Name: development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos
Uploaded: 2017-11-30T15:00:00.000+00:00
Duration: 10 min 28 s
Description: Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

Finanziamento è stato fornito dal National Institutes of Health (NIH: K01-DK087814-01A1 a D.L.S.), il California State University Program per formazione & ricerca in biotecnologia (CSUPERB: controllo molecolare della nicchia emopoietica vertebrati a D.L.S.) e dall'ufficio studi universitari presso la California State University di Chico (a A.C.B.).

Comitato consultivo istituzionale Animal Care ed uso Committee (IACUC) presso la California State University, Chico, ha approvato tutti i metodi descritti di seguito.
1. candeggina 48 embrioni di Zebrafish hpf
<ol><li>Con 15 centimetri (w) x 15 cm (l) x contenitori di plastica di 7 cm (h) creare 3 stazioni di lavaggio: 1) con 1 mL di candeggina al 5% in 1000 mL di terreno di embrione (E3; si veda tabella 1), 2) con sterile E3 e 3) con E3 sterile. Assicurarsi che ogni contenitore 500 mL di soluzione per sommergere gli embrioni in. Nota: Per ogni condizione testata, saranno necessari almeno 10 embrioni. Questa procedura di lavaggio può ospitare fino a 200 embrioni.</li>
	<li>Con una pipetta di trasferimento, posizionare gli embrioni in un colino da tè e immergere le uova nella stazione di lavaggio #1 per 5 minuti rimuovere il colino da tè (con embrioni all'interno) e inserire nella stazione di lavaggio #2 per 5 min; Ripetere per stazione di lavaggio #3 per un finale 5 min.</li>
	<li>Dopo l'ultimo lavaggio, risciacquo embrioni da colino da tè ruotandola testa in giù sopra un pulito 10 cm di Petri e risciacquo delicatamente gli embrioni fuori il colino da tè con E3 sterile e una pipetta di trasferimento.</li>
</ol>2. Dechorionate embrioni
<ol><li>Con una pipetta di trasferimento, rimuovere e scartare tanto E3 come possibile da di Petri e aggiungere 500 μL di dechorionation proteasi (10 mg/mL) agli embrioni. Incubare a temperatura ambiente per 5 min. Picchiettare delicatamente il lato di Petri per rimuovere completamente chorions.</li>
	<li>Con una pipetta sierologica, aggiungere 20 mL di sterile E3 per diluire proteasi. Embrioni di stabilirsi ed eliminarne l'E3 con una pipetta di trasferimento. Ripetere questo passaggio di lavaggio 3 volte per rimuovere tutte le tracce della proteasi dechorionation.</li>
</ol>3. preparazione dei campioni di embrione
<ol><li>Utilizzando una pipetta P1000, inserire 10 embrioni in un tubo del microcentrifuge singole sterili da 1,5 mL.</li>
	<li>Rimuovere E3 con una pipetta e scartare. Attenzione: Pipetta con cura come embrioni possono essere facilmente spostati e scartati durante le seguenti fasi di lavaggio.</li>
</ol>4. preparare gli embrioni per dissociazione
<ol><li>Trasferire campioni cappa a flusso laminare e lavare gli embrioni aggiungendo 1 mL sterile E3. Consentire gli embrioni si depositano sul fondo della provetta e rimuovere il surnatante con una pipetta P1000. Ripetere per un totale di 3 lavaggi.</li>
	<li>Dopo l'ultimo lavaggio, rimuovere E3 ed eliminare. Aggiungere 1 mL di 10 mM dithiothreitol (DTT) in E3 per rimuovere il rivestimento di muco (e spore, lievito o batteri che possono essere intrappolati in esso) che circondano il zebrafish embrionale. Posare il tubo del microcentrifuge orizzontalmente e incubare a temperatura ambiente in cappa a flusso laminare per 25 min.</li>
</ol>5. embrione dissociazione
<ol><li>Lavare il campione 3 volte con 1 mL di DPBS sterile (con Ca2 + e Mg2 +). Dopo l'ultimo lavaggio aggiungere 500 μL di DPBS (con Ca2 + e Mg2 +) e aggiungere 5 μL di proteasi di dissociazione di 5 mg/mL (26 U/mL). Attenzione: Questo enzima ha bisogno di Ca2 + e Mg2 +, in modo da garantire che il DPBS contiene questi ioni.</li>
