Name: assay for blood-brain barrier integrity in drosophila melanogaster
Uploaded: 2019-09-18T15:00:00
Description: Watch this Scientific Journal Video about Assay for Blood-brain Barrier Integrity in Drosophila melanogaster at JoVE.com

Gli autori ringraziano il Dr. F. Bryan Pickett e il Dr. Rodney Dale per l'uso di attrezzature per l'iniezione. Questo lavoro è stato finanziato da finanziamenti per la ricerca della Loyola University di Chicago a M.D., D.T., e J.J.

1. Raccolta di campioni
<ol><li>Raccolta di embrioni
	<ol><li>In ogni gabbia di raccolta embrionale, usa un minimo di 50 femmine vergini con 20-25 maschi per le collezioni. Incubare queste mosche in una bottiglia con cibo di farina di mais-agar (Tabella dei Materiali) per 1/2 giorni prima di iniziare le collezioni23. NOT:</</c2> È possibile utilizzare più mosche, ma il rapporto femminile-maschio deve essere mantenuto a 2:1.</li>
		<li>Piat...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junctions+between+intimately+apposed+cell+membranes+in+the+vertebrate+brain.">Junctions between intimately apposed cell membranes in the vertebrate brain.</a> Journal of Cell Biology. 40 (3), 648-677 (1969).</li><li>Tietz, S., Engelhardt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+barriers:+Crosstalk+between+complex+tight+junctions+and+adherens+junctions.">Brain barriers: Crosstalk between complex tight junctions and adherens junctions.</a> Journal of Cell Biology. 209 (4), 493-506 (2015).</li><li>Hindle, S. J., Bainton, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrier+mechanisms+in+the+Drosophila+blood-brain+barrier.">Barrier mechanisms in the Drosophila blood-brain barrier.</a> Frontiers in Neuroscience. 8, 414 (2014).</li><li>Schwabe, T., Bainton, R. J., Fetter, R. D., Heberlein, U., Gaul, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPCR+signaling+is+required+for+blood-brain+barrier+formation+in+drosophila.">GPCR signaling is required for blood-brain barrier formation in drosophila.</a> Cell. 123 (1), 133-144 (2005).</li><li>Stork, T., 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=Organization+and+function+of+the+blood-brain+barrier+in+Drosophila.">Organization and function of the blood-brain barrier in Drosophila.</a> Journal of Neuroscience. 28 (3), 587-597 (2008).</li><li>Unhavaithaya, Y., Orr-Weaver, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyploidization+of+glia+in+neural+development+links+tissue+growth+to+blood-brain+barrier+integrity.">Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity.</a> Genes &amp; Development. 26 (1), 31-36 (2012).</li><li>Schwabe, T., Li, X., Gaul, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+analysis+of+the+mesenchymal-epithelial+transition+of+blood-brain+barrier+forming+glia+in+Drosophila.">Dynamic analysis of the mesenchymal-epithelial transition of blood-brain barrier forming glia in Drosophila.</a> Biology Open. 6 (2), 232-243 (2017).</li><li>Von Stetina, J. R., Frawley, L. E., Unhavaithaya, Y., Orr-Weaver, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+cell+cycles+regulated+by+Notch+signaling+control+cell+size+and+ensure+a+functional+blood-brain+barrier.">Variant cell cycles regulated by Notch signaling control cell size and ensure a functional blood-brain barrier.</a> Development. 145 (3), dev157115 (2018).</li><li>von Hilchen, C. M., Beckervordersandforth, R. M., Rickert, C., Technau, G. M., Altenhein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identity,+origin,+and+migration+of+peripheral+glial+cells+in+the+Drosophila+embryo.">Identity, origin, and migration of peripheral glial cells in the Drosophila embryo.</a> Mechanisms of Development. 125 (3-4), 337-352 (2008).