572d

The Coordinated Control of EGFR Signaling by Multiple Feedback Loops: An Experimental and Computational Study In a Developing Tissue

Jeremiah J. Zartman and Stanislav Y. Shvartsman. Department of Chemical Engineering and Lewis-Sigler Institute for Integrative Genomics, Princeton University, Engineering Quadrangle, Princeton, NJ 08544

Hyperactivation of the EGFR pathway has been implicated in the majority of human epithelial cancers (1). Studying the role of EGFR signaling in developing tissues has provided a natural paradigm for controlling complex regulatory networks, and has provided new tools for the therapeutic treatment of cancer in the form of EGFR signaling antagonists (2). In particular, Drosophila oogenesis is a well established system for studying EGFR signaling feedback and is amenable to sophisticated genetic and imaging tools, as well as computational analysis (3). During Drosophila oogenesis, a two-dimensional monolayer follicular epithelium, encapsulating the developing egg, is patterned based on the integration of the Epidermal Growth Factor Receptor (EGFR) and the BMP (Bone Morphogenetic Protein) signaling pathways. The resulting differential expression of adhesion and cytoskeleton genes in the follicle cells (FCs) drives the morphogenesis of an elaborate three-dimensional eggshell, which includes a pair of tubular structures called dorsal appendages that project out from the main body of the egg. Previous research has made significant progress in elucidating the signaling and patterning events that specify the two dorsolateral patches of follicle cells that later form the roof of the dorsal appendages (roof cells). However, a qualitative and quantitative understanding of the role of EGFR inhibitory feedback in FC patterning is still lacking, and the regulatory network that specifies the dorsolateral roof cells as well as the dorsal midline cells separating the two patches of roof cells is incomplete.

We have taken a combined computational and experimental approach to identify the various roles of multiple EGFR inhibitory feedback loops in modulating the spatially distributed EGFR input (4). EGFR feedback is involved in specifying the size, shape, and relative location in the epithelium of the two dorsolateral patches of roof cells. Based on the expression and loss-of-function analysis, we show that, contrary to previously reports, the secreted EGF inhibitor, argos (aos) (5), does not play a significant role in specifying the specification of two patches of roof cells from a single peak of EGFR signaling. Instead, we find that two other EGFR inhibitors, sprouty (sty) (6) and kekkon (kek) (7) are important for modulating EGFR signaling and shaping the patches of roof cells. We present a computational model that incorporates inhibitory feedback loops, which is used to make predictions on the changes in the shape of the roof cell patches due to perturbations in the connectivity of the regulatory network. We then test the computational predictions using the sophisticated genetic tools available in Drosophila to quantitatively delineate the spatial patterning of the roof cells by sty and kek. This study provides quantitative insight into the role of inhibitory feedback for the proper patterning of a model epithelial tissue and the control of a medically important signaling pathway.

References

1. Ciardiello F, Tortora G. EGFR antagonists in cancer treatment. N Engl J Med. 2008 Mar 13;358(11):1160-74.

2. Yarden Y. The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur J Cancer. 2001 Sep;37 Suppl 4:S3-8.

3. Shvartsman SY, Muratov CB, Lauffenburger DA. Modeling and computational analysis of EGF receptor-mediated cell communication in Drosophila oogenesis. Development. 2002 Jun;129(11):2577-89.

4. Goentoro LA, Reeves GT, Kowal CP, Martinelli L, Schupbach T, Shvartsman SY. Quantifying the Gurken morphogen gradient in Drosophila oogenesis. Dev Cell. 2006 Aug;11(2):263-72.

5. Klein DE, Nappi VM, Reeves GT, Shvartsman SY, Lemmon MA. Argos inhibits epidermal growth factor receptor signalling by ligand sequestration. Nature. 2004 Aug 26;430(7003):1040-4.

6. Reich A, Sapir A, Shilo B. Sprouty is a general inhibitor of receptor tyrosine kinase signaling. Development. 1999 Sep;126(18):4139-47.

7. Ghiglione C, Carraway KL, 3rd, Amundadottir LT, Boswell RE, Perrimon N, Duffy JB. The transmembrane molecule kekkon 1 acts in a feedback loop to negatively regulate the activity of the Drosophila EGF receptor during oogenesis. Cell. 1999 Mar 19;96(6):847-56.