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. Because of its size, shape and transparency, the sea urchin embryos is an ideal system for study with light microscopy. The proposed research plan will combine these recent advances in imaging science and reporter gene activity within intact embryos; second, to characterize the post-translational modification of transcription factors thought to be involved in the patterned gene expression; third, to characterize the occupancy of cis-regulatory domains by 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 an deep penetration into tissues. The first experimental goal will be achieved by combining TPLSM with reported genes that encode gene fluorescent protein (GFP) or beta-lactamase (and a fluorescent reporter dye), resulting in quantitative assays of gene activation in intact embryos. The second goal will involved TPLSM combined with phospho-peptide specific antibodies to better define the post-translational modification thought to be critical for the regional and temporal control of transcription factor activity. The third goal will involve fusions of transcription factors with green fluorescent protein mutants to permit resonance energy transfer between and within transcription factors to be used as assays of transcription factor binding. The long-term goal of this project is use a combination of molecular and optical techniques to define of the cellular events that drive patterned gene regulation and the developmental events that are driven by patterned gene regulation.

Project Start
2001-04-01
Project End
2002-03-31
Budget Start
Budget End
Support Year
3
Fiscal Year
2001
Total Cost
$197,422
Indirect Cost
Name
California Institute of Technology
Department
Type
DUNS #
078731668
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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