</li><li>Beckervordersandforth, R. M., Rickert, C., Altenhein, B., Technau, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subtypes+of+glial+cells+in+the+Drosophila+embryonic+ventral+nerve+cord+as+related+to+lineage+and+gene+expression.">Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression.</a> Mechanisms of Development. 125 (5-6), 542-557 (2008).</li><li>Bellen, H. J., Lu, Y., Beckstead, R., Bhat, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurexin+IV,+caspr+and+paranodin--novel+members+of+the+neurexin+family:+encounters+of+axons+and+glia.">Neurexin IV, caspr and paranodin--novel members of the neurexin family: encounters of axons and glia.</a> Trends in Neurosciences. 21 (10), 444-449 (1998).</li><li>Baumgartner, 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=A+Drosophila+neurexin+is+required+for+septate+junction+and+blood-nerve+barrier+formation+and+function.">A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function.</a> Cell. 87 (6), 1059-1068 (1996).</li><li>Banerjee, S., Pillai, A. M., Paik, R., Li, J., Bhat, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+ensheathment+and+septate+junction+formation+in+the+peripheral+nervous+system+of+Drosophila.">Axonal ensheathment and septate junction formation in the peripheral nervous system of Drosophila.</a> Journal of Neuroscience. 26 (12), 3319-3329 (2006).</li><li>Bhat, 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=Axon-glia+interactions+and+the+domain+organization+of+myelinated+axons+requires+neurexin+IV/Caspr/Paranodin.">Axon-glia interactions and the domain organization of myelinated axons requires neurexin IV/Caspr/Paranodin.</a> Neuron. 30 (2), 369-383 (2001).</li><li>Faivre-Sarrailh, 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=Drosophila+contactin,+a+homolog+of+vertebrate+contactin,+is+required+for+septate+junction+organization+and+paracellular+barrier+function.">Drosophila contactin, a homolog of vertebrate contactin, is required for septate junction organization and paracellular barrier function.</a> Development. 131 (20), 4931-4942 (2004).</li><li>Salzer, J. L., Brophy, P. J., Peles, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+domains+of+myelinated+axons+in+the+peripheral+nervous+system.">Molecular domains of myelinated axons in the peripheral nervous system.</a> Glia. 56 (14), 1532-1540 (2008).</li><li>von Hilchen, C. M., Bustos, A. E., Giangrande, A., Technau, G. M., Altenhein, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predetermined+embryonic+glial+cells+form+the+distinct+glial+sheaths+of+the+Drosophila+peripheral+nervous+system.">Predetermined embryonic glial cells form the distinct glial sheaths of the Drosophila peripheral nervous system.</a> Development. 140 (17), 3657-3668 (2013).</li><li>Matzat, T., 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=Axonal+wrapping+in+the+Drosophila+PNS+is+controlled+by+glia-derived+neuregulin+homolog+Vein.">Axonal wrapping in the Drosophila PNS is controlled by glia-derived neuregulin homolog Vein.</a> Development. 142 (7), 1336-1345 (2015).</li><li>Limmer, S., Weiler, A., Volkenhoff, A., Babatz, F., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+blood-brain+barrier:+development+and+function+of+a+glial+endothelium.">The Drosophila blood-brain barrier: development and function of a glial endothelium.</a> Frontiers in Neuroscience. 8, 365 (2014).</li><li>Ho, T. 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=Expressional+Profiling+of+Carpet+Glia+in+the+Developing+Drosophila+Eye+Reveals+Its+Molecular+Signature+of+Morphology+Regulators.">Expressional Profiling of Carpet Glia in the Developing Drosophila Eye Reveals Its Molecular Signature of Morphology Regulators.</a> Frontiers in Neuroscience. 13, 244 (2019).</li><li>DeSalvo, M. K., 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+Drosophila+surface+glia+transcriptome:+evolutionary+conserved+blood-brain+barrier+processes.">The Drosophila surface glia transcriptome: evolutionary conserved blood-brain barrier processes.