Projects
In these key projects there are many research
opportunities—please visit our Positions
Available page for
a current listing.
Project 1
IN TOTO IMAGING OF EMBRYOGENESIS
We are developing techniques for performing in toto imaging that
allow us to systematically image multiple vertebrate embryos throughout
their embryonic development at single-cell resolution using in vivo
time-lapse, confocal microscopy. We are also developing an advanced
software package called GoFigure to recognize and track cells in
our 4-dimensional, xyzt image sets. The goal of in toto imaging is
to be able to image and track all the cell movements and divisions
during the formation of whole organs and eventually the whole zebrafish
embryo and to digitize this data quantitatively at the level of the
cell. We will use this data to reconstruct cell lineage models that
can serve as an armature for computer modeling of development.
Project 2
Comprehensive spatiotemporal analysis of gene
expression and function of the developing vertebrate embryo We will generate a large collection
of zebrafish lines using a transposon-based gene-trapping technology
called 'flip trapping'. The FlipTrap cassette forms a functional
fusion protein with a green fluorescent tag (GFP variant) when
inserted into an intron. When exposed to cre-recombinase, the cassette
assumes a second conformation, and generates a red fluorescent
tag (RFP variant) gene trap and a mutant allele for the trapped
gene. Thus, each flip trap line can be used to reveal the protein
expression pattern (using the GFP fusion trap) and the mutant phenotype
(using the RFP gene trap). Using in toto imaging, this information
can be read out from the embryo in vivo and non-invasively. Once
validated in the zebrafish, our goal is to apply this strategy
to quail, an amniote that much more closely resembles the developing
human embryo.
 Project
3
Design of multiplexed in situ amplifiers
for in vivo imaging with active background suppression We
propose to design, validate and apply a new in situ amplification
approach based on the mechanism of hybridization chain reaction (HCR),
in which fluorescently labeled DNA monomers self-assemble into tethered
polymers when triggered by probes bound to target mRNAs or proteins.
The design principles underlying this approach conceptually address
two major challenges to existing in situ amplification methods: elevated
background signal due to non-specific probe binding, and simultaneous
multiplexing of multiple target molecules. Our objective is to develop
in situ HCR amplifiers that will enable the simultaneous and specific
detection of multiple low-copy targets in fixed and living embryos.
Reference: Triggered
amplification by hybridization chain reaction.
R.M. Dirks and N.A. Pierce. Proc Natl Acad Sci, 101(43):15275-15278,
2004.
Project 4
Data analysis and integration of technologies
to produce "digital" fish
and bird We will use in toto imaging to upload data fluorescently
marked using FlipTraps and HCR onto a Digital Fish and Quail. We
will generate 4-dimensional atlases that portray the positions of
cells, mRNA expression patterns, protein expression and subcellular
distribution patterns, and mutant phenotypes over time quantitatively
and at high resolution. These will be compiled into a publicly available
database that generates a "digital" organism that can be
analyzed and experimented on by others. Anatomically-based, cell-centric
computer modeling will be performed to investigate how various biological
networks process information to effect development.
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