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The Notch pathway is ubiquitous in metazoans, found
in virtually all species in all stages of development. Despite its
universality, many specifics of the Notch pathway are still unknown.
Certain proteins such as Notch, Delta, Suppressor of Hairless (Su(H)),
and Mastermind (Mam) have all been identified as playing key roles
in the function of the pathway, but it is likely that not all proteins
involved have been uncovered. A genetic screen was developed in
order to discover these unknown proteins. This screen is based on
the fact that a mutation in the structure of Mam causes nicks along
the periphery of the wing in the adult fly. A truncation of Mam
protein, termed MamH, was produced containing just a basic domain
near one end of the protein. Its expression results in slightly
nicked wings. A genetic screen was used to look for modifiers of
the MamH associated wing phenotype. This was accomplished through
the utilization of a yeast gene overexpression system (GAL4-UAS).
509 single genomic hops to random sites of a DNA element (EP) were
obtained. Each hop allowed a unique Drosophila sequence to be overexpressed
through the GAL4-UAS system. Overexpressed hops that modified the
MamH wing phenotype were screened for. In using the Mam truncation
it is possible not only to detect an enhanced phenotype, but a suppressed
phenotype as well, because control MamH wings are already slightly
nicked. If the overexpression of a gene leads to the enhancement
or suppression of the wing phenotype then the random gene may be
involved in the Notch pathway. If any wing modification was detected,
further studies were performed involving the classification of the
modifier gene. Through this study a more thorough understanding
of the specific effects of the Notch pathway in the development
of metazoans is made possible.
Notch is an evolutionarily conserved pathway that
plays a significant role in the development of metazoans. This pathway
controls cell fate through the interaction of adjacent cells in
a process called lateral inhibition (Brody, 1999). In this pathway,
two different cells produce different transmembrane proteins, Notch
and Delta, that physically interact and cause proteolysis of the
cytoplasmic domain of Notch. The domain is then able to migrate
to the nucleus where it acts as a transcription factor and binds
Su(H), Mam, and other proteins. These complex interactions lead
to the migration of cells and the eventual formation of tissues
and organs (Artavanis-Tsakonas, 1999). Notch is found in all stages
of development and affects many different tissues. In adult Drosophila,
Notch has an active role in the development of the wings, bristles,
and eyes. During wing formation, Notch functions at the wing imaginal
disc in the developing larvae. The imaginal disc is flat and made
up of one layer of cells with the future wing margin across the
middle of the disc. The disc folds in half to create a fully formed
adult wing with a dorsal and ventral layer. Because the pathway
acts in the development of the wing margin of the imaginal disc,
the outside edge of the adult wing is affected by Notch pathway
mutations. Depending on the severity of the mutation, the wing may
have subtle nicks or may be extremely jagged. A mutation in Mam,
one of the proteins of the Notch transcription complex, causes nicks
around the periphery of the wing in the adult fly. The role of Mam
as a transcription factor is thought to involve the binding of other
proteins in the Notch pathway. Fully functional Mam has 3 charged
domains. The basic domain at the N-terminus binds Notch while two
acidic carboxy domains bind additional proteins in the complex.
Thus, a truncation of Mam that removes the acidic domains would
still allow Mam to bind Notch, but would not permit additional proteins
to bind Mam. When expressed, this truncated Mam, labeled MamH, results
in a faulty Notch pathway which causes nicks around the wing margin.
Using this information, a screen was developed to detect unknown
proteins that might be involved in the Notch Pathway. Using a yeast-based
Gal4-UAS system, the MamH truncation was specifically expressed
across the wing margin, creating a strain of flies with moderately
nicked wings. The system was further utilized to overexpress random
genes and look for modification of the basal MamH wing phenotype.
If the wing phenotype of the MamH flies is enhanced or suppressed
through this overexpression, then that random gene may be required
for the function of the Notch pathway. This experiment shows the
progress of the screen.
Gal4-UAS is a yeast-based overexpression system that
was used to overexpress genes in the fly. In this system a promoter
drives Gal4. Gal4 protein then physically interacts with a UAS site
engineered in the genome. This interaction leads to the UAS site
expressing its target gene. If Gal4 protein is not present then
the UAS site does not act as a promoter. This Gal4-UAS system is
modified in order to express random genes in this project. A transposase
is used to insert EP, a UAS site and a transcriptional start site,
at random locations. The UAS site expresses a unique gene with the
Hsp70 promoter. This gene is expressed only when Gal4 is present.
