Our lab investigates genes and networks regulating muscle identity, muscle size, and muscle organelle positioning, particularly myonuclei movement and placement.
Figure 5: Representation of Functional classes of genes enriched in Founder Cells. -- Eleven new transcription factors and chromatin regulators that show muscle phenotypes were identified. Additional cell functions were also suggested as critical for muscle identity.
Figure 7: Myonuclear positioning in control and ensconsinswoosh mutants. -- High magnification view of muscles (green) and their nuclei (red) at listed stages. While the initial morphogenetic events are normal in a swoosh mutant, the nuclei do not spread and position normally. While the embryos hatch into larvae, these larvae do not move normally.
Figure 8: Time-lapse sequence of myonuclear movement in Drosophila LT muscles from Control and ensconsinswoosh mutants. -- These stills are from a timelapse of a whole mount embryo, showing the movement of myonuclei in the 4 Lateral Transverse muscles in one hemisegment over 6h of development. The nuclei start as a cluster, split into two groups that move towards the muscle end. These nuclei spread to fill the muscle cell prior to hatching. In ensconsinswoosh mutants, the nuclei fail to break into two groups.
In the Drosophila embryo, a repeated pattern of 30 distinct muscle fibers is present in each abdominal hemisegment. Despite similarities, such as shared expression of contractile proteins, each muscle fiber can be distinguished by properties like its size, shape, orientation, number of nuclei, innervation, and tendon attachment site (see Figure 4).
Muscle fibers arise by the iterative fusion of two types of myoblasts, called founder cells (FCs) and fusion competent myoblasts (FCMs), to form a syncytium. FCs are thought to contain all the information needed to make a muscle of a particular size and shape. This information is encoded by DNA-binding transcription factors, such as Krüppel, Slouch, and Apterous, expressed in incompletely overlapping subsets of FCs, known as the FC identity genes.
Using a novel molecular screen, we have identified new transcription factors, such as Alhambra and Charlatan, and, for the first time, chromatin regulators such as Sin3a and Kdm2 that are enriched in different populations of FCs (see Figure 5). We have found that the chromatin regulators buffer the function of the transcriptional regulators, assuring specific muscle identities. Current investigation is directed at finding how morphological information is translated from the identity regulators to the cellular processes that control muscle size and shape.
As in humans, muscle cells in Drosophila form by the fusion of myoblasts. Thirty FCs seed the abdominal muscle pattern and recruit neighboring FCMs to form multinucleate muscle fibers. Certain muscles stereotypically contain as few as three nuclei (indicative of two fusion events), whereas others consistently incorporate up to 25 nuclei (24 fusion events).
Using genetic screens and time-lapse videography, we have identified an actin structure — the F-actin focus — that marks the fusion site. The F-actin focus forms the backbone of an invasive podosome-like structure that is required for fusion to progress. Using different genetic screens, we have shown that this structure can be modulated by several additional actin regulators as well as by PI(4,5)P2 signaling.
Current experiments are aimed at identifying additional components of the fusion process as well as the mechanism by which muscle cells “count” and determine the characteristic number of fusion events for a particular muscle.
Muscle subcellular organization (nuclear positioning, cytoskeletal organization, other organelle placement)
Highlighting the importance of proper intracellular organization, many muscle diseases are characterized by mispositioned myonuclei. We have found that proper positioning of myonuclei is dependent upon microtubule organization, microtubule-associated proteins such as Ensconsin, and the microtubule motor proteins Kinesin-1 and cytoplasmic Dynein. Mutations in these proteins affect myonuclear position and muscle function (see Figure 7, 8).
We have identified at least two distinct mechanisms by which these proteins move and position myonuclei. These motors exert forces both directly on the nuclear surface and from the cell cortex via microtubules. Current projects investigate additional components required for myonuclear movement, the contributions of other tissues to nuclear placement, and the physiological changes that occur in muscle with displaced nuclei.