HEDGEHOG SIGNALING AND LAMININ PLAY UNIQUE AND SYNERGISTIC ROLES IN MUSCLE DEVELOPMENT

HEDGEHOG SIGNALING AND LAMININ PLAY

UNIQUE AND SYNERGISTIC ROLES

IN MUSCLE DEVELOPMENT

By

Matthew T. Peterson

Thesis Advisor: Dr. Clarissa A. Henry

An Abstract of the Thesis Presented

in Partial Fulfillment of the Requirements for the

Degree of Master of Science

(in Zoology)

August, 2010

            Muscle development is a robust process that has many steps between a muscle precursor, which is a cell that will become a muscle fiber, into a fully functioning muscle fiber.  Any perturbation in these steps can result in a muscle dystrophy.  This thesis explores functions of two proteins in zebrafish muscle development.  Laminin is an extracellular matrix protein, which muscle cells attach to and conduct contraction forces through.  Hedgehog signaling is another protein that instructs certain cells to become a particular type of muscle.  I examined the interaction between these two proteins during embryonic muscle development and found that despite deficiencies in either one of these two proteins individually, muscle development was delayed and was not efficient, but did eventually recover producing elongated muscle fibers.  However, the combined deficiencies of these two proteins produced a failure of muscle development, resulting in a majority of short, round muscle cells with no contractile potential.    In order to determine exactly when and where during the process of muscle development these two proteins were needed together I examined different stages of muscle development.  Starting from before muscle development begins I find that the embryos development is not drastically disrupted by the lack of these two proteins.  I also find that muscle regulatory factors, genes that specify muscle cells, are present and in the correct pattern.  I then looked at myosins, a collection of molecular motor proteins that convert chemical energy to force during muscle contractions.  Although there was a shift in the distribution of two different myosins in embryos deficient for both Laminin and Hedgehog signaling it was still present.  In order to determine if these muscle cell were actually elongating, but perhaps in a different direction than normal, I labeled certain cells so that I could generate a three dimensional projection of individual cells.  I found that the cells were indeed short and round in all dimensions.  During zebrafish muscle development a particular subset of muscle cells, slow twitch muscles, undergo a migration from the medial portion of the embryo to the lateral portion.  This migration is instructive to other muscle cells to elongate.  These slow twitch muscle fibers are specified by the Hedgehog signaling protein, so I investigated if the lack of these cells and Laminin were responsible for the failure of muscle development in these zebrafish.  By treating the embryos with a Hedgehog inhibitor at a stage when part of the embryo has developed slow twitch muscle I could determine if the development failure matched up with the loss of slow twitch muscle.  I found that indeed the loss of slow twitch muscle to coincide with the failure of other muscle cells to elongate.