Project description:Lymph nodes and other secondary lymphoid organs play critical roles in immune surveillance and immune activation in mammals, but the deep internal locations of these organs makes it challenging to study dynamic cellular mechanisms of immune cell interaction with pathogens or other host tissues such as the vasculature in living animals. Here, we describe a previously uncharacterized external immune organ located near the base of the zebrafish pectoral fin, the pectoral axillary lobe (PAL), with a variety of features that make it ideally suited for studying immune cell dynamics in vivo. This small, transparent organ consists of an outer cortex teeming with immune cells and an inner medulla with a mesh-like network of fibroblastic reticular cells along which immune cells migrate, and a network of lymphatic vessels draining to a large adjacent lymph sac. Noninvasive high-resolution imaging of transgenically marked immune cells can be carried out in the lobes of living animals, and the PAL is readily accessible to external treatment with antigens or pathogens. This newly discovered tissue provides a superb model for dynamic live imaging of immune cells and their interaction with pathogens and surrounding tissues, including blood and lymphatic vessels.
Project description:Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. While genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using for example in situ hybridization, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. We also demonstrate that our technique is suitable for spatially-resolved differential expression analysis in wildtype and Gli3 mutant mouse forelimbs. Importantly, our method enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.
Project description:Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. While genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using for example in situ hybridization, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. We also demonstrate that our technique is suitable for spatially-resolved differential expression analysis in wildtype and Gli3 mutant mouse forelimbs. Importantly, our method enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs. To generate spatially-resolved RNA-seq data for zebrafish embryos (shield stage, 10 somites, 15 somites, 18 somites) and mouse forelimbs (E10.5), we cryosectioned samples, extracted RNA from the individual sections, and amplified and barcoded mRNA using the CEL-seq protocol (Hashimshony et al., Cell Reports, 2012) with a few modifications. Libraries were sequenced on Illumina HiSeq 2500 using 50bp paired end sequencing. Selected zebrafish libraries were sequenced on MiSeq 250bp paired-end to improve 3' annotations.