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The Genetics of Runt-dependent Regulation in the Drosophila Embryo
Tianyu Zhan, Sharon Huang, Nallammai Muthiah, Evangeline Giannopoulos, J Peter Gergen
Stony Brook University, Department of Biochemistry and Cell Biology and the Center for Developmental Genetics, Stony Brook University, Stony Brook, NY, 11790
1. Introduction:
Our approach for studying runt function takes advantage of a
system that allows for genetically controlled quantitative
manipulation of runt activity in all cells of the blastoderm stage
Drosophila embryo. Central to this work is the observation that
the engrailed expression correlates with embryo lethality. If the
females used to generate these embryos had reduced dosage
of a factor that worked with runt to repress engrailed, that the
repression of engrailed would be somewhat relieved, resulting
in an increase in the viability. Results mentioned has included a
further dissection of six regions identified in the previous
screen by Wheeler et al, a systematic screen of the entire III
chromosome ,in situ experiments to examine the effects of
selected interacting deficiencies on engrailed expression, and
the specificity of these interaction with runt.
3. Results:
My project in 2012 Spring involved re-testing seven regions initially identified by Wheeler
in 2002 for which they did not have the tools to identify single gene mutation responsible
for the interaction with runt. Using a collection of 30 different deficiency chromosomes that
span these seven regions I have confirmed interaction with six different regions and also
have been able to refine the location of potential runt interacting genes.
Viability: Runt 232 Females > Runt 17
Females > Runt 232 Males > Runt 17 Males
2. Background:
The results shows that 30D1-E1, 14E1-F2,
15A3-B1, 62A11B7, and 66A17-C8 may contain
maternal factors that are potentiating runt to
repress engrailed.
Gap, pair-rule, and segmentpolarity genes act in a
hierarchical fashion to set up
the segmented pattern in
Drosophila blastoderm
embryo.
5D
Use NGT to achieve
quantitative expression of
runt. GAL4, a transcription
factor from yeast, can bind
the cis-regulatory factor
UAS to activate the
expression of target gene
(runt).
Ectopic runt repress engrailed, and let it express in oddnumbered stripes. In the wild type embryo, engrailed will
express in the place where runt does not express (pointed by
the red arrow). However, ectopic expression of runt, then the
odd number stripes of engrailed disappear (pointed by the red
arrow).
NGT->runt
Wild-Type
Runt
mRNA
En
mRNA
Repression of engrailed
correlates with embryo
lethality. If the females
used to generate these
embryos had reduced
dosage of a factor that
work with runt to repress
engrailed, then the
repression would be
somewhat relieved,
resulting in increasing
viability.
In the summer we further sub divided these six specific regions (25427, 27407, 556, 9715,
24388, 24412), with the goal of identifying single genes responsible for the interaction.
Deficiency
×
Balancer
NGT 40
NGT 40
Deficiency
×
NGT 40
UAS-runt232
CyO
Deficiency (UAS-runt 232; CyO)
NGT 40
UAS-runt
Control
Progeny is 1:1 ratio. Measure the
effect of maternal genotype on the
relative viability.
14C
30C
62
57
65
93
Viability: Runt 232 Females > Runt 17 Females > Runt 232 Males > Runt 17 Males
In the summer, we did in situ hybridization, to examine the maternal effects of two
interesting deficiencies (556, 9715) on the mRNA expression of engrailed. The ectopic
expression of runt blocks expression of the odd-numbered engrailed stripes in all gastrula
stage embryos (a), and this repression is maintained through germband extension (b).
If we reduce the dosage of factors
in 556 or 9715 region, the oddnumber engrailed stripes in gastrula
stage embryos are blocked (c, e),
and this repression recovery
through gerband extension (d, f)
(Approximately 76% embryos in
556, and 79% embryos in 9715).
Therefore, the factors in 556 and
9715 are involved in the
maintenance of runt repression, but
not in the establishment stage.
We also did systematic and comprehensive screen of the entire third chromosome
(approximately 40% of the genome, involved screening about 112 deficiencies). The
brown regions were tested by Wheeler ten years old. The black, red, and green regions
were tested in this summer. The black regions are negative. The red regions are positive.
The green regions are in progress.
3L:
Germband extension
Gastrula
“W T”
556
9715
c
d
e
f
The deficiencies we have identified as interesting were selected based on the effect of the
lethality produced by ectopic runt.
We have also began
experiments to determine if
these deficiencies also
affect the lethality
associated with ectopic
expression of other pair-rule
transcription factors: eve, ftz,
hairy, prd, odd, opa. In the
initial experiments, the
levels of ectopic expression
are too low to see effects
for most of these pair-rule
factors, with the exception
of eve. 9715 is working
specifically with runt.
3R:
4. Conclusions and Future Directions:
The deficiency screens have candidate regions that consist genes that interact with runt.
Two regions (556, 9715) most like they affect maintenance step.
Preliminary experiments suggest their specificity.
The systematic and comprehensive screen of 3L have confirmed 9 regions that are
interacting with runt.
In the future, we will do the specificity tests with NGT40+A system to increase the ectopic
expression of runt. We will also do the in situ hybridization on the other interesting regions
to see their molecular phenotypes. We will finish the genetic screen of entire 3R
chromosome this August to identify other candidate regions that are interacting with runt.
We will continue to dissect these six specific regions to identify single genes.
References:
Wheeler J, Gergen JP et al. (2002) “Distinct in
vivo requirements for establishment versus
maintenance of transcriptional repression”
Naure 32: 206-210.
Acknowledgments:
This work was supported by a URECA
Biology Alumni Research Award to TZ.
We thank Saiyu Hang for his valuable
assistance.