	<li>Incubare i campioni a 37 ° C in un agitatore orbitale orizzontale a 180 giri/min per 60 min. campioni di posto nella cappa a flusso laminare e triturare gli embrioni con un P1000 fino a quando i campioni sono completamente dissociati. Attenzione: Controllare gli embrioni periodicamente per determinare quando essi diventano dissociate. La soluzione di embrione dovrebbe avere certa quantità di tessuto presente; non dovrebbe essere completamente omogeneo. È possibile sovra-digest, che distruggerà la HSPCs (Vedi Supplemental figura 1).</li>
</ol>6. preparazione degli embrioni dissociati
<ol><li>Embrioni di pipetta il 10 dissociato sul serbatoio superiore di un polistirolo da 5 mL tubo rotondo basso con un tappo di filtro cella 35 μm.</li>
	<li>Con una pipetta, sciacquare il tubo di Petri con PBS sterile (con nessun Ca2 + o Mg2 +) e soluzione di trasferimento a 5 mL provetta polistirolo filtrati celle dal punto 6.1. Ripetere fino a 4 mL di liquido è presente nella provetta polistirolo da 5 mL. Nota: PBS, DPBS o altre soluzioni isotoniche tampone possono essere utilizzati in questa fase, purché non contengono Ca2 + o Mg2 +
</li>
	<li>Centrifugare le provette a 4 ° C e 300 x g per 5 min a pellet campione omogeneizzato cellulare. Con una pipetta, rimuovere e scartare il surnatante dal tubo fondo tondo. Fare attenzione a non frantumare le cellule pellettate nella parte inferiore del tubo. Risospendere le cellule in 100 μL di PBS.</li>
</ol>7. preparazione di metilcellulosa
<ol><li>Generare la soluzione di riserva completa metilcellulosa (tabella 1): 1x e aggiungere 2,5 mL in provette sterili round-fondo 14 mL con 16 G aghi e siringhe da 3 mL. Utilizzare un tubo per ogni condizione testata.</li>
	<li>
Aggiungere citochine, molecole piccole o altri agenti per essere studiato per ogni campione.
	<ol>
<li>Per differenziazione mieloide, aggiungere 1% carpa del siero e 0,3 mg/mL ricombinante zebrafish stimolante le colonie granulocitarie fattore (Gcsf)34 per mezzo di metilcellulosa. Vedi Svoboda et al. 37 per la descrizione completa su come generare carpa citochine del siero e ricombinante.</li>
		<li>Per differenziazione eritroide, aggiungere 1% carpa del siero e 0,1 mg/mL zebrafish ricombinante dell'eritropoietina (Epo)38 a mezzo di metilcellulosa.</li>
		<li>Per esaminare i progenitori multilineare, aggiungere Epo e Gcsf. Attenzione: Citochine e additivi non dovrebbero totale più del 10% del volume totale, come il mezzo non sarà abbastanza viscoso a discernere le diverse colonie.</li>
	</ol>
</li>
</ol>8. aggiunta di embrioni dissociati di metilcellulosa
<ol><li>Utilizzando una pipetta, aggiungere 100 μL di dissociata 10 embrioni alla superficie della metilcellulosa preparata con ricombinante del siero di citochine e la carpa. Coperchio tappo e agitare delicatamente per omogeneizzare completamente campione. Ogni tubo deve contenere il tessuto dissociato di 10 embrioni.</li>
	<li>Utilizzando 3 mL siringhe ed aghi di 16 G, aliquotare 1 mL di campione in 2 capsule di Petri separata 35 mm. Assicurarsi che il campione è completamente dispersa in tutto il piatto di Petri. Ripetere per ogni campione.</li>
	<li>Consente di inserire un 15cm di Petri 35mm di Petri con cellule in metilcellulosa. Ad ogni piatto di 15 cm, aggiungere una piastra di Petri di 35 mm con 5 mL di acqua sterile per umidificare i campioni. Incubare a 32 ° C e 5% CO2 per 7-10 giorni.</li>
</ol>9. visualizzazione dei derivati HSPC Colony Forming Units (CFU)
<ol><li>Dopo 7 giorni di incubazione, posizionare il campione sul microscopio invertito a 40-100 X per visualizzazione ed enumerazione. A questo punto, diverse colonie possono essere isolate con una pipetta e sottoposti ad analisi di colorazione e/o gene.</li>
</ol>

