Signal Transduction in Drosophila development.
Our general interest is to understand how cell behaviors are regulated by communications between cells in development. We approach those objectives principally by using Drosophila molecular genetics supplemented by suitable biochemical and microscopy approaches. Our efforts are focused on two major projects, described below, that are of central relevance to human medicine because (i) aberrant Hedgehog signaling is a major cause of cancer and (ii) manipulation of stem cells provides a key path for many potential cell therapies.
1. Mechanism of Hedgehog Signal Transduction.
Hedgehog (Hh) signaling controls a multitude of developmental processes in Drosophila and other organisms, including humans. This is accomplished through carefully regulated expression and transport of Hh signaling molecules, followed by transduction of the Hh signal and induction of transcriptional responses that alter cell fates or cell proliferation. Our overall objective is to understand the Hh signal transduction process principally by studying Hh signaling in developing Drosophila wing discs, where detailed and incisive genetic tests are possible. Hh signaling is also being studied elsewhere in vertebrate model organisms (mice and zebrafish), independently and taking prompts from Drosophila studies. Central elements of the signal transduction process are conserved between Drosophila and mammals but there are also interesting apparent differences.
Drosophila cells are responsive to Hh only if they express the receptor Patched (Ptc) and the transcription factor Cubitus interruptus (Ci). In such cells Ptc is active in the absence of Hh; it inhibits a seven transmembrane domain protein, Smoothened (Smo) and consequently keeps the signaling pathway off. In this state, full-length Ci protein (Ci-155) is held inactive in the cytoplasm by binding partners that include the kinesin-family protein Costal 2 (Cos2) and Suppressor of fused (Su(fu)). Furthermore, Ci-155 is phosphorylated at a large number of clustere sites by Protein Kinase A (PKA), Glycogen Synthase Kinase 3 (GSK3) and Casein Kinase I (CK1). This creates a binding site for Slimb, which is the substrate-recognition component of an E3 ubiquitin ligase. Following ubiquitination, Ci-155 is proteolytically processed by the proteasome to a truncated form, Ci-75, that retains the DNA binding domain but lacks a transcriptional activation domain and acts as a repressor of transcription. A very similar mechanism involving orthologs of all of the components mentioned above operates in mammalian cells to ensure that Ci orthologs (Gli1-3) are held inactive and subject to partial or complete proteolysis in the absence of ligand.
In the presence of Hh, Ptc no longer inhibits Smo and Drosophila Smo becomes hyper-phosphorylated at a cluster of PKA and CK1 sites as it accumulates at increased concentration at the plasma membrane and undergoes some conformational changes. Activated Smo leads to several further changes that block conversion of Ci-155 to Ci-75 repressor and allow a small proportion of Ci-155 to accumulate in the nucleus and activate target gene transcription. The intermediate steps include (i) reduced interactions among Cos2, Ci-155 and the three protein kinases, PKA, CK1 and GSK3, that target Ci-155 for proteolytic processing and (ii) activation of the protein kinase Fused (Fu). Fu contributes to the regulation of Ci-155 processing but it is also critical for activation of Ci-155. In mammals Hh causes Smo to accumulate in the primary cilium, which is essential for normal Hh signal transduction, rather than at the plasma membrane. Also, it is not presently clear whether there is a protein kinase activity that plays a key role analogous to that of Fu in Drosophila. Important current objectives include identification of the critical targets of Fu kinase activity, investigating whether either the activation mechanism or targets of Fu are conserved in mammals and dissecting the key changes among Ci-155, Cos2, Fu, PKA, CK1 and GSK3 that regulate Ci-155 phosphorylation and processing.
