Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from α-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish

Telomerase Is Required for Zebrafish Lifespan

CMH and MCC are supported by the Portuguese Fundacao para a Ciencia e a Tecnologia (FCT) fellowships. This work was supported by the FCT (PTDC/SAU-ORG/116826/2010 and PTDC/SAU-ONC/116821/2 010) and the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We indebted to Prisca Chapouton, Susanne Sprungala, and Laure Bally-Cuif for communicating results and sharing reagents prior to publication. We thank Drs. Cuppen and Plasterk (Hubrecht Laboratory) and Dr. Stemple (Welcome Trust Sanger Institute) for providing the zebrafish knockout mutant, which was generated as part of the ZF- MODELS Integrated Project in the 6th Framework Programme (Contract No. LSHG-CT-2003-503496) funded by the European Commission. We are grateful to Joana Nabais, Sofia Esteves, Rita Mateus, Sofia Azevedo, Susana Lopes, and Leonor Saude for help at the initial stages of our work; Tania Carvalho for histopathological analysis; Clara Melo, Graeme Hewitt, and Joao Passos for help with the Telo-FISH. We thank Joao Passos and Lea Harrington for critically reading the manuscript. MGF is a HHMI International Early Career Scientist.Telomerase activity is restricted in humans. Consequentially, telomeres shorten in most cells throughout our lives. Telomere dysfunction in vertebrates has been primarily studied in inbred mice strains with very long telomeres that fail to deplete telomeric repeats during their lifetime. It is, therefore, unclear how telomere shortening regulates tissue homeostasis in vertebrates with naturally short telomeres. Zebrafish have restricted telomerase expression and human-like telomere length. Here we show that first-generation tert-/- zebrafish die prematurely with shorter telomeres. tert-/- fish develop degenerative phenotypes, including premature infertility, gastrointestinal atrophy, and sarcopaenia. tert-/- mutants have impaired cell proliferation, accumulation of DNA damage markers, and a p53 response leading to early apoptosis, followed by accumulation of senescent cells. Apoptosis is primarily observed in the proliferative niche and germ cells. Cell proliferation, but not apoptosis, is rescued in tp53-/-tert-/- mutants, underscoring p53 as mediator of telomerase deficiency and consequent telomere instability. Thus, telomerase is limiting for zebrafish lifespan, enabling the study of telomere shortening in naturally ageing individuals.publishersversionpublishe

Telomerase depletion leads to a time- and tissue-dependent degeneration.

<p>A) Representative images of tissue sections of <i>tert^−/−</i> Zebrafish and <i>tert^+/+</i> siblings, stained with hematoxilin-eosin. <i>tert^−/−</i> zebrafish show progressive tissue deterioration. Severe histological abnormalities are first evident in proliferative tissues (testes, gut and head kidney marrow) and later in non-proliferative (muscle). <i>tert^−/−</i> Zebrafish show reduced sperm in testes lumen (L) (Ab and d; p<0.001). The head kidney shows progressive defects in the marrow area (white asterisks) (Ag and i, which correlates with a decrease in total blood (p = 0.0228) cells when compared to <i>tert^+/+</i> siblings from an early age (3 months, N≥5) (Aj). Mesonephric tubules in the head kidney also degenerate in <i>tert^−/−</i> (Ag and i dashed outlines). Gut atrophy in <i>tert^−/−</i>, reflected as decreased villi length (Al, n, o), becomes significant from the age of 6 months (p<0.001). Muscle fibres are significantly thinner (p<0.001) at terminal time-points (c.12 months) (Aq, s, t, dashed outline); N≥5. B) <i>tert^−/−</i> display progressive thickening of gut lamina propria, indicative of inflammation (Bb, c, yellow bar and arrow, N≥4). Data are represented as mean +/− SEM. Scale bar = 50 µm.</p

Telomerase mutant zebrafish have shorter telomeres than WT siblings.

