Tianyu Zhan, Sharon Huang, Nallammai Muthiah, Evangeline Giannopoulos, J Peter GergenStony Brook University, Department of Biochemistry and Cell Biology and the Center for Developmental Genetics, Stony Brook University, Stony Brook, NY, 11790

The Genetics of Runt-dependent Regulation in the Drosophila Embryo

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).

2. Background:Gap, pair-rule, and segment-polarity genes act in a hierarchical fashion to set up the segmented pattern in Drosophila blastoderm embryo.

3. Results: 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.

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.

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.

References:Wheeler J, Gergen JP et al. (2002) “Distinct in vivo requirements for establishment versus maintenance of transcriptional repression” Naure 32: 206-210.

Ectopic runt repress engrailed, and let it express in odd-numbered 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).

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.

3R:

Acknowledgments:This work was supported by a URECA Biology Alumni Research Award to TZ. We thank Saiyu Hang for his valuable assistance.

3L:

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).

Progeny is 1:1 ratio. Measure the effect of maternal genotype on the relative viability.

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.

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.

The deficiencies we have identified as interesting were selected based on the effect of the lethality produced by ectopic runt.

ControlUAS-runt

Runt mRNA

EnmRNA

NGT->runtWild-Type

5D 9362 655730C14C

c

e f

d

“W T”

556

9715

Gastrula Germband extensionIf we reduce the dosage of factors in 556 or 9715 region, the odd-number 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 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.

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.

Viability: Runt 232 Females > Runt 17 Females > Runt 232 Males > Runt 17 Males

Deficiency NGT 40Balancer NGT 40

Deficiency UAS-runt232NGT 40 CyO

Deficiency (UAS-runt 232; CyO)NGT 40

×

×

Viability: Runt 232 Females > Runt 17 Females > Runt 232 Males > Runt 17 Males