Genetic basis of intrinsic and extrinsic contributions to the establishment of muscle pattern in the developing limb

If you open a book showing the inside of the human body one of the most striking things is how the tissues and organs are present in the right place and at the correct shape and size. One of the questions that we want to find the answer to is: how do tissues and organs develop in this way? We know that this is controlled to an extent by genes, because mutations, which cause genes to be defective, can make tissues and organs to develop abnormally. We are looking at how the muscles of the body develop in the embryo. The human body has about 650 muscles, some muscles are very large (in the leg), others are very small (in the eye). We know that each of the 40 muscles in the arm has a particular place, and it is connected by tendons to particular places on bones at each end. Because this is the same in different people, we can give a name to each muscle. The question we ask is: how does this happen? For the muscles of the arms and legs this is particularly interesting as these muscles have the strange behaviour in that they grow from muscle cells that move into the arm or leg as the embryo develops. How do these muscles know where to go in the limb and make a particular muscle, why don¿t they get all mixed up and make a jumbled muscle, and how do they make the specific attachments to bones? There are different possible solutions to this problem. One way might be that the muscle cells are programmed to know where they have to go. This is like going for a car drive and you know where you will be going and the route you will take before you start. Alternatively, muscle cells may have no clue where they are going but just follow the directions they are given. This is like a train that can go forwards, but where it goes depends on the train tracks and signals being set correctly by someone else. Scientists think that the train on the tracks model probably is a better explanation of how muscle cells move to their correct locations, and cells in the limb give signals to the muscle cells such as: move here; don¿t go there; keep moving; stop now; make a muscle now. But this is not 100 per cent certain. We wish to do a project to look into this and see if we can find some answers. We shall use a mutant mouse that has a mutation in a gene that makes a protein that switches on other genes. It is what we call a regulator gene. Mice which have a mutation in this regulator gene have very peculiar problems in the way muscles in their arms and legs form. We have noticed that some muscles are completely missing, other muscles form in places that they should not, and some muscles although they are in the right place have split into two muscles. On top of this we have seen that the tendons of the muscles are also abnormal, and many of them are much smaller than normal. We want to find out if these muscle problems are because the muscle cells that make this muscle are defective in their programme (as if some of them cannot read the road map correctly), or is this because the tendon cells and others which may signal to the muscle cells are defective (as if the switches on the rail track have been left in the wrong position, so the train goes in the wrong direction).

There is a paucity of knowledge of the basis of muscle and muscle connective tissue patterning. The aim of the project is to study molecular and cellular interactions between immigrant myoblasts from somites and resident limb mesoderm-derived connective tissue precursors. We shall test the hypothesis that the coordinated function of the Meox2 homeobox gene is essential within both the myogenic and connective tissue lineages to determine the muscle pattern of the limb. There is evidence that the muscle pattern is not pre-specified intrinsically within the myogenic progenitors, but is a property of limb mesoderm. There is a close temporal and spatial association of muscle and tendon morphogenesis and initial morphogenesis occurs autonomously of each other, but later development requires reciprocal interactions. However, there are indications that myogenic precursors may not be completely passive in this process. At present it is unclear the degree of contribution of each component to the development of the final muscle-tendon pattern. Our previous studies have revealed an essential and unique role for the Meox2 homeobox gene in regulating this process. Meox2 is expressed in both the migratory limb myoblasts and also the limb mesoderm. Meox2 mutants have defects in myogenic differentiation; and also extensive defects in muscle patterning and tendon differentiation. This mutant provides a model system to investigate and decipher the interactions between the precursors of these tissues, and thereby to identify and characterise the molecular and cellular components that underlie this process. To investigate the contribution of Meox2 in both the myogenic and tendon lineages we shall use the transgenic and mutant mice, as well as the developing chick embryo. There are four main objectives (1) Investigate the relationship between Meox2 and Scleraxis expression using reporter tagged alleles (LacZ and GFP respectively) of these genes within connective tissue precursors and identify the cellular behaviour of these progenitors in the context of normal and mutant Meox2 function. (2) Remove Meox2 mutation in the muscle precursors (by crossing a conditional floxed allele of Meox2 with a Pax3-MCre deletor line) and similarly remove Meox2 from connective tissue progenitors (by crossing the floxed allele with a mesoderm-specific Prx1-CRE line), and so determine the tissue intrinsic and extrinsic component of Meox2 to the patterning of muscle and connective tissue. We shall also investigate the development of connective tissue precursors in the presence and absence of limb muscles, by use of Splotch embryos. (3) Use in vitro culture assays of CFP and lacZ tagged transgenic limbs to recombine mutant and normal myoblasts with normal and mutant limb mesoderm in different combinations to test for the intrinsic and extrinsic requirements for the differentiation of these tissues. (4) Test whether Meox2 is sufficient to induce tendon progenitors or whether it has a later role in tendon differentiation/maintenance; and testing the epistatic relationship of Meox2 other genes expressed in the muscle-associated mesoderm.