Gdf5). This suggests that Erg has a role to play in the establishment and differentiation of articular chondrocytes. To explore the role of Erg in vivo an Erg knockout mouse was generated but found to have an early embryonic lethal phenotype. To study the role of Erg in vivo on articular chondrocytes would therefore require the generation of mouse lines with chondrocyte specific knockout of Erg and subsequent studies were carried out in vitro in the C3H10T1/2 micromass culture system.Erg splice forms were found to be expressed in micromass cultures and their expression was altered by the introduction of Gdf5 in a context dependent manner. Overexpression of splice variant Erg-10β caused downregulation of Col2a1 and Col10a1. This demonstrated that Erg-10β overexpression consistently impaired the process of chondrogenesis in micromass cultures. Microarray analysis following Erg-10β overexpression identified potential downstream targets regulated by Erg-10β, including Ube2b, Klf5 and Osr2. Of these, Osr2 may be acting either by directly downregulating Col2a1 or by driving cells down an alternative lineage. Ube2b may be involved in ubiquitin pathway reduction of BMP2 signalling, a key part of micromass formation. Lastly, Klf5 may be breaking down the cartilaginous matrix deposited in culture thereby interfering with chondrogenesis. Studies of knee joint formation in the mouse found N-cadherin expression at the joint line prior to cavitation and in the developing meniscus. Its expression pattern was found to be reciprocal to that of pERK, a key component of mechanotransduction pathways.The studies documented implicate novel pathways and provide insights into the molecular mechanisms controlling key events in chondrocyte differentiation and knee joint development.

Date of Award	31 Dec 2012
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Gillian Wallis (Supervisor)