In order to isolate random inserts that affect just the wings, a
promoter was used that only expressed genes in the wing margin.
This promoter was C96. Because Gal4 could only be promoted in the
wing margin, then the expression of the random genes by UAS could
only occur in the wing margin.
509 hops were detected in the offspring of the crosses
shown in figure 3.
Each of these hopped males was crossed with female C96RH2/sb. The
offspring of this cross were screened for enhanced and suppressed
wing phenotypes, but very few showed modified wings. Of these 500,
14 vials were found to have a more severe wing phenotype. These
vials were retested, crossing enhanced males to W1118 females and
crossing the normal males with the hop with W1118, C96RH2/sb, and
309RH/cy. These retests indicate which chromosome contains the hop
and help determine whether the hop is actually a wing modifier through
further enhancement of the wing phenotype or through the tufting
of specific bristles.
Figure
1. The Notch Pathway. This figure shows a simplified Notch pathway.
Delta and Notch, two transmembrane proteins, interact. This interaction
leads to a protease cutting Notch, liberating a cytoplasmic domain
of the protein. The severed Notch then binds Su(H) and Mam and travels
to the nucleus of the cell where it acts as a transcription factor.
This pathway leads to the formation of the wing from an imaginal
disc through action across the dorsal ventral margin.
Figure
2. Mam Truncations. This figure shows Mam truncations that have
been used in the past to affect the Notch pathway. MamN and MamH
have both been shown in previous research to cause slight mutations
in the wings of Drosophila, while MamR does not. MamH is used in
this experiment.
Figure
3. Gal4-UAS and random hops. Figure 3a shows the general concept
and the system used to express MamH. Figure 3b depicts the yeast
system used to cause random hops in the fly genome. The promoter
expresses Gal4, which in turn, interacts with the EP element containing
the UAS site. This interaction drives the overexpression of the
random gene.
Figure
4. The Screen. This figure depicts the crosses that were performed
to produce the strain of fly desired in the experiment. In the first
cross females with the EP (hop) element and a gene conferring red
(w+) eye color on the X chromosome were crossed with males with
Ä2-3, a transposase gene. Males were selected from this cross
and mated with white-eyed homozygous females. These two crosses
allowed the eye color of the fly to indicate the presence of the
EP gene at a new random site. Males with non-mottled eye color were
chosen from the second cross. The solid eye color was an indication
that the EP element had hopped from the X chromosome, but that the
transposase source was no longer present. The non-mottled males
were crossed with females with the MamH gene on chromosome 3 driven
across the wing margin via the C96 Gal4 element. These flies combined
the MamH truncation expression with the overexpression of a random
gene. The offspring of this cross were screened for a wing phenotype
modification. If there was a modification, then the random gene
may play a role in the Notch pathway.
Figure
5. Wing Modifier. This figure shows two images of nicked wings.
The control wing is C96RH/W1118, which has a very slight wing phenotype.
The enhanced wing is the identical phenotype except for the presence
of an EP hop. The more severe phenotype is one of the 14 modifier
candidates detected in the screen from over 500 different vials.
This modification could be the result of a random hop to a gene
that affects the Notch pathway. Additional crosses will be performed
in order to determine if this phenotype is due to further mutation
in the Notch pathway.
Further experiments will be performed on those candidates
that show a modified wing phenotype. These experiments will likely
include PCR and sequencing in order to locate the UAS site and to
determine which gene that site affected. This is facilitated by
the recent completion of sequencing the Drosophila genome.
This material is based on work supported by the National
Science Foundation REU under Grant #9820356.
The Notch pathway is found in all animals and is
very important in the development of organs and tissues in those
animals. In fruit flies, Notch helps in the development of the wings.
In this pathway a protein known as Mastermind is very important.
If Mastermind is mutated the pathway functions, but functions abnormally.
The mutated Mastermind causes little nicks around the edges of the
wings of the adult fly. Our goal in lab was to overexpress as many
random genes in the fly as we could and see if that over expression
either increased or decreased the mutation caused by the faulty
Mastermind in the wing. If we could find a random gene that either
made the more jagged or fixed the wing so that it was back to normal,
then we might have found a gene that played a significant role in
the Notch pathway.
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