A major question in developmental biology is how precursor cells give rise to diverse sets of differentiated cell types. This proposal tackles the question of multipotency and migratory behavior of neural crest cells, focusing on the cranial neural crest due to its broad ability to contribute to numerous and diverse cell types, as distinct as neurons and cartilage. Although classical grafting experiments have elucidated the derivatives of the neural crest, comparatively little is known about the developmental potential of individual cranial neural crest cells in vivo. Here, we propose to use replication incompetent avian retroviruses encoding different fluorescent fluorophores to label dorsal neural tubes in order to perform clonal analyses. The goal is to examine the developmental potential, movement and morphogenesis of individual or small populations of cranial neural crest cells. Experiments will be performed on avian embryos because of several advantages. Chick embryos are easily accessible to retroviral infection and experimental perturbation at early stages of development, allowing temporally and spatially controlled manipulation. Birds like humans are amniotes but, unlike mice, develop outside the mother. Therefore, they are much more accessible at early stage, while developing in a manner that is morphologically nearly identical to human embryos at comparable stages. Aim 1: Retrovirally mediated clonal analysis of the chick cranial neural crest: The cranial neural tube of chick embryos will be infected with replication incompetent avian retroviruses that encode four different fluorophores. Clonality will be established by visual observation of single cells a few hours after infection. We will then follow the long term fate of clonally related cells as a function of time by examining their localization and differentiation using antibody markers characteristic of various cell fates. Aim 2: Coupling lineage analysis with single molecule Fluorescent In Situ Hybridization to examine multiplex gene expression of clonally related cells. We will couple lineage analysis with a novel adaptation of smFISH that we have recently developed that allows multiplex analysis of gene expression at single cell resolution. Spatial Genomic Analysis (SGA) enables simultaneous analysis of the expression of 35 or more genes on tissue sections at migratory and post-migratory stages. We will combine clonal analysis with SGA to determine the genes co- expressed by clonally related cells using markers of various lineages together with neural crest and pluripotency genes to characterize the transcriptional profile of clonally related genes. Aim 3: Analysis of migratory interactions between clonally related cells: We will examine the migratory behavior of clonally related cells both in whole mount, using in ovo imaging, as well as in slice tissue sections to visualize interactions between sister cells and unrelated neighbors. Once normal migratory patterns and cell interactions are established, we will examine the effects of perturbing cell-cell interactions in individual clones migrating through an otherwise normal environment.