<ol>
	<li>Kondo, M., Weissman, I. L., Akashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+clonogenic+common+lymphoid+progenitors+in+mouse+bone+marrow.">Identification of clonogenic common lymphoid progenitors in mouse bone marrow.</a> Cell. 91, 661-672 (1997).</li><li>Akashi, K., Traver, D., Miyamoto, T., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clonogenic+common+myeloid+progenitor+that+gives+rise+to+all+myeloid+lineages.">A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.</a> Nature. 404, 193-197 (2000).</li><li>Bertrand, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cells+derive+directly+from+aortic+endothelium+during+development.">Haematopoietic stem cells derive directly from aortic endothelium during development.</a> Nature. 464, 108-111 (2010).</li><li>Kissa, K., Herbomel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+stem+cells+emerge+from+aortic+endothelium+by+a+novel+type+of+cell+transition.">Blood stem cells emerge from aortic endothelium by a novel type of cell transition.</a> Nature. 464, 112-115 (2010).</li><li>Bertrand, J. Y., Kim, A. D., Teng, S., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD41++cmyb++precursors+colonize+the+zebrafish+pronephros+by+a+novel+migration+route+to+initiate+adult+hematopoiesis.">CD41+ cmyb+ precursors colonize the zebrafish pronephros by a novel migration route to initiate adult hematopoiesis.</a> Development. 135, 1853-1862 (2008).</li><li>Bertrand, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitive+hematopoiesis+initiates+through+a+committed+erythromyeloid+progenitor+in+the+zebrafish+embryo.">Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo.</a> Development. 134, 4147-4156 (2007).</li><li>Ma, D., Zhang, J., Lin, H. F., Italiano, J., Handin, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+identification+and+characterization+of+zebrafish+hematopoietic+stem+cells.">The identification and characterization of zebrafish hematopoietic stem cells.</a> Blood. 118, 289-297 (2011).</li><li>Lam, E. Y., Hall, C. J., Crosier, P. S., Crosier, K. E., Flores, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+Runx1+expression+in+the+dorsal+aorta+tracks+the+emergence+of+blood+progenitors+from+endothelial+cells.">Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells.</a> Blood. 116, 909-914 (2010).</li><li>Lam, E. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+runx1+promoter-EGFP+transgenics+mark+discrete+sites+of+definitive+blood+progenitors.">Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors.</a> Blood. 113, 1241-1249 (2009).</li><li>Lin, H. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+thrombocyte+development+in+CD41-GFP+transgenic+zebrafish.">Analysis of thrombocyte development in CD41-GFP transgenic zebrafish.</a> Blood. 106, 3803-3810 (2005).</li><li>Driever, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+screen+for+mutations+affecting+embryogenesis+in+zebrafish.">A genetic screen for mutations affecting embryogenesis in zebrafish.</a> Development. 123, 37-46 (1996).</li><li>Weinstein, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+mutations+in+the+zebrafish.">Hematopoietic mutations in the zebrafish.</a> Development. 123, 303-309 (1996).</li><li>Ransom, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+zebrafish+mutants+with+defects+in+embryonic+hematopoiesis.">Characterization of zebrafish mutants with defects in embryonic hematopoiesis.</a> Development. 123, 311-319 (1996).</li><li>Amsterdam, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+insertional+mutagenesis+screen+in+zebrafish.">A large-scale insertional mutagenesis screen in zebrafish.</a> Genes Dev. 13, 2713-2724 (1999).</li><li>Gaiano, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertional+mutagenesis+and+rapid+cloning+of+essential+genes+in+zebrafish.">Insertional mutagenesis and rapid cloning of essential genes in zebrafish.</a> Nature. 383, 829-832 (1996).</li><li>Yeh, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+chemical+modifiers+of+oncogene-regulated+hematopoietic+differentiation.">Discovering chemical modifiers of oncogene-regulated hematopoietic differentiation.</a> Nat Chem Biol. 5, 236-243 (2009).</li><li>Paik, E. J., de Jong, J. L., Pugach, E., Opara, P., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+genetic+screen+in+zebrafish+for+pathways+interacting+with+cdx4+in+primitive+hematopoiesis.">A chemical genetic screen in zebrafish for pathways interacting with cdx4 in primitive hematopoiesis.</a> Zebrafish. 7, 61-68 (2010).</li><li>Ridges, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+screen+identifies+novel+compound+with+selective+toxicity+against+leukemia.">Zebrafish screen identifies novel compound with selective toxicity against leukemia.</a> Blood. 119, 5621-5631 (2012).</li><li>North, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+E2+regulates+vertebrate+haematopoietic+stem+cell+homeostasis.">Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis.</a> Nature. 447, 1007-1011 (2007).</li><li>Astuti, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Functional+Bioluminescent+Zebrafish+Screen+for+Enhancing+Hematopoietic+Cell+Homing.">A Functional Bioluminescent Zebrafish Screen for Enhancing Hematopoietic Cell Homing.</a> Stem Cell Reports. 8, 177-190 (2017).</li><li>Kiel, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121, 1109-1121 (2005).</li><li>Spangrude, G. J., Heimfeld, S., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+mouse+hematopoietic+stem+cells.">Purification and characterization of mouse hematopoietic stem cells.