Tools and methods for studying the Drosophila JAK/STAT pathway

doi:10.1016/j.ymeth.2014.03.023

Methods

Volume 68, Issue 1, 15 June 2014, Pages 160-172

https://doi.org/10.1016/j.ymeth.2014.03.023 Get rights and content

Highlights

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The JAK/STAT pathway is an essential, evolutionarily conserved signaling cascade.
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Drosophila JAK/STAT has diverse cellular, developmental, and homeostatic functions.
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Many molecular and genetic tools and reagents are available for JAK/STAT studies.
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These tools make Drosophila a powerful model for JAK/STAT signaling investigations.

Abstract

The Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) signaling pathway is one of a limited number of signaling cascades that is extensively utilized for many developmental and homeostatic functions. The JAK/STAT pathway is evolutionarily conserved from insects to mammals, with homologous transduction machinery in each. Yet the mammalian pathway is composed of multiple members for each family of proteins, while flies have only a single homologue of most pathway components. This simplicity and the abundance of genetic, biochemical, and developmental tools make Drosophila an attractive model to investigate this important signaling pathway. This review provides a basic description of the Drosophila JAK/STAT cascade and summarizes currently available reagents and tools to study and manipulate the pathway.

Introduction

The twentieth anniversary of the discovery of the JAK/STAT pathway was celebrated only recently [1]. While still relatively new, the JAK/STAT cascade has been the subject of intense investigation over that period due to its central role in an extensive array of biological processes and relevance to development and disease. The pathway was uncovered in mammals nearly simultaneously by distinct molecular and functional genetic approaches. The first JAK family members were molecularly identified through a PCR-based screen for new tyrosine kinase genes. Two clones encoding proteins with both a conserved tyrosine kinase domain and an adjacent second kinase-like domain were isolated and named Janus kinases, after the Roman two faced god. At the same time, somatic cell genetics and biochemistry were used to dissect the signaling cascade for interferon. These approaches converged as it was recognized that JAKs and STATs comprise an intracellular signaling cascade required for response to cytokines (see reviews, [1], [2]). We now know that the mammalian pathway is comprised of four JAKs and seven STATs, and is stimulated by dozens of cytokines and growth factors through several distinct families of receptor subunits (see reviews, [3], [4], [5]). This array of signaling molecules can promote various cellular responses: proliferation, differentiation, migration, survival, or death, depending on context. These responses are critical to physiological roles that include blood cell development, immune system regulation, mammary development, gametogenesis, and general growth [6]. Aberrant pathway activity has detrimental health consequences, including immune deficiency, inflammatory disease, and leukemia [7].

The JAK/STAT pathway has been evolutionarily conserved between vertebrates and insects. The signal transduction mechanism has remained the same, but invertebrates have the advantage of fewer family members of each pathway component. Combined with an extensive collection of genetic tools and reagents, Drosophila has been a powerful system for analysis of the JAK/STAT pathway. An overview of the fly pathway and a description of some important reagents and tools for its study are described here.

Section snippets

Components of the Drosophila JAK/STAT pathway

The JAK/STAT pathway is a relatively simple signaling cascade, with only a transmembrane receptor plus two essential intracellular components required to transduce an extracellular signal to a transcriptional response (Fig. 1). In Drosophila, the pathway is activated by the three ligands of the Unpaired (Upd) cytokine family, as compared with the dozens of cytokines and growth factors that stimulate the vertebrate cascade. Binding of ligand to the Domeless (Dome) receptor initiates

Roles of the JAK/STAT pathway

Throughout the life of the fly, JAK/STAT signaling contributes to a long list of developmental events and homeostatic processes. The pathway directs a multitude of cellular and tissue responses. A small set of specific examples for these functions are briefly described below. An in-depth discussion of these roles is beyond the scope of this work, but detailed reviews of JAK/STAT involvement in many functions can be found elsewhere.

Tools for in vitro studies

JAK/STAT pathway activity has been examined in cell culture by many groups. Some of the most extensive of these studies include several RNAi-based screens for genes that influence the pathway [23], [34], [36], [92]. These groups used different tools and methods, but each has been well-validated, including identification of established pathway components in the screens, in addition to novel interactors. Summarized here are common reagents used in these and other studies that are generally useful

Tools for in vivo studies

Over the past two decades, many researchers have made important contributions to the understanding of the Drosophila JAK/STAT pathway, developing valuable tools and reagents along the way. A small selection of these resources are described here. These are divided into reporters of pathway activation or component gene transcription, mutants and transgenes for manipulating the pathway, and genetic tools and phenotypic assays for interaction studies. A much more comprehensive list of available

Conclusion

The evolutionary conservation and extensive developmental utilization of the JAK/STAT pathway have made it a target of great interest for investigation. Studies in Drosophila have provided insights into the mechanisms of JAK/STAT pathway activation, regulation, interactions, and functions, as well as creating tools and reagents that accelerate discovery. Combined with new additions to mutant and transgenic allele collections, better imaging instrumentation and methods, new genome and proteome

Acknowledgments

The authors thank Dr. S.M.W. Harrison for comments and suggestions on this manuscript. Research in the lab has been supported by the National Science Foundation (IOB-0920432).

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Tools and methods for studying the Drosophila JAK/STAT pathway

Highlights

Abstract

Introduction

Section snippets

Components of the Drosophila JAK/STAT pathway

Roles of the JAK/STAT pathway

Tools for in vitro studies

Tools for in vivo studies

Conclusion

Acknowledgments

Immunity

Semin. Cell Dev. Biol. (Engl.)

Cell

Dev. Biol.

Cell

Curr. Biol.

Curr. Biol.

Biochem. Biophys. Res. Commun.

Mech. Dev.

Dev. Biol.

Curr. Biol.

Dev. Biol.

Dev. Biol.

Cell Stem Cell

Cell

Dev. Cell

Curr. Biol.

Curr. Biol.

Mol. Cell

Dev. Biol.

Curr. Biol.

Dev. Cell

Mech. Dev.

Dev. Biol.

Dev. Cell

Cell

Trends Cell Biol.

Curr. Opin. Cell Biol.

Cell Host Microbe

Dev. Biol.

Cell

Cell

Cell

J. Biol. Chem.

Cell. Signal.

Science

Cold Spring Harbor Perspect. Biol.

J. Cell Sci.

Immunol. Rev. (Denmark)

Genes Cancer

Development

JAKSTAT

JAKSTAT

Roux’s Arch. Dev. Biol.

Genes Dev.

Nature

Development

Genes Dev.

Proc. Natl. Acad. Sci. U.S.A.

FASEB J.