The fundamental objective of developmental biology is to understand how organisms grow to adulthood. In the past 40 years, studies using genetically tractable model systems have led to a detailed understanding of the genetic mechanisms involved in the control of developmental events, as illustrated by our intimate knowledge of patterning and morphogenesis. The next significant questions address how complex phenotypes arise in the context of the whole organism and how the programs regulating development and function of these phenotypes are influenced by genetic background and environment. For example, little is understood about how the simultaneous growth and differentiation of different tissues and organs are coordinated and how the development of different cell types and tissues is integrated within an organ. In addition, many of the mechanisms by which growth factor–triggered signaling events intersect with cell metabolism, which is regulated by nutrients, hormones, and stress, remain to be identified. Acquiring answers to these fascinating questions requires a deep understanding of the mechanisms by which cells integrate signals received from their inner selves, their neighboring cells, other organs, and the environment.
We are using Drosophila as a model system to characterize the mechanisms underlying communication between cells, tissues, and organs. We are investigating several questions:
- What is the composition and organization of signaling networks?
- What are the mechanisms by which cells compute incoming signals in time and space?
- What are the signals that mediate communication between organs and how do they influence the development, regenerative properties, and physiology of individual organs?
- How do environmental factors, such as nutrients and stress, influence homeostasis?
We are studying these fundamental problems in Drosophila because of its unrivalled arsenal of tools for both in vivo and in vitro functional studies.
Composition and Organization of Signaling Networks
To dissect signaling pathways, we use combinations of genome-wide RNAi screening and large-scale mass spectrometry to identify components of signaling networks and to characterize their activities using functional readouts such as phosphorylation changes, transcriptional outputs, and cellular phenotypes. In particular, we are interested in identifying points of intersection, or "cross-talk nodes," between signaling pathways because cells within tissues are exposed to multiple signals that are somehow integrated into coherent responses. We are applying these approaches to our ongoing studies of the insulin/TOR pathway and Hippo networks, which play fundamental roles in coordinating growth and proliferation. More recently, to probe the functional redundancies within networks, we have begun using CRISPR-based knockout strategies, which in combination with RNAi, provide a powerful platform for synthetic functional screens. Finally, we are extending our signaling network studies beyond cell lines to complex tissues and are implementing a proximity-labeling approach, based on the engineered peroxidase APEX, that allows characterization of subcellular proteomes in live tissues.
Cell Signaling in Time and Space
Understanding how intercellular signaling is able to produce defined cell fates or behavioral outcomes is a critical question in cell and developmental biology. Many years of studies have identified a small number of core signaling pathways (EGF, FGF, cytokine JAK-STAT, JNK, Hedgehog, Hippo, Notch, NF-κB, retinoic acid, TGFβ, and Wnt/Wingless) that are used in many different contexts to generate cell and tissue complexity. These studies have shown that (1) individual pathways can produce distinct responses and (2) signaling cross talk is key to generating diverse outcomes. It is now clear that the two work as an ensemble to generate the mature, functional organism. While a great deal is known about which signaling pathways are involved in which processes, it remains unclear how a combination of pathways functions to affect cell fate and behavior.
Manipulation of individual pathways over a timescale of hours or days gives an indication of how a specific pathway affects cell fate, and epistasis analyses can identify some relationships between pathways. However, primary signaling responses at the transcriptional level can occur on a timescale of minutes, so secondary responses and sequential pathway cross talk complicate interpretation of data from single, late time points. Dissecting the relationships between pathways and the flow of information that regulates cell fate requires a level of temporal and spatial resolution that can best be achieved by either time courses or, ideally, by live imaging. To observe signaling at high spatiotemporal resolution in vivo, we are developing a number of tools and approaches that involve tagging endogenous genes with dynamic fluorescence reporters that improve dramatically the temporal visualization of gene activities. We are particularly interested in using these tools to analyze in the Drosophila gut how interactions between pathways are orchestrated to control stem cell proliferation during homeostasis and regeneration.
Communication Between Organs
Organ-to-organ communications are critical to living systems and play major roles in homeostasis. For example, the vertebrate central nervous system receives information regarding the status of peripheral metabolic processes via hormonal signaling and direct macromolecular sensing. In addition, skeletal muscles produce various myokines that influence metabolic homeostasis, life span, and the progression of age-related diseases and aging in nonmuscle tissues. Drosophila is a prime system for systematically identifying mechanisms involved in organ communication because libraries of transgenic RNAi lines are available that allow knockdown of any gene in an organ- or tissue-specific manner.
We have already used such genetic screens to characterize a number of secreted factors (ImpL2/IGFBP, Myostatin/GDF11, Upd2/Leptin, Activin-β) by which organs communicate their physiological state to other organs. These genetic screens are combined with RNA sequencing of specific organs to define the transcriptional signatures corresponding to their homeostatic states and with mass spectrometry analyses from blood to characterize secreted factors. It is anticipated that these studies will reveal how subnetworks in one tissue influence subnetworks in a second tissue. Ultimately, this knowledge will generate testable hypotheses related to disease states (e.g., diabetes, cancer, and aging) to answer how biological processes affected in one tissue/organ (e.g., decreased cellular metabolism, mitochondrial dysfunction) influence processes in a different tissue/organ.
This work is also partially supported by grants from the National Institute of General Medical Sciences, the National Cancer Institute, the National Institute of Diabetes and Digestive and Kidney Diseases, and the National Human Genome Research Institute.
As of March 23, 2016