The transcription factor NF-κB plays an important role in the immune system, apoptosis and inflammation. Dorsal, a homolog of NF-κB, patterns the dorsal-ventral axis in the blastoderm embryo. During this stage, Dorsal is sequestered outside the nucleus by the IκB homolog Cactus. Toll signaling on the ventral side breaks the Dorsal/Cactus complex, allowing Dorsal to enter the nucleus to regulate target genes. Fluorescent data show that Dorsal accumulates on the ventral side of the syncytial blastoderm. Here, we use modeling and experimental studies to show that this accumulation is caused by facilitated diffusion, or shuttling, of the Dorsal/Cactus complex. We also show that active Toll receptors are limiting in wild-type embryos, which is a key factor in explaining global Dorsal gradient formation. Our results suggest that shuttling is necessary for viability of embryos from mothers with compromised levels. Therefore, Cactus not only has the primary role of regulating Dorsal nuclear import, but also has a secondary role in shuttling. Given that this mechanism has been found in other, independent, systems, we suggest that it might be more prevalent than previously thought.

SummaryThe transcription factor NF-κB plays an important role in the immune system as an apoptotic and inflammatory factor. In the Drosophila melanogaster embryo, a homolog of NF-κB called Dorsal (dl) patterns the dorsal-ventral (DV) axis in a concentration-dependent manner. During early development, dl is sequestered outside the nucleus by Cactus (Cact), homologous to IκB. Toll signaling at the ventral midline breaks the dl/Cact complex, allowing dl to enter the nucleus where it transcribes target genes. Here we show that dl accumulates on the ventral side of the embryo over the last 5 cleavage cycles and that this accumulation is the result of facilitated diffusion of dl/Cact complex. We speculate that the predominant role for Cact in DV axis specification is to shuttle dl towards the ventral midline. Given that this mechanism has been found in other, independent systems, we suggest it may be more prevalent than previously thought.

Optimal imaging conditions are of critical importance in developmental biology, as much of the data in the discipline is acquired through microscopy. However, imaging deep sections of tissue, especially live tissue, can be a technical challenge due to light scattering and difficulties in mounting the sample. In particular, capturing high-quality images of dorsal-ventral cross sections requires "end-on" mounting to orient the anterior-posterior axis vertically. Here we present methods to mount and image dorsal-ventral cross sections of both live and fixed Drosophila melanogaster embryos. Our methods have the advantages of being rapid, allowing deep optical sections, and not requiring expensive, specialized equipment.