Clonal Analysis of the Cranial Neural Crest using RIA Viruses

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Project Summary The neural crest is a population of cells that are critical for vertebrate embryonic development. These cells are known for their migratory behavior, and contribution to a wide range of derivatives such as the craniofacial skeleton, epidermal pigment cells, and much of the peripheral nervous system. This proposal tackles a fundamental question in developmental biology: whether individual cranial neural crest cells in vivo are multipotent, and retain the ability to differentiate into numerous fates, or alternatively, if they represent a population of fate-restricted cells, each confined to a unique cellular fate. To tackle this question, replication incompetent avian retroviruses encoding different fluorescent fluorophores will be used to label neural fold cells and perform clonal analyses to examine the developmental potential, movement and morphogenesis of individual or small populations of cranial neural crest cells in vivo. 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, making them more accessible at early stage, while developing in a manner that is morphologically nearly identical to that of 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 limiting dilutions of replication incompetent avian retroviruses that encode fluorophores. Four different viruses reflecting different colors will be used per embryo and clonality will be established by visual observation a few hours after infection. The fate of clonally related cells will then be tracked using antibody staining as well as a novel method of single molecule fluorescent in situ hybridization that allows simultaneous analysis of up to 35 transcripts in single cells. Aim 2: Analysis of migratory interactions between clonally related cells: The migratory behavior of clonally related cells will be examined in whole mount, using in ovo imaging, as well as in slice tissue sections to visualize interactions between sister cells, cousin cells and unrelated neighbors. Once normal migratory patterns and cell interactions are established, the effects of perturbing cell-cell and cell-substrate interactions in individual clones of migratory cells will be examined within an otherwise normal environment.