</a> Frontiers in Neuroscience. 8, 346 (2014).</li><li>. BDSC Cornmeal Food Available from: <a target="_blank" href="https://bdsc.indiana.edu/information/recipes/bloomfood.html">https://bdsc.indiana.edu/information/recipes/bloomfood.html</a> (2017)</li><li>Le, T., 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+new+family+of+Drosophila+balancer+chromosomes+with+a+w-+dfd-GMR+yellow+fluorescent+protein+marker.">A new family of Drosophila balancer chromosomes with a w- dfd-GMR yellow fluorescent protein marker.</a> Genetics. 174 (4), 2255-2257 (2006).</li><li>Casso, D., Ramirez-Weber, F. A., Kornberg, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP-tagged+balancer+chromosomes+for+Drosophila+melanogaster.">GFP-tagged balancer chromosomes for Drosophila melanogaster.</a> Mechanisms of Development. 88 (2), 229-232 (1999).</li><li>Halfon, M. 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=New+fluorescent+protein+reporters+for+use+with+the+Drosophila+Gal4+expression+system+and+for+vital+detection+of+balancer+chromosomes.">New fluorescent protein reporters for use with the Drosophila Gal4 expression system and for vital detection of balancer chromosomes.</a> Genesis. 34 (1-2), 135-138 (2002).</li><li>Miller, D. F., Holtzman, S. L., Kaufman, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Customized+microinjection+glass+capillary+needles+for+P-element+transformations+in+Drosophila+melanogaster.">Customized microinjection glass capillary needles for P-element transformations in Drosophila melanogaster.</a> BioTechniques. 33 (2), 366-372 (2002).</li><li>Luong, D., Perez, L., Jemc, J. C. <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+raw+as+a+regulator+of+glial+development.">Identification of raw as a regulator of glial development.</a> PLoS One. 13 (5), e0198161 (2018).</li><li>Pinsonneault, R. L., Mayer, N., Mayer, F., Tegegn, N., Bainton, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+models+for+studying+the+blood-brain+and+blood-eye+barriers+in+Drosophila.">Novel models for studying the blood-brain and blood-eye barriers in Drosophila.</a> Methods in Molecular Biology. 686, 357-369 (2011).</li><li>Love, C. R., Dauwalder, B., Barichello, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+Model+to+Study+the+Blood-Brain+Barrier.">Drosophila as a Model to Study the Blood-Brain Barrier.</a> Blood-Brain Barrier. , 175-185 (2019).</li><li>Lin, D. M., Goodman, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+and+increased+expression+of+Fasciclin+II+alters+motoneuron+growth+cone+guidance.">Ectopic and increased expression of Fasciclin II alters motoneuron growth cone guidance.</a> Neuron. 13 (3), 507-523 (1994).</li><li>Sepp, K. J., Schulte, J., Auld, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+glia+direct+axon+guidance+across+the+CNS/PNS+transition+zone.">Peripheral glia direct axon guidance across the CNS/PNS transition zone.</a> Developmental Biology. 238 (1), 47-63 (2001).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li><li>Devraj, K., Guerit, S., Macas, J., Reiss, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vivo+Blood-brain+Barrier+Permeability+Assay+in+Mice+Using+Fluorescently+Labeled+Tracers.">An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers.</a> Journal of Visualized Experiments. 132, e57038 (2018).</li><li>Fairchild, M. J., Smendziuk, C. 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Questo protocollo fornisce una descrizione completa dei passi necessari per testare l'integrità di BBB durante gli stadi larvali tardoembrionici e terzi instar dello sviluppo di D. melanogaster. Approcci simili sono stati descritti altrove per testare l'integrità della BBB durante lo sviluppo, così come nelle fasi adulte5,7,29,30. Tuttavia, le descrizioni delle procedure nelle sezio...