</a> Science. 241, 58-62 (1988).</li><li>Morrison, S. J., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long-term+repopulating+subset+of+hematopoietic+stem+cells+is+deterministic+and+isolatable+by+phenotype.">The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype.</a> Immunity. 1, 661-673 (1994).</li><li>Osawa, M., Hanada, K., Hamada, H., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+lymphohematopoietic+reconstitution+by+a+single+CD34-low/negative+hematopoietic+stem+cell.">Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.</a> Science. 273, 242-245 (1996).</li><li>Goodell, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye+efflux+studies+suggest+that+hematopoietic+stem+cells+expressing+low+or+undetectable+levels+of+CD34+antigen+exist+in+multiple+species.">Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.</a> Nat Med. 3, 1337-1345 (1997).</li><li>Goodell, M. A., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo.">Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.</a> J Exp Med. 183, 1797-1806 (1996).</li><li>Gansner, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sorting+zebrafish+thrombocyte+lineage+cells+with+a+Cd41+monoclonal+antibody+enriches+hematopoietic+stem+cell+activity.">Sorting zebrafish thrombocyte lineage cells with a Cd41 monoclonal antibody enriches hematopoietic stem cell activity.</a> Blood. 129, 1394-1397 (2017).</li><li>Smith, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+cell+transplantation+establishes+that+tumor-initiating+cells+are+abundant+in+zebrafish+T-cell+acute+lymphoblastic+leukemia.">High-throughput cell transplantation establishes that tumor-initiating cells are abundant in zebrafish T-cell acute lymphoblastic leukemia.</a> Blood. 115, 3296-3303 (2010).</li><li>Mizgirev, I., Revskoy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+clonal+zebrafish+lines+and+transplantable+hepatic+tumors.">Generation of clonal zebrafish lines and transplantable hepatic tumors.</a> Nat Protoc. 5, 383-394 (2010).</li><li>de Jong, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+immune-matched+hematopoietic+transplantation+in+zebrafish.">Characterization of immune-matched hematopoietic transplantation in zebrafish.</a> Blood. 117, 4234-4242 (2011).</li><li>Wolf, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Caudal+Haematopoietic+Embryonic+Stromal+Tissue+(CHEST)+Cells+Support+Haematopoiesis.">Zebrafish Caudal Haematopoietic Embryonic Stromal Tissue (CHEST) Cells Support Haematopoiesis.</a> Sci Rep. 7, 44644 (2017).</li><li>Campbell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+embryonic+stromal+trunk+(ZEST)+cells+support+hematopoietic+stem+and+progenitor+cell+(HSPC)+proliferation,+survival,+and+differentiation.">Zebrafish embryonic stromal trunk (ZEST) cells support hematopoietic stem and progenitor cell (HSPC) proliferation, survival, and differentiation.</a> Exp Hematol. 43, 1047-1061 (2015).</li><li>Svoboda, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+vertebrate+hematopoiesis+using+zebrafish+thrombopoietin.">Dissection of vertebrate hematopoiesis using zebrafish thrombopoietin.</a> Blood. 124, 220-228 (2014).</li><li>Stachura, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+granulocyte+colony-stimulating+factors+(Gcsfs):+2+paralogous+cytokines+and+their+roles+in+hematopoietic+development+and+maintenance.">The zebrafish granulocyte colony-stimulating factors (Gcsfs): 2 paralogous cytokines and their roles in hematopoietic development and maintenance.</a> Blood. 122, 3918-3928 (2013).</li><li>Stachura, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+hematopoietic+progenitor+cells+in+the+zebrafish.">Clonal analysis of hematopoietic progenitor cells in the zebrafish.</a> Blood. 118, 1274-1282 (2011).</li><li>Stachura, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+kidney+stromal+cell+lines+support+multilineage+hematopoiesis.">Zebrafish kidney stromal cell lines support multilineage hematopoiesis.</a> Blood. 114, 279-289 (2009).</li><li>Svoboda, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+tools+for+the+clonal+analysis+of+zebrafish+hematopoiesis.">Ex vivo tools for the clonal analysis of zebrafish hematopoiesis.</a> Nat Protoc. 11, 1007-1020 (2016).</li><li>Paffett-Lugassy, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+conservation+of+erythropoietin+signaling+in+zebrafish.">Functional conservation of erythropoietin signaling in zebrafish.</a> Blood. 110, 2718-2726 (2007).</li><li>Stachura, D. L., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+dissection+of+zebrafish+hematopoiesis.">Cellular dissection of zebrafish hematopoiesis.</a> Methods Cell Biol. 133, 11-53 (2016).</li><li>Traver, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat Immunol. 4, 1238-1246 (2003).</li><li>Kobayashi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+localization+of+side+population+(SP)+cells+in+zebrafish+kidney+hematopoietic+tissue.">Characterization and localization of side population (SP) cells in zebrafish kidney hematopoietic tissue.</a> Blood. 111, 1131-1137 (2008).</li><li>Henninger, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+fate+mapping+quantifies+the+number+of+haematopoietic+stem+cells+that+arise+during+development.">Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development.</a> Nat Cell Biol. 19, 17-27 (2017).</li><li>Tamplin, O. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+arrival+triggers+dynamic+remodeling+of+the+perivascular+niche.">Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.</a> Cell. 160, 241-252 (2015).</li></ol>

