RESEARCH ABSTRACT SUMMARY:
Norbert Perrimon is using functional genomic approaches to identify molecular
mechanisms that link physiology, cell biology and cell differentiation.
The central thesis of Developmental Biology is to understand how organisms grow and develop. In the past 30 years, studies using genetically tractable model organisms have led to a detailed understanding of the genetic mechanisms involved in the control of developmental events as illustrated by our intimate knowledge of patterning and morphogenesis. The next big questions are to understand how complex phenotypes arise in the context of the whole organism, and how the programs regulating their development and function are influenced by genetic background and environment. For example, little is understood on how the simultaneous growth and differentiation of different tissues is coordinated and on how within an organ the development of different cell types and tissues is integrated.
To address some of these questions, we have initiated a number of studies on muscle growth during Drosophila larval development as this system provides unique opportunities to identify molecular mechanisms that link physiology, cell biology and cell differentiation. During larval growth, individual muscles grow almost 100 fold in a period of four days. This tremendous growth occurs in the absence of change in the number of nuclei and is easily visualized by the addition of new sarcomeres to the pre-existing myofibrils. This process relies on an increase in ploidy of the existing nuclei, protein synthesis and mitochondriogenesis, and is thought to be regulated by the Tor pathway that links amino acid and growth factor levels with muscle cell growth. During growth phase, new sarcomeres are added to “assembly centers” at the muscle edge. Starvation reverses this process, as the sarcomeres are degraded by proteolysis, and the cytoplasm consumed by autophagy. Myofibrillar protein degradation occurs primarily through the proteosome while autophagy breaks down the rest of the cytoplasm and organelles. The dynamic regulation of muscle growth provides a unique paradigm to address how physiology influences aspects of cell biology and differentiation. In addition, analysis of muscle growth and homeostasis may also shed light on the control of muscle wasting that is commonly associated with lack of exercise and a number of human diseases, and that is somehow prevented in hibernating animals.
We are currently addressing a number of questions with regards to muscle growth and its maintenance. First, although many of the proteins that constitute the sarcomeres are known, the mechanisms that regulate their assembly at the muscle edges are not. Second, we are characterizing the extent to which muscle growth can adjust to the physiological milieu and how this homeostatic response correlates with a change in protein translation and gene expression. In particular, as microRNAs have been implicated in muscle growth we wish to understand the role of these small non-coding RNAs in protein translation. Third, to address how muscles sense fluctuations in hemolymph nutrients (amino acids, glucose, triglycerides) that accompany changes in diet, we are focusing on the role of the Insulin pathway in the control of the assembly of sarcomeres and autophagy in muscle cells.
To address these issues, we are using a variety of state of the art technologies. In particular, to identify genes involved in sarcomere organization and growth we are conducting RNA interference (RNAi) screens using our high-throughput genome-wide screening platform (http://flyrnai.org/) in cultured Drosophila primary muscle cells under various nutrient conditions. Cells isolated from gastrula differentiate into muscles within hours and are sensitive to RNAi such that genes that affect growth and sarcomere differentiation can be readily identified. To facilitate the analysis of the genome-wide screens, we are developing algorithms for automated image analyses that provide quantitation of cellular features describing muscle growth and shape. Further, to complement the tissue culture approach and provide validation of the genes identified in the RNAi screens, we are building a large collection of transgenic UAS-hairpin RNAi lines to knockdown gene function exclusively in larval muscles using muscle-specific Gal4 drivers. Finally, we are building a comprehensive network of the genes involved in the regulation of growth and homeostasis to provide a "system level" understanding. Our approach is to integrate datasets from the functional screens with those from the time course gene expression microarrays in muscle under different physiological conditions, allowing us to correlate reduction of given components by RNAi with their corresponding transcriptional consequences.