Durante lo sviluppo, la comunicazione cellulare-cellula e le interazioni sono fondamentali per la creazione della struttura e della funzione dei tessuti e degli organi. In alcuni casi, queste interazioni cellula-cellula sigillano gli organi dall'ambiente circostante per garantire la corretta funzione dell'organo. Questo è il caso del sistema nervoso, che è isolato dalla barriera emato-encefalica (BBB). La disfunzione della BBB nell'uomo è stata collegata a disturbi neurologici tra cui l'epilessia, e la rottura della barriera è stata osservata nelle malattie neurodegenerative tra cui la sclerosi multipla e la sclerosi laterale amiotrofica1. Nei mammiferi, la BBB è formata da strette giunzioni tra le cellule endoteliali2,3. Altri animali, tra cui la mosca della frutta, Drosophila melanogaster, hanno un BBB composto da cellule gliali. Queste cellule gliali formano una barriera selettivamente permeabile per controllare il movimento di sostanze nutritive, prodotti di scarto, tossine e grandi molecole dentro e fuori il sistema nervoso4. Ciò consente la manutenzione del gradiente elettrochimico necessario per sparare potenziali di azione, consentendo la mobilità e la coordinazione4. In D. melanogaster, la glia proteggere il sistema nervoso dal potassio-ricco, emolymph sangue-come5.
Nel sistema nervoso centrale (CNS) e nel sistema nervoso periferico (PNS) di D. melanogaster, due strati gliali esterni, la glia subperineuriale e la glia perineuriale, così come una rete esterna di matrice extracellulare, la lamella neurale, formano barriera emolymph-brain e emolymph-nerve6, indicata come BBB in tutto questo articolo. Durante lo sviluppo glia subperineurial diventano poliploidi e ingrandiscono per circondare il sistema nervoso5,6,7,8,9,10,11 . La glia subperineuriale forma giunzioni di septate, che forniscono la barriera di diffusione principale tra l'emolina e il sistema nervoso5,6,12. Queste giunzioni sono molecolarmente simili alle giunzioni simili a septate che si trovano nei paranodi della glia mielinata nei vertebrati, e svolgono la stessa funzione delle giunzioni strette nella BBB dei mammiferi13,14, 15 Mi lasa del sistema , 16 , 17.La glia perineuriale si divide, cresce e avvolge la glia subperineuriale per regolare la diffusione di metaboliti e grandi molecole6,10,18,19. La formazione di BBB è completata da 18,5 h dopola deposizione delle uova (AEL) a 25 . Studi precedenti hanno identificato geni che sono regolatori critici della formazione di BBB20,21,22. Per comprendere meglio i ruoli esatti di questi geni, è importante esaminare l'effetto della mutazione di questi potenziali regolatori sull'integrità BBB. Mentre studi precedenti hanno delineato approcci per affermare l'integrità della BBB negli embrioni e nelle larve, un protocollo completo per questo test deve ancora essere descritto5,7. Questo protocollo passo-passo descrive i metodi per il test di integrità BBB durante gli stadi della larva embrionale e terza nella stella di D. melanogaster.

<table>
	<tbody>
		<tr>
			<td>10 kDa sulforhodamine 101 acid chloride (Texas Red) Dextran</td>
			<td>ThermoFisher Scientific</td>
			<td>D1863</td>
			<td>Dextran should be diluted in autoclaved ddH2O to a concentration of 25 mg/mL.</td>
		</tr>
		<tr>
			<td>20 &#956;L Gel-Loading Pipette Tips</td>
			<td>Eppendorf</td>
			<td>22351656</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol (200 proof)</td>
			<td>Pharmco-Aaper</td>
			<td>11000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Active Dry Yeast</td>
			<td>Red Star</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1423</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Air Compressor</td>
			<td>DeWalt</td>
			<td>D55140</td>
			<td></td>
		</tr>
		<tr>
			<td>Apple Juice</td>
			<td>Mott&#39;s Natural Apple Juice</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Household Bleach</td>
			<td></td>
			<td>1-5% Hypochlorite</td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle Plugs</td>
			<td>Fisher Scientific</td>
			<td>AS-277</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td>Samples imaged using 20x objective (UPlanSApo 20x/ 0.75)</td>
		</tr>
		<tr>
			<td>Cotton-Tipped Applicator</td>
			<td>Puritan</td>
			<td>19-062614</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-Sided Tape 1/2&#34;</td>
			<td>Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers; Pattern #5; .05 X .01mm Tip</td>