Gli autori non hanno nulla a rivelare.

Il sistema di modello di zebrafish è diventato un modello efficiente, efficace ed economico per studiare l'ematopoiesi vertebrato primitivo e definitiva. Generazione di saggi che sono veloci, poco costoso e presenta alcune difficoltà tecniche può essere utilizzato per test di piccole molecole, analizzando gli embrioni mutanti e delucidare i meccanismi molecolari importanti per la biologia HSPC. Placcatura in vitro di HSPCs dallo zebrafish adulto è un metodo efficace per studiare la mutagenesi e citochine ema...

L'ematopoiesi è il processo di fare il gran numero di cellule ematiche mature, necessaria per la sopravvivenza di un organismo. È un processo chiave di sviluppo che coinvolge la differenziazione di cellule staminali ematopoietiche (HSCs) in una varietà di tipi cellulari inerente allo sviluppo limitato che comprendono sangue maturo. Questi HSCs devono rinnovarsi affinché il sistema non è mai esaurito e si deve mantenere dal primo sviluppo embrionale fino alla morte. Nei vertebrati, costante differenziazione e la proliferazione delle cellule staminali e progenitrici ematopoietiche (HSPCs) sono necessari per alimentare adeguatamente la maggior parte delle cellule del sangue che sono post-mitotici e viene riciclato ogni giorno. HSC generano cellule ematiche mature differenziando prima in sottoinsiemi di cellule progenitrici limitato; comuni progenitori linfoidi (CLPs)1, che alla fine producono le cellule T, B e NK e comuni dei progenitori mieloidi (CMPs)2 che generano i granulociti, eritrociti, macrofagi e megacariociti. Questi progenitori si sono impegnati a generare cellule specifiche e ulteriormente differenziano in altre cellule progenitrici inerente allo sviluppo limitato come progenitori eritroidi mieloide (MEP) che generano gli eritrociti e piastrine o granulociti progenitori del macrofago (GMP) che generano i basofili, eosinofili, neutrofili e macrofagi2. Identificare e isolare questi progenitori permette l'identificazione di importanti vie molecolari coinvolti nel differenziamento ematopoietico e molte malattie ematopoietiche come leucemia sorgono quando queste cellule progenitrici non correttamente differenziare.
Negli ultimi decenni, il sistema di modello di zebrafish (Danio rerio) è diventato uno strumento chiave della ricerca per gli studi emopoietici embrionali ed adulti. Zebrafish sono suscettibile di analisi genetica e le specie di vertebrati modello filogeneticamente più basse che hanno un simile sistema vascolare e sistema ematopoietico agli esseri umani. Embrioni di zebrafish sviluppano ex utero, ed entro 48 ore post fertilizzazione (hpf) generare HSPCs3,4,5,6,7,8. Zebrafish sono altamente fertile, anche con le femmine posa oltre 100 uova in un singolo frizione, permettendo per campioni di grandi dimensioni e replica sperimentale. Embrioni di zebrafish sono otticamente trasparenti, consentendo per visualizzazione microscopica del sistema ematopoietico. Parecchie linee transgeniche fluorescente di zebrafish marcatura HSCs come runx1: EGFP pesce9, cd41: EGFP pesce10, e kdrl:mCherry; cMyb: animali GFP3 double-positivo, consentire