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| Introduction |
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a developing embryo of any imaginable type, common themes
occur from the smallest insect to man, starting from the first
divisions of the fertilized egg. The fertilized egg, or single
celled embryo, contains all the genetic information necessary
to manufacture all the myriad parts of that particular organism.
And it’s all in one cell! Each subsequent division of
cells within the developing embryo results in a shift from
the potential of the cell to become anything to the ability
to become a more limited but much more specialized cell or
groups of cells. This process is called cell fate specification.
One very interesting question is how cells differentiate
to form a specialized tissue or organ. Each of these specialized
cells, tissues or organs gain their identity through the expression
of a unique set of gene products. Thus, differentiation of
cells results in altered gene expression. So, we can use the
power of genetics to address this question. |
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| I
have chosen to examine this problem in the model organism,
Caenorhabditis elegans, a free-living soil nematode
about 1 mm in length. C. elegans, as a model system,
is particularly amenable to study this process due to its
simple structure, excellent genetics, and ease of handling.
My studies focus on the identification and characterization
of the genes required to specify the cell fate of the gonadal
sheath cells using a new technique called RNA interference
or RNAi (Montgomery and Fire, 1998). |
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| Background |
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C.
elegans is a hermaphroditic
species (Brenner 1974), which means that it produces both
sperm and eggs. Its gonad consists of two U-shaped arms surrounded
by the gonadal sheath and germ cells develop within each arm.
Approximately the first 300 germ cells produced become sperm
and the remainder form oocytes. The gonadal sheath is a tissue
comprised of five pairs of gonadal sheath cells that encircle
the proximal half of each gonadal arm (Hall et al, 1999) and
that have been implicated in gametogenesis, spermatogenesis,
meiotic progression of the germ cells and ovulation of the
mature oocytes into the spermatheca, the storage organ for
the hermaphrodite’s sperm (McCarter et al, 1999). When
one or more cell pairs within this tissue is absent, aberrations
in germline maintenance, oocyte maturation and/or ovulation
occur (McCarter et al 1999). Thus, in addition to understanding
underlying mechanisms to common developmental mechanisms,
my research may contribute to underlying mechanisms common
for fertility in many organisms. |
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| In
1998, C. elegans was the first multicellular organism
to have its genome sequenced (Ainscrough et al, 1998) in the
massive genome sequencing project begun about 10 years ago
with the ultimate goal of sequencing the human genome, a goal
that was in large part achieved two years ago. Access to this
watershed of data has led to the development of new technologies
that has facilitated research in recent years. One such on-going
development has been the creation of collections or “libraries”
of all the genes in an ordered fashion. One such library is
an RNA-mediated interference library discussed below (Fraser
et al, 2000). |
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| Current
Research |
Search for genes necessary for the formation of the gonadal sheath using RNAi |
One
way to identify key components in a biological process is
to look for genes that, when functionally disable, lead to
a visible defect in the processes under investigation. One
way this screening can be done is a new way available in C.
elegans: RNA-mediated interference (RNAi). RNAi can create
the null mutant phenotype of any gene for which the sequence
is available (all genes in this case, since the worm genome
is sequenced). Thus, it is possible to examine each gene for
its effect on the particular cell fate. Using RNAi as an initial
approach I am determining the components necessary for specification
of the gonadal sheath. Such a collection of genes in vectors
that induce double-stranded RNA is available as an RNAi library
for each of the chromosomes of C. elegans (Fraser
et al, 2000; Kamath et al, 2003).
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To
visualize the gonadal sheath, I am using a green fluorescent
protein (GFP) reporter system that auto-fluoresces in the
gonadal sheath. Green fluorescent protein is a protein that
is normally produced by jellyfish and fluoresces green in
ultraviolet light. Use of GFP is a common tool to mark the
position of individual protein components in an organism or
to make a cell or subset of cells. Genetic manipulations that
affect gonadal sheath cell development will display an alteration
the GFP expression pattern. |
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I
am screening for genes that are required for gonadal sheath
cell development by looking for worms fed with bacteria expressing
the RNAi construct for individual genes that either no longer
fluoresce green or show an altered pattern of expression.
The worms that lose fluorescence will be further examined
for altered fertility, as that is one of the defects that
is anticipated in an animal that does not have one or more
gonadal sheath cell pairs. To date, I and undergraduate students have done screens using a chromosomes I and II RNAi library.
A number of interesting candidates have been recovered that will be explored in the near future. |
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Regulation of lim-7
The GFP reporter system mentioned above that auto-fluoresces in the gonadal sheath pairs 1-4 is fused to the first two exons and first intron of lim-7, a gene encoding a member of the LIM homeodomain family of transcription factors. In addition to the gonadal sheath, LIM-7 is expressed in many neurons and perhaps some muscle and intestinal cells as well. More is known about the neuronal role(s) of LIM-homeodomain proteins throughout evolution. Therefore we would like to elucidate the role that these proteins play in the non-neuronal cells, particularly using the gonadal sheath as a model. An in-frame deletion in lim-7 that removes most of both LIM domains causes early larval lethality and so the gene is very important not only for the gonadal sheath but also for early embryonic decisions, which is not surprising given the neuronal expression. However, nothing is known about its role in the gonadal sheath, in the embryo or what its cellular role its. We are currently trying to understand these mechanisms that LIM-7 controls.
To begin these studies we made a full-length lim-7 genomic construct to rescue the deletion mutation and found that it rescued the lethality that was seen; however, all the adult hermaphrodites were sterile (see Voutev, Keating, Hubbard, Vallier 2009). We wish to make another deletion allele of lim-7 to see if these results can be duplicated. Ultimately we want to understand which genes LIM-7 is controlling in the genome and for what pruposes as well as what controls lim-7 expression.
The genomic structure of lim-7 is unusual in that it has several large introns. The average size of a C. elegans intron is <100 bp; those genes that contain large introns, particularly large first introns have been noted to have regulatory information residing within the introns. Likewise lim-7 has an enhancer element in its large first intron (Voutev, Keating, Hubbard, Vallier 2009) that is necessary and sufficient for gonadal sheath expression. We are interested in parsing out the other tissue-specific regulatory information that may exist in intron 1 or its other large introns and work is on-going to determine this. In addition, we are further exploring the relationship between intron size and the presence of regulatory information for all genes, using conserved intron elements among closely related Caenorhabditis species as a criterion for possible regulatory elements. |
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