Noninvasive In Toto Imaging of the Thymus Reveals Heterogeneous Migratory Behavior of Developing T Cells

Abstract

The migration of developing T cells (thymocytes) between distinct thymic microenvironments is crucial for their development. Ex vivo studies of thymus tissue explants suggest two distinct migratory behaviors of thymocytes in the thymus. In the cortex, thymocytes exhibit a stochastic migration, whereas medullary thymocytes show confined migratory behavior. Thus far, it has been difficult to follow all thymocytes in an entire thymus and relate their differentiation steps to their migratory dynamics. To understand the spatial organization of the migratory behavior and development of thymocytes in a fully functional thymus, we developed transgenic reporter lines for the chemokine receptors ccr9a and ccr9b, as well as for rag2, and used them for noninvasive live imaging of the entire thymus in medaka (Oryzias latipes). We found that the expression of these two chemokine receptors in the medaka juvenile thymus defined two spatially distinct subpopulations of thymocytes. Landmark events of T cell development including proliferation, somatic recombination, and thymic selection can be mapped to subregions of the thymus. The migratory behavior of thymocytes within each of the subpopulations is equally heterogeneous, and specific migratory behaviors are not associated with particular domains in the thymus. During the period when thymocytes express rag2 their migratory behavior was more homogeneous. Therefore, the migratory behavior of thymocytes is partly correlated with their developmental stage rather than being defined by their spatial localization.

A high-sensitivity bi-directional reporter to monitor NF-κB activity in cell culture and zebrafish in real time

“…To visualize NF-κB activation upon infection or injury, time-lapse experiments were carried out with an Ultraview ERS spinning disk (PerkinElmer) confocal microscope using a 40× water-immersion objective. Images were analyzed with Imaris software as described previously (Bajoghli et al, 2015).…”

Making Thymus Visible: Understanding T-Cell Development from a New Perspective

“…Both zebrafish and medaka embryos are transparent, offering an opportunity to directly monitor T-cell development. A range of transgenic fluorescence-based reporter lines have been developed to visualize lymphoid progenitors ( 41 , 42 ), thymocyte subsets ( 41 – 45 ), naïve T-cells ( 42 , 46 ), DCs ( 47 ), and TECs ( 43 ) in both model systems. Most of these transgenic lines carry a construct in which the expression of a fluorescent protein is under the control of cis-regulatory elements of a cell-specific gene.…”

“…Alternatively, a fluorescent protein can be integrated into a gene locus using the CRISPR/Cas9 technique. We recently used this method to introduce green fluorescent protein (GFP) directly into the autoimmune regulatory ( aire ) gene locus to visualize the thymic medullary region in juvenile medaka ( 42 ). In fish models, fluorescent-based reporter lines have two advantages.…”

“…The small body size of fish larvae offers the opportunity to monitor cell trafficking at the whole-organism level using laser-scanning confocal ( 48 ) or light-sheet fluorescence microscopy ( 42 ). In vivo imaging of zebrafish and medaka larvae is generally noninvasive and can be continued for more than 10 h ( 41 , 43 , 49 ).…”

Intravital microscopy: Imaging host–parasite interactions in lymphoid organs

“…Finally, another alternative for imaging the thymus, is the use of teleost fish models including zebrafish and medaka. These models have allowed performing time-lapse imaging of the entire thymus, and visualization of T cell dynamics at different stages of development(Bajoghli et al, 2015;Hess & Boehm, 2012). Window types, and their advantages and limitations, are summarised inTable 1.To our knowledge, IVM has not been used to study the thymus in the context of parasitology.…”