			<td>Roboz</td>
			<td>RS-5015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly Food Bottles</td>
			<td>Fisher Scientific</td>
			<td>AS-355</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly Food Vials</td>
			<td>Fisher Scientific</td>
			<td>AS-515</td>
			<td></td>
		</tr>
		<tr>
			<td>Foot Pedal</td>
			<td>Treadlite II</td>
			<td>T-91-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Caster</td>
			<td>Bio-Rad</td>
			<td>1704422</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Tray</td>
			<td>Bio-Rad</td>
			<td>1704436</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pipette</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Granulated sugar</td>
			<td></td>
			<td></td>
			<td>Purchased from grocery store.</td>
		</tr>
		<tr>
			<td>Halocarbon Oil</td>
			<td>Lab Scientific, Inc.</td>
			<td>FLY-7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Source</td>
			<td>Schott</td>
			<td>Ace I</td>
			<td></td>
		</tr>
		<tr>
			<td>Manipulator Stand</td>
			<td>World Precision Instruments</td>
			<td>M10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>KITE-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>World Precision Instruments</td>
			<td>MPH310</td>
			<td></td>
		</tr>
		<tr>
			<td>Nightsea Filter Sets</td>
			<td>Electron Microscopy Science</td>
			<td>SFA-LFS-CY</td>
			<td>For visualization of YFP</td>
		</tr>
		<tr>
			<td>Nightsea Full Adapter System w/ Royal Blue Color Light Head</td>
			<td>Electron Microscopy Science</td>
			<td>SFA-RB</td>
			<td>For visualization of GFP</td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td>Simply Simmons</td>
			<td>Chisel Blender #6</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetter</td>
			<td>Fisher Scientific</td>
			<td>13-683C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic Pump</td>
			<td>World Precision Instruments</td>
			<td>PV830</td>
			<td>This is also referred to as a microinjector or pressure regulator. Since the model used in our study is no longer available this is one alternative.</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP363-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Embryo Collection Cages</td>
			<td>Genesee Scientific</td>
			<td>59-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic Anhydrous</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel Base Plate</td>
			<td>World Precision Instruments</td>
			<td>5052</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000</td>
			<td>Used for tissue dissection.</td>
		</tr>
		<tr>
			<td>Stereomicroscope with transmitted light source</td>
			<td>Baytronix</td>
			<td></td>
			<td>Used for injection.</td>
		</tr>
		<tr>
			<td>Tegosept (p-hydroxybenzoic acid, methyl ester)</td>
			<td>Genesee Scientific</td>
			<td>20-258</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td>Nonionic surfactant</td>
		</tr>
		<tr>
			<td>Vial Plugs</td>
			<td>Fisher Scientific</td>
			<td>AS-273</td>
			<td></td>
		</tr>
	</tbody>
</table>

assay for blood-brain barrier integrity in drosophila melanogaster

Un corretto sviluppo del sistema nervoso include la formazione della barriera emato-encefalica, la barriera di diffusione che regola strettamente l'accesso al sistema nervoso e protegge il tessuto neurale dalle tossine e dagli agenti patogeni. I difetti nella formazione di questa barriera sono stati correlati con le neuropatie, e la ripartizione di questa barriera è stata osservata in molte malattie neurodegenerative. Pertanto, è fondamentale identificare i geni che regolano la formazione e il mantenimento della barriera emato-encefalica per identificare potenziali bersagli terapeutici. Per comprendere i ruoli esatti che questi geni svolgono nello sviluppo neurale, è necessario testare gli effetti dell'espressione genica alterata sull'integrità della barriera emato-encefalica. Molte delle molecole che funzionano nella creazione della barriera emato-encefalica sono state trovate per essere conservate attraverso le specie eucariotiche, tra cui la mosca della frutta, Drosophila melanogaster. I moscerini della frutta hanno dimostrato di essere un ottimo sistema modello per esaminare i meccanismi molecolari che regolano lo sviluppo e la funzione del sistema nervoso. Questo protocollo descrive una procedura passo-passo per testare l'integrità della barriera emato-encefalica durante le fasi embrionali e larve dello sviluppo di D. melanogaster.

