title: The Role of RhoA in Early Heart Development
Creators: name: Kaarbo, Mari
Publisher: name: Griffith University
Year: 2005
Subjects: subject: RhoA
subject: GTPase
subject: gene expression
subject: cardiogenesis
subject: cardiomyogenesis

description: RhoA is a small GTPase that acts as a molecular switch to control a variety of signal transduction pathways in eukaryotes. From an initial established role in the regulation of the actin cytoskeleton, RhoA has now been implicated in a range of functions that include gene transcription and regulation of cell morphology. In earlier studies from this laboratory that employed differential display and in situ hybridisation, RhoA was indicated as being up-regulated during the stages of early heart development in the developing chick embryo. Given the important effects of RhoA on both gene expression and morphology in other systems, it was hypothesised that RhoA plays a central role in the molecular mechanisms controlling cardiogenesis. This thesis describes investigations undertaken to elucidate the role of RhoA in these processes. As an initial approach to corroborate the earlier gene expression findings and provide further evidence for a role in tissue developmental mechanisms, RhoA proteins levels in the developing chick embryo were analysed using immunocytochemistry. These experiments demonstrated that RhoA is most abundant in heart-forming regions, findings compatible with the earlier gene expression studies and the proposed role of this protein in early heart development. Preliminary studies from this laboratory had also suggested that chick RhoA is expressed as different length mRNA transcripts that vary only in the 3' untranslated region (UTR). This thesis presents additional evidence for the existence of these different RhoA transcripts from experiments using Northern hybridisation and RT-PCR analyses. These analyses also serve to demonstrate that the second shortest RhoA transcript (designated RhoA2) is the most abundant transcript in developing heart tissue, in contrast to the situation in other embryonic tissues, findings that could be taken to suggest a possible role for this 3'UTR in developmental mechanisms that is yet to be elucidated. One potentially informative approach for testing the function of a protein in a biological system is to inhibit its expression and/or activity and observe the changes induced. The effects of inhibiting RhoA in early heart development and early organogenesis in the chick embryo model were investigated using small interfering RNAs (siRNA). Reduction in RhoA expression by siRNA treatment, as confirmed by real-time PCR, resulted in loss of heart tube fusion and abnormal head development, the former result providing further direct evidence of a role for RhoA in heart developmental processes. In order to investigate the function of RhoA specifically during the process of cardiomyocyte differentiation, an inducible model of cardiomyogenesis, P19CL6 cells, was used in combination with over-expression of different forms of mouse RhoA. The striking result from these investigations was that over-expression of the dominant negative mutant of mouse RhoA (mRhoAN19) prevented the differentiation of induced P19CL6 cells to the cardiomyocyte phenotype, results consistent with an essential role for RhoA in this cellular transition. The mechanism by which RhoA mediates its different cellular functions is unclear, however some studies have implicated RhoA in the regulation of transcription factors. To investigate such a mechanism as a possible explanation for the requirement of RhoA in cardiomyocyte differentiation, the P19CL6 inducible cell system over-expressing different forms of RhoA was analysed through real-time PCR to quantify the levels of transcription of genes known to play an important role in early heart development. These investigations indicated that RhoA inhibition causes an accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac-cx-actin. The expression of a protein is controlled by, among other factors, regulatory proteins that control transcription. To investigate factors in heart that potentially regulate RhoA expression at the molecular level, the chick RhoA gene organisation was analysed. The gene was shown to contain three introns that interrupt the protein coding sequence and at least one intron in the 5'UTR. Comparative RhoA gene studies indicated both an almost identical organisation and coding sequence of the chick, mouse and human RhoA genes, indicative of strict conservation of this gene during evolution. The putative promoter region of RhoA was predicted by computer analyses and tested for promoter activity using luciferase reporter analyses in non-differentiated and differentiated cardiomyocytes, using the inducible P19CL6 cell system. These investigations served to define a putative core promoter region that exhibited significantly higher promoter activity in differentiated cardiomyocytes than in non-differentiated cells, and other elements upstream of this core region that appear to be required for transcriptional regulation of RhoA. The majority of the consensus transcription factor sites identified in this putative promoter have been previously implicated in either heart development and/or organogenesis. These results therefore provide further, although indirect, evidence for an important role for RhoA in the molecular mechanisms controlling both cardiogenesis and embryogenesis in general. In summary, this thesis provides novel information on the role of RhoA in the processes of cardiogenesis and provides a firm foundation for continuing investigations aimed at elucidating the molecular basis of this contribution.