This document summarizes a presentation on a study investigating the role of MED13 and Wingless signaling in muscle on obesity in Drosophila. The study found that knocking down MED13 or its downstream target Wingless in muscle tissue led to increased fat accumulation and triglyceride levels. Overexpressing Wingless in muscle suppressed obesity. Genetic interaction experiments supported Wingless acting downstream of MED13 in muscle to regulate obesity by signaling to fat tissue. The findings reveal a conserved MED13-Wingless signaling pathway in muscle that controls whole-body energy homeostasis and protects against obesity.

1. Presentation on
Heart- and muscle-derived signaling system dependent
on MED13 and Wingless controls obesity in Drosophila
Presented by : Kutub Ashraf
Erasmus Mundus BioHealth
Molecular Biotechnology Center (MBC)

2. Objectives of the Study
Grueter et al.(2012) reported previously that cardiac deletion of
MED13 confers an obese phenotype and that cardiac-specific
overexpression of MED13 prevents obesity in mice.
To further explore the generality and mechanistic basis of this
observation, Lee et al. investigated the potential influence of
MED13 expression in heart and muscle on the susceptibility of
Drosophila to obesity.
Because Drosophila provides a powerful model system for the genetic
analysis of obesity. (Pospisilik JA, et al. (2010)

3. Experimental Outline
To identify possible muscle-secreted obesity regulators, they performed an
RNAi-based genetic screen of 150 genes using UAS/Gal4 system that encode
secreted proteins and found that Wingless inhibition caused obesity
Consistent with these findings, muscle-specific inhibition of Armadillo, the
downstream transcriptional effector of the Wingless pathway also evoked an
obese phenotype in flies.
Finally, Epistasis experiments demonstrated that Wingless functions
downstream of MED13 within a muscle-regulatory pathway.
So, a skd-null mutation dominantly enhanced the arm phenotype in muscle
and that wg is epistatic to skd, suggesting that Wg is a downstream effector of
MED13 in muscle.

4. MED13 expression in muscle regulates obesity.
A) By 3 wk of age, they observed
increased abdominal fat bodies in
skd RNAi flies (FigA) in comparison
to control.
(C–E) Relative triglyceride amounts of
adult females with muscle-specific
knockdown of skd, kto, or luciferase
(control)
Using a different fly muscle-specific driver Mef2-Gal4( C), Mhc-Gal4(D) or
Tin-Gal4(E) with RNAi knockdown to target skd mRNA.They observed that
Mhc>skd(figD) & Tin>skd RNAi flies ( Fig E) showed a significant increase
in total triglycerides levels similar to the effect of Figure C.

5. Three-week-old flies with Mef2-Gal4 driven knockdown of skd or kto
survived substantially longer under starvation conditions (Fig. 1F).
However, their overall lifespans under normal conditions did not change
(Fig. 1G).
**Mef2>skd RNAi (median survival, 108 h, n = 120); Mef2>kto
RNAi(median survival, 96h,n = 80); Control, (median survival, 72 h, n=72

6. Flies with MED13 or MED12 knockdown display increased susceptibility to obesity.
Flies with muscle specific
knockdown of skd or kto showed no
obvious changes in 2 wk but a
substantial increase was observed by
3wk &4wk when by 5wk of age, fat
accumulation reached a maximum
level irrespective of genotype.
(B) Effects of high-fat diet on
total triglyceride amounts.12 days
old flies grown in normal food
were transferred high-fat food.
With normal food, Mef2>skd
RNAi& Mef2>kto RNAi
triglycerides were 22% and 23%
but with high-fat food, it was 57%
and 33%

7. The Wg signal in muscle supresses obesity.
The expression of arm RNAi increased fat
bodies(A)but due to muscle specific
overexpression(D-E) Mhc-Gal4&Tin-Gal4 of
armS2(Wild)/S10 form reduced total triglyc.
Total triglyceride
amounts of the flies
were increased but
using UAS-wg cDNA
lines decreased
whereas again
increased by muscle-
specific knockdown of
armRNAi( FigC)
Flies expressing
either wg RNAi or
armRNAi with Mef2-
Gal4 showed
increased abdominal
fat body mass
containing enlarged
lipid droplets.** (luc)
RNAi (control)

8. Genetic Interaction Between skd (MED13) and Arm( arm) in
Muscle.
To test the functional interaction of the Wg pathway and MED13 in muscle,
They performed genetic-interaction experiments between arm & skd.
Flies with arm knockdown using Mef2-Gal4 are viable without morphological
defects.
skdT606 is a null allele that is homozygous lethal but skdT606 heterozygotes are
viable without morphological defects.
However, arm knockdown using Mef2-Gal4 in a skdT606 heterozygous (skd−/
+) background caused complete lethality, which was fully penetrant.
To better understand the nature of this lethality, the somatic muscle
structure of the embryos was examined.

9. Genetic interaction between MED13 and Arm in muscle.
Embryos expressing luciferase RNAi in muscle in a skd+/+ or skd−/+
background maintained intact muscle patterns (Fig. A and B) but a portion
of embryos expressing arm RNAi in muscle in a skd+/+ had intact muscle
patterns (Fig.C) whereas other embryos with the same genotype(C′) displayed
patterning defects in the somatic musculature However, all observed embryos
expressing arm RNAi using Mef2-Gal4 in the skd−/+ background patterning
defects in their somatic musculature (Fig. D&D′).Red asterisks for dorsal
muscle defects &dotted boxes normal A, B, and C) or abnormal LT (C′, D& D′)

10. Epistatic relationship between Wg & skd in muscle for obesity control
Flies expressing either skd RNAi or wg cDNA or both
with Mef2-Gal4 were grown & their total triglyceride
levels were compared. Flies with both skd knockdown
and wg overexpression were decreased compared with
those of flies with skd knockdown, but indistinguishable
from those flies with wg overexpression alone(FigA)
They also did the same experiments using Mhc-Gal4
and Tin-Gal4 obtained consistent results B and C).So,
these data strongly support that muscle-secreted Wg
acts as downstream effector of skd function in muscle to
suppress fat deposition.
Finally, they tested
the effect of Wg
activation in the fat
body. When wg was
overexpressed using
the fat body specific
Dcg-Gal4 it caused
lethality at the pupal
stage & severe
reduction of fat
body mass in the
abdominal region of
third instarlarva(D).

11. All of previous finding support a model in which muscle secreted wg acts on the fat body to inhibit OBESITY
Expression of MED13 or Wg in muscle suppresses fat accumulation
in the fat body .
Wg acts downstream effector of MED13.and activates the Wg signal
in both muscle and fat body ultimately to achieve systemic inhibition
of obesity.

12. Future Insights
Previously, they reported that cardiac expression of MED13 controlled
systemic energy homeostasis in mice such that increased or decreased
expression of MED13 caused leanness or obesity, respectively.
Here, they reported that MED13 also acts within muscle of Drosophila to
control obesity. Moreover, the secreted peptide Wingless acts as a
downstream effector of MED13 to mediate cross-talk with adipose tissue
and suppress obesity.
In short, This publication revealed a conserved signaling system in muscle
in which MED13 and Wingless act as key controllers of obesity.

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