I metodi qui descritti consentono la visualizzazione dell'integrità della BBB in tutto il CNS in embrioni e larve di D. melanogaster (Figura 1). Al termine della formazione di BBB nella tarda embriogenesi, il BBB funziona per escludere grandi molecole dal cervello e VNC5. Questo protocollo sfrutta questa funzione per affermare la formazione di BBB. Quando furono iniettati embrioni di tipo selvaggio (Oregon R) in fase avanzata 17 (20-21 h- ) con 10 kDa dextra...

Watch this Scientific Journal Video about Assay for Blood-brain Barrier Integrity in Drosophila melanogaster at JoVE.com

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

L'integrità della barriera emato-encefalica è fondamentale per la funzione del sistema nervoso. Nella Drosophila melanogaster, la barriera emato-encefalica è formata da cellule gliali durante l'embriogenesi tardiva. Questo protocollo descrive i metodi per la formazione e la manutenzione delle barriere emato-encefalice negli embrioni di D. melanogaster e delle larve instar terze.

Analisi per l'integrità della barriera del cervello edelico nella Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

Questo metodo può aiutare a rispondere a domande chiave riguardanti la regolazione genetica della formazione e il mantenimento della barriera ematica-encefalica durante più fasi dello sviluppo della drosofila. Questo metodo può anche essere adattato per esaminare la permeabilità di altre barriere, come l'epitelio intestinale in una varietà di organismi. Il principale vantaggio di questa tecnica è che consente la valutazione della funzione di barriera eto-encefalica nei tessuti vivi.La dimostrazione visiva di questo metodo è fondamentale, in quanto le manipolazioni di esempio possono essere difficili e molti passaggi richiedono molta attenzione ai dettagli. Per la raccolta degli embrioni posizionare un minimo di 50 femmine vergini con da 20 a 25 maschi per bottiglia con cibo Corn Meal Agar e incubare queste mosche per uno o due giorni prima di iniziare le collezioni. Il giorno prima della raccolta, scaldare i piatti di agar di succo di mela a 25 gradi Celsius durante la notte.La mattina seguente trasferire le mosche anestetizzate di anidride carbonica in una gabbia di raccolta e posizionare un piatto di agar di succo di mela prerifavorato con un piccolo striscio di pasta di lievito all'estremità aperta della gabbia. Quindi fissare il piatto alla gabbia con la manica rossa. Per cancellare gli embrioni più vecchi, consenti alle mosche di deporre le uova su un piatto di agar di succo di mela per un'ora a 25 gradi Celsius.Alla fine dell'incubazione, invertire il lato della rete della gabbia verso il basso e toccare le mosche sul fondo della gabbia. Sostituire l'agar succo di mela con un nuovo piatto di agar di succo di mela prerifamato con un piccolo striscio di pasta di lievito. Consenti alle mosche di deporre le uova sul nuovo piatto per un'altra ora a 25 gradi Celsius.Per raccogliere 17 embrioni in fase avanzata per iniezione, sostituire la piastra di agar del succo di mela con un nuovo piatto pre-riscaldato con uno striscio di pasta di lievito. E consentire alle mosche di deporre le uova sul piatto a 25 gradi Celsius. Dopo un'ora, sostituire la piastra e invecchiare la piastra con embrioni raccolti per 19 ore a 25 gradi Celsius in modo che gli embrioni avranno dai 20 ai 21 ore al momento dell'imaging.Al termine dell'incubazione di 19 ore, rimuovere la piastra di raccolta dell'embrione dall'incubatrice e coprire la superficie della piastra con PbTx. Utilizzare un pennello per allentare gli embrioni dalla superficie della piastra in un filtro a rete di nylon da 70 micrometri. E mettere il colino con embrioni in una soluzione di candeggina al 50% in