Single Molecule Fluorescence In Situ Hybridization Using RNAscope to Study Hematopoietic and Vascular Interactions in the Zebrafish Embryo and Larva
Our goal is to understand how hematopoietic stem cell precursors emerge from vessels and to visualize their settling in developmental and more definitive niches that will persist in the adult. For this, we use as a biological model the zebrafish, which offers invaluable advantages owing to its trans...
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Bio-protocol LLC
2025-04-01
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| author | Léa Torcq Anne Schmidt |
| author_facet | Léa Torcq Anne Schmidt |
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| description | Our goal is to understand how hematopoietic stem cell precursors emerge from vessels and to visualize their settling in developmental and more definitive niches that will persist in the adult. For this, we use as a biological model the zebrafish, which offers invaluable advantages owing to its transparency and small size, allowing high-resolution imaging and investigations of the entire animal. In vertebrate species, precursors of hematopoietic stem cells emerge from arterial vessels, mainly from the ventral side of the dorsal aorta. From there, they can either reside in the underlying vascular niche and/or pass through the vein to enter the blood circulation and conquer the caudal hematopoietic tissue, a functional equivalent to the fetal liver in mammals. Here, we provide experimental details of a protocol we have recently optimized to identify, based on mRNA in situ hybridization, precursors of hematopoietic stem cells while still embedded in the aortic wall (at the embryonic stage) as well as when they reside in specific niches a few days after emergence (at the early larval stage). Our experimental approach uses RNAscope technology, which allows combining high-sensitivity mRNA detection with high-resolution fluorescence confocal imaging to achieve spatial transcriptomics. Importantly, the small size of the probes allows better penetration inside tissues, which is a significant improvement in comparison to long mRNA probes; this is an invaluable advantage for reaching deeply embedded niches such as the ones of the pronephros region in the larva and, in addition, provides an increased signal-to-noise ratio. |
| format | Article |
| id | doaj-art-12265dc699db460a8da438947c03c818 |
| institution | OA Journals |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-04-01 |
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| spelling | doaj-art-12265dc699db460a8da438947c03c8182025-08-20T02:26:55ZengBio-protocol LLCBio-Protocol2331-83252025-04-0115710.21769/BioProtoc.5269Single Molecule Fluorescence In Situ Hybridization Using RNAscope to Study Hematopoietic and Vascular Interactions in the Zebrafish Embryo and LarvaLéa Torcq0Anne Schmidt1Institut Pasteur, Université Paris Cité, CNRS UMR3738, Department of Developmental and Stem Cell Biology, Paris, FranceSorbonne Université, Collège doctoral, Paris, FranceInstitut Pasteur, Université Paris Cité, CNRS UMR3738, Department of Developmental and Stem Cell Biology, Paris, FranceOur goal is to understand how hematopoietic stem cell precursors emerge from vessels and to visualize their settling in developmental and more definitive niches that will persist in the adult. For this, we use as a biological model the zebrafish, which offers invaluable advantages owing to its transparency and small size, allowing high-resolution imaging and investigations of the entire animal. In vertebrate species, precursors of hematopoietic stem cells emerge from arterial vessels, mainly from the ventral side of the dorsal aorta. From there, they can either reside in the underlying vascular niche and/or pass through the vein to enter the blood circulation and conquer the caudal hematopoietic tissue, a functional equivalent to the fetal liver in mammals. Here, we provide experimental details of a protocol we have recently optimized to identify, based on mRNA in situ hybridization, precursors of hematopoietic stem cells while still embedded in the aortic wall (at the embryonic stage) as well as when they reside in specific niches a few days after emergence (at the early larval stage). Our experimental approach uses RNAscope technology, which allows combining high-sensitivity mRNA detection with high-resolution fluorescence confocal imaging to achieve spatial transcriptomics. Importantly, the small size of the probes allows better penetration inside tissues, which is a significant improvement in comparison to long mRNA probes; this is an invaluable advantage for reaching deeply embedded niches such as the ones of the pronephros region in the larva and, in addition, provides an increased signal-to-noise ratio.https://bio-protocol.org/en/bpdetail?id=5269&type=0 |
| spellingShingle | Léa Torcq Anne Schmidt Single Molecule Fluorescence In Situ Hybridization Using RNAscope to Study Hematopoietic and Vascular Interactions in the Zebrafish Embryo and Larva Bio-Protocol |
| title | Single Molecule Fluorescence In Situ Hybridization Using RNAscope to Study Hematopoietic and Vascular Interactions in the Zebrafish Embryo and Larva |
| title_full | Single Molecule Fluorescence In Situ Hybridization Using RNAscope to Study Hematopoietic and Vascular Interactions in the Zebrafish Embryo and Larva |
| title_fullStr | Single Molecule Fluorescence In Situ Hybridization Using RNAscope to Study Hematopoietic and Vascular Interactions in the Zebrafish Embryo and Larva |
| title_full_unstemmed | Single Molecule Fluorescence In Situ Hybridization Using RNAscope to Study Hematopoietic and Vascular Interactions in the Zebrafish Embryo and Larva |
| title_short | Single Molecule Fluorescence In Situ Hybridization Using RNAscope to Study Hematopoietic and Vascular Interactions in the Zebrafish Embryo and Larva |
| title_sort | single molecule fluorescence in situ hybridization using rnascope to study hematopoietic and vascular interactions in the zebrafish embryo and larva |
| url | https://bio-protocol.org/en/bpdetail?id=5269&type=0 |
| work_keys_str_mv | AT leatorcq singlemoleculefluorescenceinsituhybridizationusingrnascopetostudyhematopoieticandvascularinteractionsinthezebrafishembryoandlarva AT anneschmidt singlemoleculefluorescenceinsituhybridizationusingrnascopetostudyhematopoieticandvascularinteractionsinthezebrafishembryoandlarva |