per la visualizzazione in tempo reale di HSC emersione ed espansione in vivo3,4,7,8, 9. Generazione veloce tempo e sviluppo ex utero di zebrafish ha condotto al relativo uso in droga e mutagenesi studi11,12,13,14,15 16,17,18,19,20 per composti che contengono promessa terapeutica per malattie del sangue umano di screening. Nel complesso, conservazione del sistema ematopoietico, la presenza e il facile sviluppo di linee transgeniche e il tempo di rigenerazione rapida ha reso zebrafish un modello economico, veloce, flessibile e ideale per studi ematopoietici.
Sono stati sviluppati numerosi metodi di isolamento e test HSCs nei sistemi mammiferi ematopoietici. Gli investigatori possono utilizzare una combinazione di recettori di superficie per Marco HSCs21,22,23,24, nonché di sfruttare la capacità di HSCs di efflusso tintura25,26. Dopo sono etichettati, fluorescenza-attivato delle cellule ordinano (FACS) consente loro separazione fisica. Dimostrare che una cella è un HSC richiede irradiando un animale ospite per distruggere HSPCs endogena, trapianto di HSC putativo e osservando la ricostituzione a lungo termine, multi-lignaggio di tutto maturo tipi di cellule del sangue. Questi test funzionano bene in topi, come ci sono numerosi cellula-superficie anticorpi contro le cellule ematopoietiche e ceppi inbred del mouse che facilitano immuni per il trapianto. Tuttavia, pochi anticorpi di superficie delle cellule ematopoietiche zebrafish sono stati generati27, ostacolando l'identificazione e l'isolamento di HSCs. Il modo più comune per contrassegnare e isolare HSCs zebrafish è con animali transgenici, per cui una sequenza di cellula-specifico promotore sta guidando la espressione di una proteina fluorescente. Studi hanno visualizzato ed enumerato HSCs nella parete ventrale dell'aorta dorsale con microscopia che utilizzano questa tecnica3,4,8,9. Altri laboratori hanno generato ceppi clonali di zebrafish28,29 e sono effettuati trapianti di successo negli animali MHC-abbinato30. Tuttavia, queste tecniche sono costi proibitivi per molti laboratori, sono tecnicamente difficili e richiedono molto tempo. Per risolvere questi problemi, laboratori hanno generato diversi saggi in vitro per verificare la presenza, i tassi di proliferazione e la capacità di differenziazione di HSPCs31,32,33, 34,35,36. Queste analisi mostrano la proliferazione e la differenziazione di HSPCs in vitro31,32,33,34,35,36, il salvataggio di ematopoietiche difetti36e un metodo efficiente per l'individuazione e sperimentazione di citochine33,34,35. Essi sono stati utilizzati anche per identificare i geni responsabili HSPC biologia31,32. In questo studio, prendiamo queste analisi un passo avanti, consentendo la quantificazione di HSPCs in un embrione di zebrafish sviluppo. Queste analisi possono essere utilizzate anche per quantificare il numero di HSPCs in animali mutanti e gli animali trattati con farmaci emopoietici dirompente. In sostanza, queste analisi sono veloce, presenti alcune sfide tecniche e sono modi economici per quantificare i numeri HSPC, esaminare la loro proliferazione e indagare blocchi nella differenziazione.

