| Jeffrey Trimarchi , Ph.D.
Harvard Medical School
77 Ave Louis Pasteur, Genetics NRB Room 360
Boston, MA 02115
Tel.: (617) 432-7758
trimarc@genetics.med.harvard.edu
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Lineage analyses in the vertebrate retina have shown that the
progenitor cells that give rise to differentiated retinal cell types
are multipotent throughout development and, as development
progresses, these progenitor cells can be induced to generate only
the temporally appropriate cohort of retinal cell types. One current
model proposes that a heterogeneous pool of progenitor cells passes
through distinct states of responsiveness (competence states) during
which time different populations are biased to produce distinct
retinal cell types. Relatively little is known, however, about the
genetic programs responsible for retinal cell fate decisions,
particularly concerning how progenitor information is passed on to
daughter cells. Expression studies of several different factors
indicate that there is some degree of heterogeneity among retinal
progenitor cells. However, important unresolved questions remain.
First, how many different types of retinal progenitor cells are
there? Second, what is the transcriptional program that must be
activated in these cells to produce the differentiated cell types?
Third, how do progenitor cells move from one pattern of gene
expression to another? My project focuses on setting up a framework
to answer all of these questions, while directly answering question
one.
Using a protocol modified from Catherine Dulac’s laboratory at
Harvard University, I am isolating single retinal cells from three
distinct stages of mouse development (E12.5, E16.5 and P0). cDNAs
from each single retinal cell are compared on cDNA microarrays
containing approximately 12,000 genes from the collection of Dr.
Bento Soares (http://genome.uiowa.edu) and PCR products of clones of
interest from our laboratory. To identify cells with common patterns
of gene expression, the resulting microarray data has been clustered
using available software packages and the assistance of several
bioinformatics groups here at Harvard Medical School. The clustering
analyses should reveal potential similarities and differences among
subsets of retinal cells and at the same time, identify novel genes
expressed in distinct subsets of retinal progenitor cells. In fact,
the method has worked very robustly so far. I have been able to
distinguish mitotic cycling progenitor cells from postmitotic retinal
neurons by the single cell gene expression profiling protocol. In
addition, I have identified numerous novel genes whose expression is
specific either to cycling progenitor cells or postmitotic neurons.
To further validate these results, I am performing double fluorescent
in situ hybridizations on dissociated retinal cells and
quantitatively verifying the existence of certain retinal progenitor
cell subsets as predicted from the microarray analysis.
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