A Field Guide to The Limb Progenitor

Abstract

The primary goal of this thesis was to characterize the embryonic limb progenitor, a cell type that populates the early limb bud during the onset of limb morphogenesis. Two features of the limb progenitor were explored 1) the identity of a potential set of minimal transcription factors that are sufficient to instantiate limb progenitor identity and 2) the limb progenitor gene expression response to signaling molecules that are critical for regulating the ultimate morphology of the developing limb.

The limb bud consists of mesenchymal progenitor cells, or limb progenitors, that originate from the lateral plate mesoderm (LPM). In addition to the limb progenitors of the limb bud, the LPM also gives rise to mesenchyme that will form the mesodermal components of the trunk, flank and neck. However, only the cells of the limb are capable of forming the patterned skeletal structures observed in arms and legs. The other LPM-derived mesenchymal progenitor cells are unable to form such structures, even when placed in the context of the organizing signals found in the limb bud. To determine which genes are responsible for specifying a limb progenitor identity that is distinct from the identity of neighboring mesenchymal tissues, a screen of transcription factors capable of imparting limb progenitor-like properties on non-limb embryonic and post embryonic fibroblasts was conducted. A minimal subset of genes was identified with the capability of producing limb progenitor-like cells that are similar in gene expression and functional capacity as endogenous limb progenitors.

During embryonic development, fields of progenitor cells form complex spatial structures through a dynamic interaction with external signaling molecules (morphogens). It has been posited that morphogens instruct cells to adopt morphogenetic fates through the activation of gene expression in a graded manner, a so called morphogen gradient model. The morphogen Sonic hedgehog (Shh) has been extensively studied in the developmental context of limb patterning and has been thought to be a dose dependent regulator of skeletal number. In this study, a limb progenitor culture system was used to directly and quantitatively study the limb progenitor gene expression response to Shh and Fgf signals. The limb progenitor response to Shh was found to be a simple ON/OFF switch, a response that is far simpler than those predicted by morphogen gradient models. However, additional complexity in the limb progenitor response was uncovered when the Shh response was assessed in conjunction with Fgf dose. These results highlight the importance of studying morphogen mediated response in context with other signals.