<p>A) Representative image of TRAP assay showing that telomerase is not active in the <i>tert^−/−</i> zebrafish, as compared to <i>tert^+/+</i> siblings. Here shown are caudal fin and skin protein extracts. Hela cell extract is shown as positive control. N = 4. B) Representative image of restriction fragment analysis of caudal fin genomic DNA of 3 different individuals at different ages, by southern blot (random primer-labelled telomeric probe (CCCTAA)₁₂³²P-dCTP). <i>tert^+/+</i> Zebrafish have heterogeneous telomeres, with two distinct peaks of different lengths. In <i>tert^+/+</i> the highest peak (∼16 Kb, top red arrow) becomes more distinct after 1 months of age and decreases in length over-time (B and D). The lowest peak of telomere intensity also decreases in length (bottom red arrow, B and D). <i>tert^−/−</i> zebrafish have shorter telomeres than <i>tert^+/+</i> siblings in different tissues (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003214#pgen.1003214.s001" target="_blank">Figure S1A and S1B</a>), observed by the decrease in length of the higher TRF peak. The shortest TRF peaks accompany those of <i>tert^+/+</i> siblings, and decrease over-time at similar rates. C) Testes fractionation in <i>tert^+/+</i> reveals the two-telomere length populations in whole testes, whereas mature sperm only shows the shorter TRF smear of about 6 Kb, suggesting different telomere lengths in different cells within a tissue. D) TRF mean sizes were calculated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003214#pgen.1003214-Kimura1" target="_blank">[50]</a>. E) Telomere PNA-FISH in 6-month-old gut tissue shows cells with different telomere intensities in the wild type, mainly localizing to the proliferative niche. In contrast <i>tert^−/−</i> mutants display cells with less bright and more homogeneous telomere intensity.</p

Proliferative tissue degeneration is accompanied by a sustained decrease in proliferation, acute apoptotic responses, and progressive accumulation of DDR foci.

<p>Representative immunofluorescence images of tissue sections in F1 <i>tert^−/−</i> and <i>tert^+/+</i> zebrafish show levels of proliferation (PCNA), apoptosis (TUNEL), DNA damage (53BP1) and senescence-associated β–galactosidase at the ages of 3 to c.12 months. Proliferative tissues such as A) testes, B) head kidney and C) gut sections show sustained significant decrease in proliferation in <i>tert^−/−</i> as compared to <i>tert^+/+</i> siblings (panels b, d and e) (p<0.001) and an acute apoptotic response at 3 months of age (p<0.001), which clears by c. 12 months (panels g, i and j). This is accompanied by a progressive increase in 53BP1 foci, reaching maximum significance at c. 12 months (panels l, n and o; p<0.001). This coincides with the presence of senescence-associated β –galactosidase at c.12 months (panels s and t). Note in the testes that most of apoptosis (TUNEL) seems to localize to the spermatogenic zone (Ag, dashed outline) and panel E, where we see an increase in TUNEL-labelled germ cells, labelled with the specific marker PLZF. Most of DNA damage (53BP1) locates to the proliferative zone of maturing spermatocytes (Al, uniform outline). Note in the head kidney B), both the proliferative haematopoietic tissue (Ba–d, arrows) and the non-proliferative mesonephric tubule epithelium (Ba–d, dashed outline) are affected by increased apoptosis (Bg, I, j), DNA damage (Bl, n and o) and senescence (Bs and t) in the <i>tert^−/−</i>. D) Muscle, a largely non-proliferative tissue (Da–d) shows significant accumulation of DNA damage foci at in <i>tert^−/−</i> by the age of c.12 months (Dn and o; p<0.001), when the muscle fibres are already atrophic (Dn, dashed outline). Quantifications were performed in at least 3 different fields of view of at least 3 different individuals of each genotype at the different time-points indicated in the graphs. Gut IF quantifications were calculated as number of positive cells per “crypt” zone (C) uniform square outline exemplified). Other tissues' IF was quantified as overall % positive cells. β-galactosidase was quantified as % area stained blue, per field of view. Data are represented as mean +/− SEM. Scale bar = 50 µm.</p

Elimination of <i>tp53</i> function partially rescues <i>tert^−/−</i> degeneration in proliferative tissues.

