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The larvae/pupae developmental transition provides an excellent platform to
study LD dynamics in a single tissue undergoing distinct physiological processes
in terms of energy homeostasis. Larval development is largely associated with
continuous growth of lipid storage at the site of fat body LDs, while during pupal
development these LDs are steadily mobilized to support animal survival and
development as the major energy source ( Chien et al., 2011 ).
The larval fat body cells persist 3 days into adulthood, until the adult fat body is
fully differentiated. Adult fat body arises from a different mesodermal lineage, likely
from imaginal disk-associated adepithelial cells ( Aguila, Hoshizaki, & Gibbs, 2013;
Aguila, Suszko, Gibbs, & Hoshizaki, 2007; Hoshizaki, Lunz, Ghosh, & Johnson,
1995 ). The adult fat body is also a multifunctional organ. In addition to its role in
metabolic regulation as the major energy depot, the adult fat body is essential for
regulating innate immunity, aging, courtship behavior, and detoxification ( Arrese
& Soulages, 2010 ). Because it is less accessible for imaging, we will focus the meth-
odology of LD studies on larval fat body cells in the following sections.
4.2.1 Genetic manipulations of fat bodies for LD study
The tissue-specific binary expression system makes Drosophila a very appealing
model to study the effect of perturbation of a given gene in physiology and devel-
opment ( Southall, Elliott, & Brand, 2008 ). In short, to manipulate gene expression
in the larval fat body, a fat-body specific Gal4 driver (made by putting the Gal4 open
reading frame under the control of a fat-body specific enhancer) can be combined
with one or multiple UAS-transgenes to either overexpress the transgenes or down-
regulate the targeted genes by RNA interference.
Several fat-body specific Gal4 lines have been established ( Asha et al., 2003;
Benes, Spivey, Miles, Neal, & Edmondson, 1990; Colombani et al., 2003 ). We
use the CgGal4 line as an example here. All the other lines could be used in the
same manner. CgGal4 is made by fusing the 2.7-kb regulatory region between
the Cg25C and vkg genes to Gal4 ( Asha et al., 2003 ) and drives high expression
in both larval and adult fat bodies. A CgGal4, UAS-mCherry recombined strain is
used to observe fat-body morphology changes during development. Because the fat
body in this strain strongly expresses red fluorescent protein, the gross morphology
of fat bodies in live flies can be easily observed under a standard fluorescent ste-
reoscope ( Fig. 4.2 A).
We can also use this CgGal4, UAS-mCherry strain to cross to transgenic
libraries of UAS-RNAi strains. These libraries contain flies carrying UAS-RNAi
transgenes targeting most genes in the fly genome. A few independent genome-
wide libraries are publically available through several stock centers. The ones
available at the Vienna Drosophila RNAi Center (VDRC, Austria) and those at
the National Institute of Genetics (NIG-Fly, Japan) make long dsRNAs of up
to several hundred base pairs long, while the Transgenic RNAi Project lines gen-
erated at Harvard Medical School make short hairpin RNAs ( Dietzl et al., 2007;
Ni et al., 2008 ).
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