Mechanism and implications of morphogen shuttling: Lessons learned from dorsal and Cactus in Drosophila.

In a developing animal, morphogen gradients act to pattern tissues into distinct domains of cell types. However, despite their prevalence in development, morphogen gradient formation is a matter of debate. In our recent publication, we showed that the Dorsal/NF-κB morphogen gradient, which patterns the DV axis of the early Drosophila embryo, is partially established by a mechanism of facilitated diffusion. This mechanism, also known as "shuttling," occurs when a binding partner of the morphogen facilitates the diffusion of the morphogen, allowing it to accumulate at a given site. In this case, the inhibitor Cactus/IκB facilitates the diffusion of Dorsal/NF-κB. In the fly embryo, we used computation and experiment to not only show that shuttling occurs in the embryo, but also that it enables the viability of embryos that inherit only one copy of dorsal maternally. In this commentary, we further discuss our evidence behind the shuttling mechanism, the previous literature data explained by the mechanism, and how it may also be critical for robustness of development. Finally, we briefly provide additional experimental data pointing toward an interaction between Dorsal and BMP signaling that is likely affected by shuttling.

Robustness of the Dorsal morphogen gradient with respect to morphogen dosage.

In multicellular organisms, the timing and placement of gene expression in a developing tissue assigns the fate of each cell in the embryo in order for a uniform field of cells to differentiate into a reproducible pattern of organs and tissues. This positional information is often achieved through the action of spatial gradients of morphogens. Spatial patterns of gene expression are paradoxically robust to variations in morphogen dosage, given that, by definition, gene expression must be sensitive to morphogen concentration. In this work we investigate the robustness of the Dorsal/NF-κB signaling module with respect to perturbations to the dosage of maternally-expressed dorsal mRNA. The Dorsal morphogen gradient patterns the dorsal-ventral axis of the early Drosophila embryo, and we found that an empirical description of the Dorsal gradient is highly sensitive to maternal dorsal dosage. In contrast, we found experimentally that gene expression patterns are highly robust. Although the components of this signaling module have been characterized in detail, how their function is integrated to produce robust gene expression patterns to variations in the dorsal maternal dosage is still unclear. Therefore, we analyzed a mechanistic model of the Dorsal signaling module and found that Cactus, a cytoplasmic inhibitor for Dorsal, must be present in the nucleus for the system to be robust. Furthermore, active Toll, the receptor that dissociates Cactus from Dorsal, must be saturated. Finally, the vast majority of robust descriptions of the system require facilitated diffusion of Dorsal by Cactus. Each of these three recently-discovered mechanisms of the Dorsal module are critical for robustness. These mechanisms synergistically contribute to changing the amplitude and shape of the active Dorsal gradient, which is required for robust gene expression. Our work highlights the need for quantitative understanding of biophysical mechanisms of morphogen gradients in order to understand emergent phenotypes, such as robustness.

Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display.

Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here, we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and nondisruptively controlling intracellular or intraembryonic processes.

Formation, interpretation, and regulation of the Drosophila Dorsal/NF-κB gradient.

The morphogen gradient of the transcription factor Dorsal in the early Drosophila embryo has become one of the most widely studied tissue patterning systems. Dorsal is a Drosophila homolog of mammalian NF-κB and patterns the dorsal-ventral axis of the blastoderm embryo into several tissue types by spatially regulating upwards of 100 zygotic genes. Recent studies using fluorescence microscopy and live imaging have quantified the Dorsal gradient and its target genes, which has paved the way for mechanistic modeling of the gradient. In this review, we describe the mechanisms behind the initiation of the Dorsal gradient and its regulation of target genes. The main focus of the review is a discussion of quantitative and computational studies of the Dl gradient system, including regulation of the Dl gradient. We conclude with a discussion of potential future directions.