(636g) Analyzing Negative Feedback and Shuttling Using a Synthetic Gene Network

Analyzing
Negative Feedback and Shuttling Using a Synthetic Gene Network

15D02 Intracellular
Processes I

Ashley
A. Jermusyk and Gregory T. Reeves

In multicellular
organisms, cellular signaling events are crucial for patterning tissues, as
well as for maintaining healthy adult tissues, while improper signaling can
lead to disease states, such as cancer.  Therefore, cellular signaling
processes must be tightly regulated.  Complex systems of gene regulatory
circuits control these signaling processes and act to buffer these systems
against noise, thereby minimizing mistakes in gene expression and preventing
patterning defects or disease states.  Despite their importance to patterning
and development, hypotheses regarding these gene regulatory circuits have been
difficult to test experimentally due to their complexity and high
connectivity.  Therefore, to better understand the fundamental processes
involved, we created a synthetic gene network in the fruit fly Drosophila
melanogaster embryo.  This approach has the advantages that (1) the gene
network is orthogonal to native Drosophila biology, and (2) the network
is designed.  These two aspects imply the connectivity of the network is
understood, and thus hypotheses regarding the designed network motif can be
experimentally tested with this system.  We are examining expression at the
syncytial blastoderm stage when the Drosophila embryo is one cell with
many nuclei.   By studying a single-cell system we are able to simplify what
can be a very complex process (patterning) as well as gain insight into
processes at varying stages of development. 

Developing tissues are
patterned using signaling proteins that determine expression of downstream
genes based on concentration thresholds.  These signaling proteins form
gradients due to localized protein production and subsequent diffusion and
degradation.  A second protein can bind to the signaling protein to inhibit
signaling (shrink the spatial gradient - negative feedback) or to facilitate
transport (broaden the morphogen gradient ? known as ?shuttling?).  Both negative feedback and shuttling have
been proposed as mechanisms to increase the robustness of gene expression
(Eldar et al., 2003; Haskel-Ittah et al., 2012), but this hypothesis is
difficult to test experimentally.  Therefore, we have created a synthetic
network to test the effects of these two systems (shuttling and negative
feedback) utilizing genes from yeast and E. coli, namely, gal4, gal80,
and lacZ.  We expressed gal4 in a graded fashion along the
anterior-posterior axis of the embryo, mimicking the intracellular diffusion of
Bicoid, an endogenous transcription factor and signaling molecule.  As seen below in Figure 1, the Gal4
protein activates expression of UAS-linked gal80 and lacZ.  Gal80
binds to Gal4, preventing Gal4 from binding to UAS and activating
expression of gal80 and lacZ; this sequestration creates a
negative feedback loop in our system.  However, due to the spatial
dynamics of the system, a shuttling mechanism can also be observed when the
level and spatial expression domain of Gal80 is altered.  The effects of this shuttling
mechanism on the network can be determined by looking at lacZ expression, specifically
changes in expression due to the addition of Gal80 to the network.  These genes were chosen
since they are not endogenous to Drosophila, so all interactions in this
network are fully understood.  Our goal is to measure the effect of
Gal80-mediated Gal4 expression on the robustness of the location of the lacZ domain
at varying levels of Gal80 (namely in both the negative feedback and shuttling
regime).
This system provides a
direct experimental test of the effects of these control mechanisms in cellular
signaling events, specifically whether this shuttling and negative feedback can
lead to increases in robustness of the system.  It is this robustness that is
important for combating diseases and defects in development and maintenance of
expression in the organism.

Figure 1. Network Diagram

Representation of the interactions in the studied network where arrows
represent activation and flat arrowheads denote repression by binding of Gal80
to Gal4.

References

Eldar A, Rosin D, Shilo B, Barkai N. 2003.
Self-enhanced ligand degradation underlies robustness of morphogen gradients.
Dev Cell 5(4):635-46.

Haskel-Ittah M, Ben-Zvi D, Branski-Arieli M, Schejter
ED, Shilo B, Barkai N. 2012. Self-organized shuttling: Generating sharp
dorsoventral polarity in the early drosophila embryo. Cell 150(5):1016-28.

The
focus of this session is the quantitative analysis of intracellular processes
at the molecular level utilizing experimental and/or modeling techniques.
Topics of interest include, but are not limited to, signal transduction,
intracellular trafficking, cytoskeletal dynamics, metabolic and transcriptional
networks, and subcellular imaging.