Single-cell imaging of normal and malignant cell engraftment into optically clear prkdc-null SCID zebrafish.
Cell transplantation into immunodeficient mice has revolutionized our understanding of regeneration, stem cell self-renewal, and cancer; yet models for direct imaging of engrafted cells has been limited. Here, we characterize zebrafish with mutations in recombination activating gene 2 (rag2), DNA-dependent protein kinase (prkdc), and janus kinase 3 (jak3). Histology, RNA sequencing, and single-cell transcriptional profiling of blood showed that rag2 hypomorphic mutant zebrafish lack T cells, whereas prkdc deficiency results in loss of mature T and B cells and jak3 in T and putative Natural Killer cells. Although all mutant lines engraft fluorescently labeled normal and malignant cells, only the prkdc mutant fish reproduced as homozygotes and also survived injury after cell transplantation. Engraftment into optically clear casper, prkdc-mutant zebrafish facilitated dynamic live cell imaging of muscle regeneration, repopulation of muscle stem cells within their endogenous niche, and muscle fiber fusion at single-cell resolution. Serial imaging approaches also uncovered stochasticity in fluorescently labeled leukemia regrowth after competitive cell transplantation into prkdc mutant fish, providing refined models to assess clonal dominance and progression in the zebrafish. Our experiments provide an optimized and facile transplantation model, the casper, prkdc mutant zebrafish, for efficient engraftment and direct visualization of fluorescently labeled normal and malignant cells at single-cell resolution.
Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129.|Cancer Center, Massachusetts General Hospital, Charlestown, MA 02129.|Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114.|Harvard Stem Cell Institute, Cambridge, MA 02139.
John C
0000-0002-3419-0151
JC
Moore
Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129.|Cancer Center, Massachusetts General Hospital, Charlestown, MA 02129.|Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114.|Harvard Stem Cell Institute, Cambridge, MA 02139.
Qin
0000-0002-9487-570X
Q
Tang
Harvard Stem Cell Institute, Cambridge, MA 02139.|Harvard University, Cambridge, MA 02138.
Nora Torres
0000-0001-6105-3446
NT
Yordán
Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129.|Cancer Center, Massachusetts General Hospital, Charlestown, MA 02129.|Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114.|Harvard Stem Cell Institute, Cambridge, MA 02139.
Finola E
FE
Moore
Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129.|Cancer Center, Massachusetts General Hospital, Charlestown, MA 02129.|Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114.|Harvard Stem Cell Institute, Cambridge, MA 02139.
Elaine G
0000-0002-4443-6477
EG
Garcia
Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129.|Cancer Center, Massachusetts General Hospital, Charlestown, MA 02129.|Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114.|Harvard Stem Cell Institute, Cambridge, MA 02139.
Riadh
0000-0002-8310-7991
R
Lobbardi
Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129.|Cancer Center, Massachusetts General Hospital, Charlestown, MA 02129.|Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114.|Harvard Stem Cell Institute, Cambridge, MA 02139.
Ashwin
0000-0003-1569-6664
A
Ramakrishnan
Cancer Center, Massachusetts General Hospital, Charlestown, MA 02129.
Dieuwke L
0000-0002-3226-7573
DL
Marvin
Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.|Department of Genetics, Harvard Medical School, Boston, MA 02115.
Anthony
0000-0002-4761-1541
A
Anselmo
Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114.|Department of Genetics, Harvard Medical School, Boston, MA 02115.
Ruslan I
RI
Sadreyev
Molecular Pathology, Massachusetts General Hospital, Charlestown, MA 02129 dlangenau@mgh.harvard.edu.|Cancer Center, Massachusetts General Hospital, Charlestown, MA 02129.|Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114.|Harvard Stem Cell Institute, Cambridge, MA 02139.
David M
0000-0001-6664-8318
DM
Langenau
D000740:Anemia / Q000473:pathology; D000818:Animals; D001483:Base Sequence; D002999:Clone Cells; D051747:DNA-Activated Protein Kinase / Q000172:deficiency* / Q000378:metabolism; D004195:Disease Models, Animal; D005720:Gamma Rays; D006720:Homozygote; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D016867:Immunocompromised Host / Q000528:radiation effects; D008164:Luminescent Proteins / Q000378:metabolism; D032342:Muscle Cells / Q000473:pathology / Q000528:radiation effects; D009154:Mutation / Q000235:genetics; D009368:Neoplasm Transplantation; D055620:Optical Phenomena; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D012038:Regeneration / Q000528:radiation effects; D059010:Single-Cell Analysis / Q000379:methods*; D014184:Transplantation, Homologous; D060152:V(D)J Recombination / Q000235:genetics; D015027:Zebrafish / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000378:metabolism; D000097573:Red Fluorescent Protein
D051747:DNA-Activated Protein Kinase; D008164:Luminescent Proteins; D016271:Proto-Oncogene Proteins c-myc; D029961:Zebrafish Proteins; D000097573:Red Fluorescent Protein; C000621724:Prkdc protein, zebrafish