Our research applies high-resolution live imaging of vertebrate model organisms to quantitatively study the multicellular dynamics of embryonic development and adult regeneration, with a focus on how stem cells migrate long distances (akin to cancer metastasis) and differentiate into a variety of specialized cell types. We determine what happens when development or regeneration is selectively perturbed via genetic, molecular, or physical manipulation.This deliberately in vivo, system-wide approach is inspired by important but challenging long-term goals of biomedical research, namely to prevent and/or repair human birth defects and cancer metastasis. To achieve these goals, we must first understand how embryonic development goes awry, which in turn requires understanding how development (and, in some cases, regeneration) takes place correctly in its natural, in vivo environment. The overall objective of our work is to elucidate the multicellular movements, interactions, and fate determination underpinning stem cell migration and differentiation, with experimental aims encompassing embryonic development, adult regeneration, and cancer metastasis. To this end, we use the easily accessed and manipulated olfactory system and surrounding neural crest-derived craniofacial mesenchyme in zebrafish as our primary model system, with the use of other model organisms as necessary. Main Research Goals: 1. Understand how cranial neural crest stem cells collectively migrate and differentiate into neurons (and related derivatives) via high-resolution live imaging and downstream quantitative analysis, eventually harnessing the knowledge gained to promote neuronal regeneration post-injury. 2. Uncover the molecular pathways responsible for neural crest differentiation into craniofacial mesenchyme versus sensory neurons and define the cell-cell signaling between these two adjacent cell populations. 3. Query a first-of-its-kind, in vivo olfactory regeneration model system to determine how rapid neuronal turnover is initiated and maintained throughout adulthood. 4. Use high-resolution imaging of neural crest migration as an apropos model for studying neural crest-derived neuroblastoma metastasis.
Details: By revealing the origins of vertebrate sensory neurons and the molecular and cellular processes driving the remarkable transformation from stem cell to neuron, we can better comprehend general mechanisms of neurogenesis and the potential for regeneration. Olfactory sensory neurons are particularly unique in their regenerative capacity throughout adulthood, with a completely new set of neurons present in the human nose every month. Thus, there is significant translational value in understanding the origins and differentiation pathways of these unusual derivatives.
The neural crest is a highly migratory, multipotent stem cell population that contributes to a variety of tissues in the developing embryo - including a significant portion of the peripheral nervous system - and is critically important for craniofacial development as a whole. In addition, the neural crest-derived sympathetic lineage can give rise to neuroblastomas, the most common type of cancer in the first year of human life. Intriguingly, neuroblastomas share many common features - both genetic and phenotypic - with the neural crest, and over 60% of neuroblastomas metastasize in a manner reminiscent of neural crest migration.
Our work has revealed a novel contribution of the cranial neural crest to olfactory sensory neurons (an unexpected fate switch as opposed to the expected formation of craniofacial mesenchyme) via dynamic, precisely orchestrated cell migration and differentiation (Saxena et al., eLife 2013). Now, we are working to further uncover the molecular, cellular, and system-wide changes underlying this intricate developmental process, including how two tightly intermingled populations (neural crest- and placode-derived) communicate and assemble during olfactory organogenesis and what role is played in olfactory development by the surrounding neural crest-derived mesenchymal nasal cavity. Additionally, we have crossed multiple transgenic lines into the pigmentation-deficient Casper background. This setup permits live imaging and precise, two-photon laser-based perturbation of stem cell movement/differentiation in juveniles and adults (as done previously with embryos using custom-designed molds). Inflicting injury and tracking recovery via live imaging allows for an in-depth look at regeneration. We are examining where adult stem cells reside, how they reenter the cell cycle and migrate to form neurons and mesenchymal derivatives, and which molecular pathways are conserved in adult regeneration vis-à-vis embryonic development. Eventually, we will also use these tools to shed light on the understudied field of in vivo metastatic migration with the goal of discovering new inhibitors of cancer metastasis. Given the poor clinical prognosis post-metastasis, inhibiting migration and thus restricting malignant cells to their point of origin may prove hugely effective at improving patient outcomes.
As suggested above, neural crest and placodal progenitor cells communicate actively with their craniofacial surroundings to cooperatively guide olfactory development. Therefore, rather than study individual cells in vitro where context is missing, our approach makes use of live zebrafish - and other species as necessary - to provide a system-wide overview of frontonasal development, i.e. how neighboring cells and signaling molecules interact with and influence each other over space and time in their natural environment. These data are compiled using molecular, genetic, and physical (laser-induced) perturbation combined with high-resolution imaging and downstream quantitative analysis. Our imaging toolset includes the use of lattice light-sheet (LLS), a novel derivative of Bessel plane SR-SIM developed by Dr. Eric Betzig's group at HHMI Janelia Research Campus. Our collaborative imaging of zebrafish neural crest and the olfactory system has produced the highest spatiotemporal resolution to date in live vertebrates (Warrier et al., In Prep). The core advantage of LLS lies in obtaining multiple scales and types of data - e.g., cell-cell interactions, subcellular localization, and cytoskeletal dynamics - while maintaining developmental fidelity, and the high resolution in all four dimensions allows for accurate tracking of cell migration and axon pathfinding, among other processes (see Videos for examples).Thus, by studying developmental and regenerative biology completely in vivo, we aim to gain translationally relevant insights into how neurons, supporting cells, and the surrounding craniofacial mesenchyme are formed, how complex three-dimensional organs are assembled in embryos and maintained in adults, and how neural crest migration and cancer metastasis are commonly regulated.