<table><tbody><tr><td>10% Bovine Serum Albumin in Iscove&#039;s MDM</td><td>StemCell Technologies&amp;nbsp;</td><td>9300</td><td></td></tr><tr><td>DPBS (10x) with Calcium&lt;sup&gt;2+&lt;/sup&gt; and Magnesium&lt;sup&gt;2+&lt;/sup&gt;</td><td>Life Technologies</td><td>14080-055</td><td></td></tr><tr><td>HyClone PBS (1x)</td><td>GE Healthcare Life Sciences</td><td>sh30256.01</td><td></td></tr><tr><td>&amp;nbsp;DMEM 500 mL</td><td>Corning CellGrow</td><td>10-017-CV</td><td></td></tr><tr><td>&amp;nbsp;Ham&#039;s F12 500 mL</td><td>Corning CellGrow</td><td>10-080-CV</td><td></td></tr><tr><td>FBS 500 mL</td><td>Gemini Bio-Products</td><td>100-108</td><td></td></tr><tr><td>HEPES 100 mL (1 M)</td><td>Gibco life technologies</td><td>15-630-080</td><td></td></tr><tr><td>Penicillin/streptomycin (5000 U/mL&lt;br/&gt; and 5000 mg/mL) with L-Glutamine (200 mM)</td><td>Corning Mediatech</td><td>30-009-CI</td><td></td></tr><tr><td>Gentamycin Sulfate 10 mL (50 mg/mL)</td><td>Corning Mediatech</td><td>30-005-CR</td><td></td></tr><tr><td>1.5 mL MCF tube</td><td>FisherBrand</td><td>05-408-129</td><td></td></tr><tr><td>3 mL 23gx1 injection needle with Luer lock</td><td>BD Safety Glide</td><td>305905</td><td></td></tr><tr><td>5 mL polystyrene round bottom tube with cell strainer cap</td><td>Corning Falcon</td><td>352235</td><td></td></tr><tr><td>Methocel MC</td><td>Sigma-Aldrich</td><td>64630</td><td></td></tr><tr><td>14 mL Polystyrene round bottom tube</td><td>Corning Falcon</td><td>352057</td><td></td></tr><tr><td>10 mm polystyrene easygrip Petri dish</td><td>Corning Falcon</td><td>351008</td><td></td></tr><tr><td>Librease TM</td><td>Roche Sigma-Aldrich</td><td>5401119001</td><td>dissociation protease</td></tr><tr><td>Pronase</td><td>Roche Sigma-Aldrich</td><td>11459643001</td><td>dechorionation protease</td></tr><tr><td>15 cm Petri dish</td><td>Corning Falcon</td><td>351058</td><td></td></tr><tr><td>AB</td><td>Zebrafish International Resource Center (ZIRC)</td><td>ZL1</td><td>zebrafish strain used</td></tr><tr><td>Dithiolthreitol (DTT)</td><td>Sigma-Aldrich</td><td>646563</td><td></td></tr></tbody></table>

