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The Notch pathway is an intercellular signaling mechanism responsible for proper development in a variety of organisms ranging from insects to mammals.
Despite extensive study, the Notch pathway is still not completely understood, and additional components of this signal transduction cascade still remain to
be discovered. A loss of function analysis of domino, a gene predicted to encode a chromatin remodeling protein and believed to function in the Notch pathway
was done. Using GAL4-UAS-driven RNAi constructs, the expression of the domino gene was reduced in tissues that require Notch pathway function. Our results
show that loss of function for the domino gene does lead to certain known Notch phenotypes and supports our hypothesis that domino is involved in the Notch
pathway.
The Notch pathway is a signal transduction cascade with various mechanisms, including lateral inhibition during metazoan development. Defects in this pathway
have been linked to disease-related processes in humans. As more activities of this pathway were recently characterized, it became apparent that there are more
genes involved in its function than have been discovered. Consequently, the genetic and biochemical details of the Notch pathway are only partially understood,
and a more complete insight into its role requires identification of additional components. Like other biological pathways, defects in components of the pathway
are expected to create mutant phenotypes.
GAL4-UAS-driven Domino RNAi vector constructs were generated in the Yedvobnick lab by Wooly Pierre. The RNAi constructs are able to produce RNA
interference, which lead to degradation of homologous RNA sequences, producing loss of function for the gene encoding that RNA. In preliminary testing, one
copy of the RNAi vector was insufficient to generate significant phenotypes; thus flies containing four copies of the RNAi vector on the same chromosome were
generated to create sufficient RNA interference. Constructs mapping to chromosome 3 were identified, and multiple constructs were recombined onto
chromosome 3 (Figure 1). These RNAi vectors were expressed in a tissue specific manner using GAL4 drivers.
Crosses were set up to recombine several transgenes onto chromosome 3. By crossing different transgenic lines and selecting for crossovers (via darker
eye color) that contain two transgenes on the same chromosome, homozygotes of two, and eventually four transgenic lines (SymP 4x Dom) were obtained
(Figure 1). The repression of Domino expression was tested on different tissues that require Notch pathway function by crossing the RNAi recombinant
chromosomes with strains that express GAL4 in the wings, eyes, and bristle forming tissues, and also embryos. Offspring from the crosses were analyzed
for Notch pathway phenotypes. Analysis of adults was performed under a dissecting microscope, whereas embryonic phenotypes are scored after CNS staining.
Digital images of all phenotypes were obtained and compared to wild type and other control crosses that should not exhibit phenotypes.
Fig. 1. Creation of strain containing four RNAi vectors on chromosome 3 (SymP 4x Dom). Two strains containing unique RNAi vectors were mated. The
heterozygotes were mated with W1118 strain and the recombinants were selected via darker eye color. Two unique strains each containing two vectors
were mated and the heterozygote were mated with W1118 again, and the recombinants were selected for in same fashion as the previous selection to obtain
flies containing four different RNAi vectors on chromosome 3. Different GAL 4 drivers were then used to express the multicopy RNAi in different tissues.
The results of the experiments are displayed in figure 2. The control for the experiments is the relevant homozygous GAL4 driver strain, which can be seen in the
first column. The experimental data are shown in the second column, which show heterozygotes of various GAL4 strains and UAS-SymP 4x Dom.
- When the RNAi was expressed in the wing margin, distinct nicks on the wings were seen, resulting in loss of hair around the margin and also shallow notches.
This cross produced significantly different wing phenotype from the control with smooth, wildtype wing margin.
- Reduced expression of domino in the interior region of the wings resulted in abnormal vein development, specifically the anterior crossvein. The experimental
phenotype is completely missing the anterior crossvein, while the anterior crossvein is clearly visible on the control strain.
- Reduced expression of domino in the head region also produced significant phenotypes. The experimental cross produced heads that lacked bristles on the
ocelli and also a fusion of the ocelli, whereas the control shows wildtype bristles and ocelli.
- RNA interference in the notum tissue produced a fusion defect of the two notum sections, resulting in a cleft. The bristles on the notum are also improperly
developed as seen by their improper orientation.
- Reduced expression of domino in the legs led to abnormal leg segmentation. While the control strain shows wildtype leg formation of 5 distinct distal segments,
the experimental cross clearly lacked the distinct segments.
Fig. 2. RNA interference effects of domino expressed in various tissues. Panels A to D show wings prepared from genotypes Vg-GAL4 (A) which exhibit wildtype
wing formation, Vg-GAL4 x SymP 4x Dom (B) which contain nicks on wing margin, Ptc-GAL4 (C) which exhibit wildtype wing formation, and Ptc-GAL4 x SymP
4x Dom (D) which is missing the anterior crossvein (arrows designate position of anterior crossvein). Panels E and F show heads prepared from genotypes
Ptc-GAL4 (E) which exhibit wildtype bristle and ocelli formation and Ptc-GAL4 x SymP 4x Dom (F) which lacks bristles and shows fused ocelli (arrows designate
position of ocelli). Panels G and H show notums prepared from genotypes Pnr-GAL4 (G) exhibiting wildtype notum formation and Pnr-GAL4 x SymP 4x Dom (H)
which exhibits a cleft in notum formation and also irregular bristle orientation (arrows designate region of notum where the cleft appears on the mutant strain.)
Panels I and J show legs prepared from genotypes BLK-GAL4 (I) exhibiting wildtype leg segmentation and BLK-GAL4 x SymP 4x Dom (J) which lacks proper leg
segmentation (arrows demonstrate proper leg segmentation lengths.)
The phenotypes produced by the RNAi strain appear to result from the loss of function of the domino gene, and not from other various background mutations.
The results are significant in that certain effects resemble known phenotypes of the Notch pathway, especially the mutations of the wing margin, wing veins, and
leg segmentation. Since a loss of function of the domino gene produced phenotypes typically associated with Notch pathway mutations, our results suggest that
domino is in fact involved in the intercellular pathway.
This material is supported by a grant from the National Science Foundation provided by Dr. Barry Yedvobnick and by the Howard Hughes
Medical Institute under Grant No.52003727.
Many biological processes depend on intricate pathways with many components. The different components work together to help the organism perform its normal
functions. One of these pathways in living organisms is the Notch pathway, which is found in many types of animals ranging from fruit flies to humans. We are
trying to find those specific components that make this pathway work. By finding a way to turn off one of these components that we think is involved, we can
observe the effects on the organism, and thus determine whether that component was involved in the pathway by comparing it to an organism with a fully
functioning pathway.
Embryo Staining, Fly cage setup, microscope preparation, phenotype scoring, and embryo collecting
Notch pathway, drosophila, domino, loss of function screening, RNAi
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