The Chen Lab studies how cell signaling regulates embryonic development and contributes to adult physiology and disease. These studies combine organic synthesis, biochemistry, cell biology, and embryology, creating a diverse environment for graduate student and postdoctoral training. Research projects in the laboratory currently focus on three major areas: the identification of small-molecule and genetic regulators of Hedgehog signaling, the development of chemical technologies for perturbing and observing the molecular programs of embryonic patterning and tumorigenesis, and the study of tissue regeneration using zebrafish as a model organism.

Small molecule modulation of Hedgehog signaling

Our interest in compounds that perturb the Hedgehog pathway stems from a need for mechanistic probes of this signaling process and the therapeutic potential of pathway agonists and antagonists. Hedgehog pathway inhibitors have a rich history, beginning with an epidemic of cyclopic sheep in Idaho during the 1950s. The United States Department of Agriculture determined that a plant-derived natural product was responsible for this outbreak, and fifty years later we and Philip Beachy discovered that this compound (appropriately named “cyclopamine”) inhibits a transmembrane signaling protein in the Hedgehog pathway called Smoothened. More recently, we have determined that the Hedgehog pathway agonists SAG and purmorphamine bind directly to Smoothened as well, activating this G protein-coupled receptor-like protein. We have also worked with other laboratories to identify new Hedgehog pathway inhibitors, including an antagonist of the Hedgehog protein (robotnikinin).

We are now investigating Hedgehog signaling modulators that act downstream of Smoothened, since these small-molecule probes will be valuable tools for determining how the Gli transcription factors are regulated. The recent discovery that certain tumors exhibit Smoothened-independent Gli activation also suggests that these compounds could be important leads for the development of Hedgehog pathway-targeting therapies that are more broadly efficacious than Smoothened inhibitors. In collaboration with the Stanford High-Throughput Bioscience Center, we have identified several compounds that are epistatic to Smoothened, including some that appear to act at the level of the Gli proteins. We are now actively pursuing their mechanisms of action and efficacies in mouse tumor models.

Mechanistic studies of Hedgehog pathway regulation

Our current understanding of the Hedgehog pathway is largely derived from genetic and biochemical studies in Drosophila. Yet vertebrate Hedgehog signaling has mechanistically diverged from its invertebrate counterpart and requires distinct signaling proteins and cellular organelles such as the primary cilium. To gain new insights into the mechanisms of vertebrate Hedgehog signaling, we are conducting both overexpression and gene-silencing screens, the latter in collaboration with Matthew Scott’s laboratory. We are currently following up several leads from these screens, and our hope is that these studies will bridge gaps in our understanding of Hedgehog pathway regulation and identify new targets for anti-cancer therapies.

Chemical technologies for perturbing and observing vertebrate embryogenesis

While many aspects of developmental signaling can be studied in cultured cells, model organisms are required to gain a comprehensive understanding of how these pathways regulate embryonic patterning, tissue regeneration, and tumorigenesis. Our laboratory studies the zebrafish as a model of vertebrate biology, taking advantage of its facile aquaculture and husbandry, rapid development, optical transparency, compatibility with forward-genetic approaches, and pharmacological accessibility. Realizing the full potential of zebrafish in biomedical research, however, will require new technologies for manipulating and visualizing the genetic programs within this model organism.

Toward this goal, we have developed chemical and genetic methods that enable spatiotemporal control of gene function in zebrafish embryos, larvae, and adults. For example, we have created a chemically inducible gene expression system that utilizes a chimeric transactivator (GV-EcR) composed of the Gal4 DNA-binding domain, the VP16 activation domain, and the ecdysone receptor ligand-binding domain. By expressing this ligand-gated transcription factor in a tissue-specific manner, one can achieve precise control of exogenous gene expression in zebrafish using the EcR agonist tebufenozide. We are now exploring the use of this system to generate zebrafish models of Hedgehog pathway-dependent tumors.

We have also developed caged morpholinos, which enable light-controlled gene silencing in zebrafish embryos. With these chemical reagents, we can create the chemical equivalent of genetically mosaic organisms; however, we can target specific tissues with far greater spatial and temporal precision. Caged morpholinos also permit gene silencing in selected tissues based on morphological cues alone. We are now using caged morpholinos to interrogate the transcriptional networks that regulate mesoderm patterning, and we are collaborating with several laboratories to study other developmental processes with these reagents.

Zebrafish as a model of vertebrate regeneration

In addition to being a genetically and chemically tractable model of vertebrate development and physiology, the zebrafish has the unique ability to regenerate several of its tissues, including those of the heart, retina, spinal cord, and fins. Understanding the molecular mechanisms that underlie this process will provide important insights into how tissue regeneration is achieved and perhaps strategies for recapitulating these events in mammals. Our research group is working with Michael Longaker’s laboratory to decipher the molecular and cellular events associated with larval tail regeneration, and we have identified several genes that are upregulated or downregulated in posterior cells after the tail is amputated. We are now using an interdisciplinary approach to determine the roles of these genes in the regenerative process.