(94c) Robustness of Cellular Signaling with Respect to Dosage

Introduction: How tissues are patterned in a natural context can serve as the foundation for our future methods of tissue engineering and regeneration. In a developing tissue, the timing and placement of gene expression is crucial to the proper assignment of cell fate, and subsequent differentiation of the cells into a reproducible pattern of tissues and organs. Proper gene expression patterns are often achieved through the action of spatial gradients of chemicals, typically a class of proteins called morphogens, which signal to cells in a concentration-dependent fashion. However, morphogen-mediated patterning models face a paradox: spatial patterns of gene expression are surprisingly robust to variations in morphogen dosage despite the fact that, by definition, cells 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-supplied dorsal (dl). Through the action of Toll receptor signaling, which is high on the ventral side of the embryo, Dorsal (Dl) becomes free of its cytoplasmic inhibitor Cactus (Cact)/IκB and is able to enter the nucleus to regulate gene expression¹. In this manner, a spatial gradient (ventral high, dorsal low) in Dl nuclear concentration is established, which acts to pattern the dorsal/ventral (DV) axis of the early embryo. Although these biophysical interactions have been characterized in detail, how their function is integrated to produce robust gene expression patterns to variations in the dl maternal dosage is still unclear.

Results: We measured gene expression of two Dl target genes: sna, which is on the ventral-most 20% of the embryo, and sog, which resides in a broad lateral region, in embryos carrying 1, 2, or 4 copies of dl. The gene expression boundaries have small, but detectable differences depending on dl dosage. However, naïve models of Dl distribution, which assume that Dl concentration globally scales with dl gene dosage, predict high sensitivity gene expression boundaries. This implies there must be mechanisms that alter the shape of the Dl gradient when dl dosage is perturbed. Previous work^2,3 has suggested three such mechanisms: facilitated diffusion of Dl by Cact, saturation of active Toll receptors, and the presence of Dl/Cact complex in the nucleus. We perform a parameter screen using a mechanistic model of the Dl/Cact system to show that these mechanisms are together necessary for robustness of gene expression boundary with respect to dl dosage.

Conclusions: In the Dl gradient system, gene expression is robust due to the action of three mechanisms: facilitated diffusion, Toll receptor saturation, and the action of Dl/Cact complex in the nucleus. Morphogen gradients must be able to determine cell fates in a concentration-dependent fashion, while at the same time maintain robustness with respect to morphogen dose. The Dl/Cact system shows an example of this engineering principle. Ultimately, we may be able to use our engineering knowledge gained from the basic study of tissue patterning to benefit the field of regenerative medicine.

This research was supported by NSF CBET-1254344 and NIH R21HD092830.

References:

1. Reeves, GT & Stathopoulos, A. Cold Spring Harb Perspect Biol (2009) 1: a000836.
2. Carrell, SN et al. Development (2017) 144: 4450-4461.
3. O’Connell, MD & Reeves GT. PLoS Comput. Biol. (2015) 11: e1004159.