development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos

L'ematopoiesi è un processo cellulare essenziale in cui cellule staminali e progenitrici ematopoietiche (HSPCs) si differenziano nella moltitudine degli stirpi differenti delle cellule che compongono il sangue maturo. Isolamento ed identificazione di questi HSPCs è difficile perché sono definiti ex post facto; possono essere definiti solo dopo la loro differenziazione in cellule specifiche. Negli ultimi decenni, il pesce zebra (Danio rerio) è diventato un organismo modello per studiare l'ematopoiesi. Embrioni di zebrafish sviluppano ex uteroe di post-fertilizzazione 48h (hpf) hanno generato definitiva HSPCs. saggi per valutare la differenziazione HSPC e sono state sviluppate capacità di proliferazione, che utilizza il trapianto e successive ricostituzione del sistema ematopoietico oltre a visualizzare linee specializzate transgenici con microscopia confocale. Tuttavia, queste analisi sono costo proibitivo, tecnicamente difficile e richiede tempo per molti laboratori. Sviluppo di un modello in vitro per valutare HSPCs sarebbe conveniente, più veloce, e presenti meno difficoltà rispetto al precedentemente descritti metodi, che permettano di laboratori valutare rapidamente le schermate di mutagenesi e droga che influiscono sulla biologia HSPC. Questo romanzo in vitro test per valutare la HSPCs viene eseguito da embrioni di zebrafish intero placcatura dissociata e aggiungendo fattori esogeni che promuovono solo HSPC differenziazione e la proliferazione. Gli embrioni sono dissociati in cellule singole e placcati con HSPC-solidale Colonia di granulociti che causano loro di generare unità formanti colonie (CFU) che derivano da una cellula progenitrice singolo. Queste analisi dovrebbero consentire un esame più attento delle vie molecolari responsabili della proliferazione, la differenziazione e il regolamento, che permetterà ai ricercatori di capire le basi della ematopoiesi vertebrato e sua disregolazione HSPC durante la malattia.

Per valutare il numero HSPC in zebrafish embrionale, 48 hpf embrioni sono stati digeriti, placcati in metilcellulosa con fattori di crescita ematopoietici-solidale esogeni e incubati per 7 giorni (Figura 1A). Dopo 7 giorni, unità formanti colonie (CFU) sono stati enumerati (Figura 1B) e immagine (Figura 1C). Controllando le diverse citochine aggiunt...

Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Qui, presentiamo un metodo semplice per quantificare le cellule ematopoietiche staminali e progenitrici (HSPCs) in zebrafish embrionale. HSPCs dallo zebrafish dissociate sono placcati in metilcellulosa con fattori solidale, differenziando in sangue maturo. Questo consente il rilevamento di sangue difetti e permette lo screening per essere facilmente condotto.

Sviluppo di un test In Vitro per quantificare progenitrici e staminali ematopoietiche cellule (HSPCs) nello sviluppo di embrioni di Zebrafish

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

development-an-vitro-assay-to-quantitate-hematopoietic-stem

Research

JoVE Journal

Developmental Biology

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

7.9K Views.  California State University, Chico. Qui, presentiamo un metodo semplice per quantificare le cellule ematopoietiche staminali e progenitrici (HSPCs) in zebrafish embrionale. HSPCs dallo zebrafish dissociate sono placcati in metilcellulosa con fattori solidale, differenziando in sangue maturo. Questo consente il rilevamento di sangue difetti e permette lo screening per essere facilmente condotto.

Video: Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

Su larga scala Zebrafish embrionali Cuore dissezione per l&#39;analisi trascrizionale

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Ricerca

Biologia dello sviluppo

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Il trapianto di midollo renale intero in Zebrafish adulti

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Biologia

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

Isolamento e la coltura di Neurosfere Zebrafish adulti Brain-derived

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

Isolamento e caratterizzazione di singole cellule da embrioni di zebrafish

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Time-lapse immagini dal vivo di relazione clonale cellule progenitrici neurali in via di sviluppo Zebrafish proencefalo

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neuroscienze

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Generazione di Parabiotic embrioni di zebrafish da fusione chirurgica di Sviluppo Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

Genotipizzazione e quantificazione della colorazione dell'ibridazione in situ nel pesce zebra

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

Utilizzo della citometria a flusso per rilevare e quantificare la formazione di sangue alterato nel pesce zebra in via di sviluppo

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

Isolamento della popolazione Side di Myc-indotta delle cellule T leucemia linfoblastica acuta in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

Immunologia e infezioni

<meta charset="utf8"></head><body>Robusto trapianto di cellule di pesce zebra per la ricerca sulla rigenerazione e lo sviluppo

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>Autore in primo piano: Mappatura della connettività cellulare nel sistema nervoso del pesce zebra durante lo sviluppo e la rigenerazione

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

Trapianto di cellule ad alta risoluzione in zebrafish embrionale e larvale

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...