Molecular and Cell Biology of Neuromesodermal Progenitors

State University New York Stony Brook, Stony Brook NY

Investigators

Linked publications & trials

Abstract

Abstract (taken from the parent grant) We aim to understand the normal biology of neuromesodermal progenitors (NMPs), as well as to use NMPs to model cellular and molecular mechanisms in vivo. NMPs are basal progenitor cells located in the tailbud of vertebrate embryos that continue to make a germ-layer decision after gastrulation to generate ectoderm and mesoderm. As a primary source of cells generating the spinal cord, skeletal muscle, and other mesodermal derivatives, NMPs are a key cell type contributing to the formation of the vertebrate body plan. Studying NMPs has advanced our understanding of how body plans are generated, improved techniques for in vitro tissue generation, and provided critical insights into signaling pathway mechanisms. I discovered zebrafish NMPs as a postdoctoral fellow and my lab continues to focus on them. Our recent work has uncovered important roles for the transcription factor Sox2 in maintaining NMPs in an undifferentiated state through interactions with the canonical Wnt signaling pathway. This, combined with the past research showing the Wnt signaling effector bcat physically interacts with Sox2, indicates Wnt signaling can modulate gene expression and biological activity directly via Sox2. Little is known about this exciting branch of the Wnt/ bcat pathway. We will interrogate this pathway of Sox2 and Wnt/ bcat interactions at a number of biological levels, focusing on direct physical interactions between Sox2 and bcat, as well as genome wide analysis of Sox2/ bcat mediated transcriptional regulation. We hypothesize that Sox2/ bcat signaling represents a new arm of the Wnt/ bcat pathway distinct from the canonical TCF/LEF transcription factor family mediated signaling. We also recently developed a new Cyclin Dependent Kinase biosensor transgenic zebrafish and observed that NMPs and some of their derivatives exist in restricted cell cycle phases. The NMPs are held primarily in the G2 phase, while mesodermal notochord progenitors are restricted to the G1 phase. Cell cycle phase is broadly implicated in various aspects of stem cell maintenance, cell differentiation, and cell migration and invasion. We will manipulate the cell cycle in NMPs and their derivatives to understand how the cell cycle phase impacts normal NMP development. We hypothesize that the G2 phase restriction of NMPs is essential for maintenance of the undifferentiated state and for receipt of Wnt signaling based on G2 dependent receptor activation. We also hypothesize that the G1 phase of notochord progenitors is essential for their morphogenetic behavior of convergence and extension. Together, our work will shed important light on not only NMP development and vertebrate body plan formation, but also basic principles of cell biology and signaling. Wnt/ bcat signaling and Sox2 are found together and play important roles in numerous normal and diseased cellular contexts, including stem cells and cancer. The cell cycle is a fundamental aspect of normal development and is dysregulated in diseases such as cancer. Understanding how cell cycle phase impacts cell fate and morphogenetic behaviors in vivo will provide essential insight into normal and disease states. Justification of the need for a new epi-fluorescence microscope There are two main areas of research in this project related to neuromeosdermal progenitor development, both of which rely heavily on the use of epi-fluorescence microscopy. In the first area, we are investigating how the canonical Wnt signaling pathway and the Sox2 transcriptional factor interact to control call fate and morphogenesis of the neuromesodermal progenitors and their descendants, including the spinal cord and the paraxial mesoderm. To do this, we use transgenic zebrafish reporter lines that allow us to visualize Sox2 expression levels and canonical Wnt signaling activity based on fluorescence levels. We also perform mosaic analyses of host embryos containing mutant or transgenic cells in which the Sox2 or Wnt signaling levels are manipulated. This involves long-term time-lapse imaging and cell tracking of morphogenetic behaviors and cell fate commitment. In the second area, we are determining how the cell cycle state of neuromesodermal progenitors influences their morphogenesis and cell fate commitment. We generated transgenic biosensor lines that detect cell cycle state based on ratiometric distribution of fluorophores between the nucleus and cytoplasm. We also perform time-lapse analysis of mosaic embryos to determine how manipulating the cell cycle impacts morphogenesis and cell fate. In both projects, we use additional reporter and biosensor lines to monitor such things as cell-type specification and F-actin regulation, among many others. These two projects require heavy daily use of epifluorescence microscopy with advanced capabilities such as fast acquisition times, multi-wavelength detection, and a scanning stage for multi-embryo time-lapse capture. When I started my lab at Stony Brook University in 2012 I used my startup to purchase an automated Leica DMI600B epi-fluorescence microscope. It has a motorized turret for multiple-color acquisition as well as a z- motorized stage and appropriate software for collection of image stacks, embryos at multiple stage positions, and time-lapse imaging. We also use this for fixed embryo analysis such as multi-color hybridization chain reaction to visualize mRNA expression in embryos. This has been the workhorse piece of equipment in the lab since I started and gets daily use. Unfortunately, this microscope has been discontinued by Leica, making it difficult to service, and has been showing the signs of many years of heavy usage. We had to replace the computer, and some of the motorized components have failed. Additionally, it cannot meet some our current needs, such as the ability to image more than 6 colors, and to rapidly acquire multiple wavelengths for live-cell colocalization. The current instrument also does not eliminate the need to also use confocal microscopes to capture high resolution subcellular images, due to out of focus light capture typical of epi-fluorescent microscopes. Proposed replacement instrument The Leica DMi8 is the current version of our DMI6000B, and it comes as part of the new Leica Thunder package. The THUNDER Imager 3D Live Cell system provides real-time imaging with exceptional clarity, even in deep areas of the sample, eliminating out-of-focus blur. This is made possible by Leica Microsystems' Computational Clearing method. It minimizes photobleaching, ensuring optimal physiological conditions while delivering high-performance imaging and efficient data throughput, enhancing workflow and statistical analysis. Thus, not only does this microscope replace the capabilities of our old microscope, it also will eliminate much of our need to utilize shared confocal instruments. The replacement instrument also has improved abilities over our current instrument that will greatly enhance our research output. These include an 8 channel LED light source that will allow rapid switching between wavelengths for fast multi-color image capture, as well as the ability to use 8 different fluorophores instead of our current limit of 6.