<p><i>tert^−/−tp53^−/−</i> show increased proliferation and apoptosis in both testes (Ac, d and e, f) and gut (Bc, d and e, f), as compared to <i>tert^−/−</i> alone. In the testis, dashed outline represents spermatogenic zone, uniform outline proliferative zone of maturing spermatocytes and L the lumen where mature sperm is located. Elimination of <i>tp53</i> function partially rescues mature sperm numbers (Aa, b) but completely rescues gut villi length (Ba, b). DNA damage as assessed by 53BP1 is maintained in <i>tert^−/−tp53^−/−</i> testes (Ag and h) and decreased in the gut (Bg and h), as compared to <i>tert^−/−</i>. N≥3. Data are represented as mean +/− SEM. Scale bar = 50 µm.</p

Tissue degeneration is accompanied by p53 induction with a <i>puma</i> acute response and sustained increase of <i>cyclin G1</i> and <i>cdkn1a</i> expression.

<p>A) Immunoblot analysis of p53 in 10-month old WT and <i>tert^−/−</i> testes and gut lysates. 6 month-old WT zebrafish were injected with the DNA damaging agent doxorubicin to serve as positive control for p53 activation. Asterisk depicts a non-specific cross-reactive band that serves as loading control. RT-qPCR analysis showing expression of B) pro-apoptotic (<i>puma</i>) and cell cycle arrest targets (C) <i>cyclin G1</i> and D) <i>cdkn1a</i>) in testes, head kidney, gut and muscle of 1, 3, 6 and c.12 months old WT and <i>tert^−/−</i> zebrafish (N = 3 to 8 fish per genotype). Data are represented as mean +/− SEM. <i>Rel. mRNA</i> refers to relative mRNA levels of each gene normalized to <i>beta-actin</i>.</p

First-generation telomerase mutant zebrafish show progressive body wasting and die prematurely.

<p>A) Representative images of <i>tert^+/+</i> and <i>tert^−/−</i> zebrafish show that <i>tert^−/−</i> fish are born and develop normally until reproductive maturity at ∼3 months of age, but progressively lose body mass since then, B) represented as an overall reduction in width/length ratios as compared to wild-type siblings N≥6 p<0.001. This progressive wasting phenotype is accompanied by increase in mortality. C) Kaplan-Meier curve showing that <i>tert^−/−</i> zebrafish have significantly reduced survival when compared to <i>tert^+/+</i> siblings (AVG lifespan 9 <i>versus</i> >22 months (p<0.005)). N = 24 <i>tert^−/−</i>; N = 45 <i>tert^+/+</i>. Data are represented as mean +/− SEM. Scale bar = 1 cm.</p

In Vivo Imaging of Tumor-Propagating Cells, Regional Tumor Heterogeneity, and Dynamic Cell Movements in Embryonal Rhabdomyosarcoma

Embryonal rhabdomyosarcoma (ERMS) is an aggressive pediatric sarcoma of muscle. Here, we show that ERMS-propagating potential is confined to myf5+ cells and can be visualized in live, fluorescent transgenic zebrafish. During early tumor growth, myf5+ ERMS cells reside adjacent normal muscle fibers. By late-stage ERMS, myf5+ cells are reorganized into distinct regions separated from differentiated tumor cells. Time-lapse imaging of late-stage ERMS revealed that myf5+ cells populate newly formed tumor only after seeding by highly migratory myogenin+ ERMS cells. Moreover, myogenin+ ERMS cells can enter the vasculature, whereas myf5+ ERMS-propagating cells do not. Our data suggest that non-tumor-propagating cells likely have important supportive roles in cancer progression and facilitate metastasis. ► Functional heterogeneity of ERMS cells can be visualized in live zebrafish ► Tumor-propagating potential is restricted to myf5+ ERMS cells ► myogenin+ ERMS cells are highly invasive and colonize new areas of growth ► Metastatic and ERMS-propagating potential resides in distinct cell subpopulation