2. Regulating Follicle Stem Cells (FSCs) in the Drosophila ovary.
How stem cells maintain tissues has recently become an important question in the context of potential regenerative therapies and cancer origins and treatments in humans. However, stem cells can be hard to identify and study in complex organisms, so invertebrate model organisms have become an important vehicle for deciphering basic principles underlying stem cell function. Currently a handful of different types of stem cell are being actively studied in Drosophila and each system is making unique and complementary contributions. We have been studying Follicle Stem Cells (FSCs) in the Drosophila ovary. FSCs self-renew within the germarium of a Drtosophila ovariole, continuously producing daughter cells that divide further as they form an epithelial layer around germline cells and produce a few stalk cells that allow egg chambers (germline cells enveloped by follicle cells) to bud from the germarium. It is expected that certain extracellular factors provide a suitable niche to sustain FSC self-renewal and that some intrinsic factors are also essential specifically for stem cell function. We have explored these issues by investigating which signaling pathways influence FSC function and by performing a screen for genetic functions that are required in FSCs to support FSC self-renewal. Those studies have shown that several signaling pathways are important for FSC function, that positive regulators of cell proliferation favor FSC function and that a key regulated property for FSC function is adhesion to the niche. Important current objectives include deciphering how signaling pathways, proliferative cues and adhesive functions intersect in FSCs.
- Reilein A, Melamed D, Park KS, Berg A, Cimetta E, Tandon N, Vunjak-Novakovic G, Finkelstein S, Kalderon D. (2017) Alternative direct stem cell derivatives defined by stem cell location and graded Wnt signalling. Nature Cell Biology 19:433-444. Article Discussion
- Zadorozny EV, Little JC, Kalderon D. (2015) Contributions of Costal 2-Fused interactions to Hedgehog signaling in Drosophila. Development 142(5):931-42. Article
- Huang J, and Kalderon D. (2014) Coupling of Hedgehog and Hippo pathways promotes stem cell maintenance by stimulating proliferation. J. Cell Biol. 205(3):325-38. Article
- Vied C, Reilein A, Field NS, Kalderon D. (2012) Regulation of stem cells by intersecting gradients of long-range niche signals. Dev Cell 23: 836-48. Article
- Wang ZA, Huang J, and Kalderon D. (2012) Drosophila follicle stem cells are regulated by proliferation and niche adhesion as well as mitochondria and ROS. Nat Commun. 3:769. Article
- Zhou, Q. and Kalderon, D. (2011) Hedgehog activates Fused through phosphorylation to elicit a full spectrum of pathway responses. Dev. Cell 20: 802-14. Article
- Marks, S.A. and Kalderon, D. (2011) Regulation of mammalian Gli proteins by Costal 2 and PKA in Drosophila reveals Hedgehog pathway conservation. Development 138: 2533-42. Article
- Wang, Z.A. and Kalderon, D. (2009) Cyclin E-dependent protein kinase activity regulates niche retention of Drosophila ovarian follicle stem cells. Proc. Natl. Acad. Sci. USA 106: 21701-6. Article
- Vied, C. and Kalderon, D. (2009) Hedgehog-stimulated stem cells depend on non-canonical activity of the Notch co-activator Mastermind. Development, in press. Article
- Smelkinson, M.G., Zhou, Q. and Kalderon, D (2007) Regulation of Ci-SCFSlimb binding, Ci proteolysis, and Hedgehog pathway activity by Ci phosphorylation Dev. Cell 13: 481-495. Article
- Zhou, Q, Apionishev, S. and Kalderon, D. (2006) The contributions of protein kinase A and smoothened phosphorylation to Hedgehog signal transduction in Drosophila melanogaster Genetics 173: 2049-2062. Article
- Smelkinson MG, Kalderon D. (2006) Processing of the Drosophila hedgehog signaling effector Ci-155 to the repressor Ci-75 is mediated by direct binding to the SCF component Slimb. Curr Biol 16: 110-116. Article
- Apionishev S, Katanayeva NM, Marks SA, Kalderon D, Tomlinson A. (2005) Drosophila Smoothened phosphorylation sites essential for Hedgehog signal transduction Nat Cell Biol. 7(1): 86-92. Article
- Price MA, Kalderon D. (2002) Proteolysis of the Hedgehog signaling effector Cubitus interruptus requires phosphorylation by Glycogen Synthase Kinase 3 and Casein Kinase 1. Cell 108: 823-35. Article
- Zhang Y, Kalderon D. (2001) Hedgehog acts as a somatic stem cell factor in the Drosophila ovary. Nature 410: 599-604. Article