The function of R-spondins during mouse limb development and chondrogenesis

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A small set of cellular signals called WNTs, hedgehogs, BMPs, and FGFs control much of embryonic development. These secreted proteins interact in a precise spatial and temporal sequence to pattern the embryo. The formation of the limbs (arms and legs) is among the best-characterized developmental pathways in vertebrates. We use the mouse limb as a system to understand how cellular signals control morphogenesis (the creation of form) and cellular differentiation. The mechanisms by which the activities of these signaling systems are precisely modulated is unknown. Recently, the R-spondin proteins (RSPOs) have been identified as important components of the WNT pathways. RSPOs are key candidates for modifying these critical pathways during limb development. The four genes that code for RSPOs (Rspo1-4) have been shown to have important developmental functions ranging from sex determination to placental, lung and limb development. As the necessary mouse mutants are becoming available, limb morphogenesis will likely be the first developmental system where a comprehensive description of Rspo gene function may be obtainable. This proposal pursues this goal by organizing two objectives around successive stages of limb development: 1) The early outgrowth and patterning of the limb bud and 2) the later process of chondrogenic differentiation and maturation that establishes the cartilage template of the limb skeleton. In Objective 1 we determine the function of Rspo4 during limb development and its genetic interactions with Rspo2 and Rspo3. By combining the Rspo4 and Rspo2/3 mutations we will obtain a more complete understanding of the function of these genes during limb development. In Objective 2 we determine the function of R-spondins during limb cartilage development, using an in vitro system called micromass cell culture. After they have participated in early limb development, we hypothesize that the R-spondins have a function during the differentiation of the cartilage-producing cells, the chondrocytes. Taken together these aims will reveal how R-spondins help control the WNT signaling pathways so that they are deployed in a precise manner during development.