Recent advancements in the development of fluorescent indicator dyes, the isolation of proteins that naturally fluoresce, and the refinement of techniques for in vivo microscopy offer unprecedented opportunities to study the cellular and molecular events within living, intact embryos. In parallel, there has been dramatic progress in the study of gene regulation during the embryonic development of the sea urchin and the ascidian, culminating in the proposal of Gene Regulatory Networks for developmental patterning. Because of their size, shape and transparency, the sea urchin embryo and ascidian embryos are ideal systems for study with light microscopy. The proposed research plan will combine these recent advances in imaging science and developmental biology to attack three related goals: first, to better define the spatio-temporal aspects of gene activity within intact embryos; second, to refine tools for the imaging and perturbation of factors thought to be involved in the patterned gene expression; third, to characterize the mobilization and activity of transcription factors during key developmental and gene regulatory events. Two-photon laser-scanning microscopy (TPLSM) will be used as the major imaging tool in these experiments, because of its high sensitivity, low photo-toxicity and deep penetration into tissues. The first experimental goal will be achieved by combining TPLSM with reporter genes that encode green fluorescent protein (GFP) and its color variants to yield quantitative assays of gene activation in intact embryos. Parallel experiments will employ fluorescent in situ hybridization technologies to assay gene activation in fixed embryos. The second goal will involve TPLSM combined with GFP fusions and specific antibodies to better define the regional and temporal redistribution of transcription factors, and caged morpholinos to permit photoactivatable perturbation of the gene regulatory network. The third goal will involve fusions of transcription factors with green fluorescent protein mutants to permit transcription factor mobilization and docking to be followed by advanced fluorescence techniques. The long-term goal of this project is use a combination of molecular and optical techniques to define the cellular events that drive patterned gene regulation and the developmental events that are in turn driven by patterned gene regulation. This will provide the tools needed to test the genetic regulatory networks proposed in the other components of this Program Project.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Program Projects (P01)
Project #
5P01HD037105-08
Application #
7213232
Study Section
Special Emphasis Panel (ZHD1)
Project Start
Project End
Budget Start
2006-03-01
Budget End
2007-02-28
Support Year
8
Fiscal Year
2006
Total Cost
$225,818
Indirect Cost
Name
California Institute of Technology
Department
Type
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
Slota, Leslie A; McClay, David R (2018) Identification of neural transcription factors required for the differentiation of three neuronal subtypes in the sea urchin embryo. Dev Biol 435:138-149
Hutchins, Erica J; Kunttas, Ezgi; Piacentino, Michael L et al. (2018) Migration and diversification of the vagal neural crest. Dev Biol :
Kerosuo, Laura; Neppala, Pushpa; Hsin, Jenny et al. (2018) Enhanced expression of MycN/CIP2A drives neural crest toward a neural stem cell-like fate: Implications for priming of neuroblastoma. Proc Natl Acad Sci U S A 115:E7351-E7360
Rogers, Crystal D; Sorrells, Lisa K; Bronner, Marianne E (2018) A catenin-dependent balance between N-cadherin and E-cadherin controls neuroectodermal cell fate choices. Mech Dev 152:44-56
McClay, David R; Miranda, Esther; Feinberg, Stacy L (2018) Neurogenesis in the sea urchin embryo is initiated uniquely in three domains. Development 145:
Roellig, Daniela; Tan-Cabugao, Johanna; Esaian, Sevan et al. (2017) Dynamic transcriptional signature and cell fate analysis reveals plasticity of individual neural plate border cells. Elife 6:
Lignell, Antti; Kerosuo, Laura; Streichan, Sebastian J et al. (2017) Identification of a neural crest stem cell niche by Spatial Genomic Analysis. Nat Commun 8:1830
Martik, Megan L; McClay, David R (2017) New insights from a high-resolution look at gastrulation in the sea urchin, Lytechinus variegatus. Mech Dev 148:3-10
Murko, Christina; Bronner, Marianne E (2017) Tissue specific regulation of the chick Sox10E1 enhancer by different Sox family members. Dev Biol 422:47-57
Peter, Isabelle S (2017) Regulatory states in the developmental control of gene expression. Brief Funct Genomics 16:281-287

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