(512h) Novel Interactions Between the NF-?B and BMP Signaling Pathways in the D. Melanogaster Embryo

Receptor-mediated signals play
a crucial role in tissue patterning of developing organisms, stem cell
maintenance, and adult homeostasis. Many diseases, most notably cancer, occur
when cells misinterpret or ignore signals regarding proliferation, migration,
and/or apoptosis, implying that these signaling pathways must be tightly
regulated. Since these same signals are highly conserved between species, we study
the regulation of signaling pathways in the context of patterning the
dorsal-ventral (DV) axis in the early Drosophila melanogaster embryo.

When the embryo is roughly
2 hours old, two conserved signaling pathways act to pattern the DV axis: the
Dorsal pathway and the Dpp pathway. On the ventral side of the embryo, the
transcription factor Dorsal, homologous to NF-κB, triggers expression
of the genes that initiate the DV pattern in the Drosophila embryo.
Dorsal is present in a nuclear gradient, with the highest concentration at the
ventral midline and a steady decay to about 40% of the embryo's circumference. Signaling
through the Toll receptor initializes the Dorsal gradient in the developing
embryo; on the ventral side of the embryo, the activated ligand Spätzle binds to Toll, which then phosphorylates the
inhibitor protein Cactus (homologous to I-κB), marking it for
degradation. In the absence of Cactus, Dorsal can enter the nuclei and activate
expression of target genes in a concentration-dependent manner. Dorsal also
acts as a transcriptional repressor, limiting some genes to expression on the
dorsal half of the embryo.  One of those dorsally-expressed genes is decapentaplegic
(dpp), which encodes Dpp, a bone morphogenic protein (BMP) ligand. Dpp
is present in a graded fashion, establishing DV gene patterns beyond Dorsal's
reach. Dpp signals through the receptor Thickveins (Tkv), phosphorylating the receptor
Smad protein MAD; phospho-MAD (pMAD) can be used as a readout of BMP signaling.
See (A) for a schematic of the two signaling pathways. Note: dorsal is up,
ventral is down. Together, the nuclear gradient of Dorsal and BMP signaling
work together to pattern the DV axis of developing Drosophila embryos. 

Because robustness of
tissue patterning is essential for proper embryonic development, regulatory
loops must exist to ensure correct placement of target genes in the face of
perturbed conditions. As an example, embryos with a half dose of Dorsal protein
survive to adulthood. We have analyzed the Dorsal gradient in these
heterozygotes by modeling the nuclear gradient of Dorsal as a Guassian, using
the equation:

C(x)
= Aexp[-x²/2σ²] + B

where C is the
concentration of nuclear Dorsal, x is the normalized DV coordinate (0 being the
ventral midline), A represents the amplitude, σ, the width, and B is
the basal level of Dorsal present in the nuclei at the dorsal midline. As shown
in (B), we found that the Dorsal gradients in these embryos are not only
shorter in amplitude as compared to wild type, as expected, but statistically
wider and fundamentally different in shape, having a broader domain of peak
nuclear concentration. These differences point to the existence of Dorsal
gradient regulation in circumstances in which protein levels are compromised. We
will present evidence that the Dpp signaling network can alter the Dorsal
gradient. When levels of MAD (the BMP signal transducer) were overexpressed, we
found that the width of the gradient (as measured by σ) expanded
significantly. Therefore, feedback through the Dpp signaling network is a prime
candidate for enhancing the robustness of patterning of DV gene expression, as
shown in (C). These interactions between the NF-κB and BMP signaling
pathways may be necessary to ensure robust gene expression in the developing Drosophila
embryo.