una piastra di Petri di 100 millimetri per cinque minuti con agitazione occasionale a temperatura ambiente per dechorionate.Al termine dell'incubazione, sciacquare gli embrioni tre volte ruotando il colino in PbTx fresco, utilizzando una nuova piastra di Petri per ogni lavaggio. Lavare gli embrioni su un lato del colino con PbTx e utilizzare una pipetta di vetro per trasferire gli embrioni su una lastra di gel di agarosio al 2%. Rimuovere il liquido in eccesso con carta da filtro.Allineare da sei a otto embrioni sulla lastra con i posteriori a destra e i lati dorsali rivolti verso l'alto e premere saldamente un pezzo di nastro bipendo apposto su una diapositiva sopra gli embrioni. Quindi essiccare gli embrioni a temperatura ambiente per circa 25 minuti prima di coprire i campioni con olio di alocarbonio. Per la raccolta larvale, impostare una croce con da 5 a 10 mosche femminili vergini del genotipo desiderato e la metà dei maschi del genotipo desiderato in una fiala con cibo Cornmeal Agar.Dopo cinque o sette giorni a 25 gradi Celsius, a seconda del genotipo, utilizzare le forcep per raccogliere delicatamente le terze larve instar errante dal flaconcino e risciacquare le larve con PBS per rimuovere qualsiasi cibo bloccato. Trasferire le larve su un piatto di agar succo di mela per genotipizzare se necessario prima di utilizzare un pennello per asciugare le larve su un tessuto. Quindi utilizzare le forcep per trasferire da sei a otto larve in una diapositiva preparata con nastro a doppia fronte.Prima dell'iniezione, utilizzare un estrattore di micropipette per tirare i tubi capillari in una forma ad ago standard per le iniezioni di D.Melanogaster e ancorare gli aghi in argilla in una piastra di Petri. Per l'iniezione embrionale, utilizzare una punta di pipetta a caricamento in gel da 20 microlitri per caricare un ago preparato con 5 microlitri di 10 kilodalton dextran coniugati in cloruro acido sulforhodamina 101 e caricare l'ago in un portaaghi. Posizionare l'ago in un micromanipolatore fissato a una base di acciaio e impostare l'apparato di iniezione a 50 libbre per pollice quadrato e da 5 a 10 millisecondi con un intervallo di 10.Posizionare lo scivolo sullo stadio del micromanipolatore e spazzolare il bordo dell'ago contro il bordo del nastro a doppia fronte con un angolo di 45 gradi per creare una punta leggermente angolata rotta quanto basta per consentire il flusso del dextran da 10 kilodalton attraverso l'apertura. Pompare il pedale fino a quando il colorante non è sulla punta dell'ago. Allineare l'ago in modo che sia parallelo all'embrione e forare l'estremità posteriore del campione.Pompare il pedale per iniettare due nanolitri di colorante nel campione. Si noti il tempo di iniezione a scopo di incubazione e continuare lungo lo scivolo per iniettare campioni aggiuntivi. Per l'iniezione larvale, fare un'apertura angolata leggermente più grande di quella utilizzata per iniettare embrioni e inclinare leggermente l'ago verso il basso verso il campione prima di iniettare le larve con 220 nanolitri simili a quelli dimostrati per i campioni embrionali.Al termine dell'incubazione a iniezione di 10 minuti, utilizzare un applicatore con punta di cotone per applicare vaselina sul lato destro e sinistro dei campioni sullo scivolo come distanziale. Posizionare lo slittamento del coperchio sopra e posizionare lo scivolo sullo stadio del microscopio confocale. Seleziona l'obiettivo 20X e immagini i campioni per tutta la profondità di ogni embrione.Quindi calcola la percentuale di campioni con una barriera emato-encefalica compromessa secondo la formula. Prima di iniziare la procedura di dissezione, utilizzare lo smalto per montare due scivolamenti di copertura distanziati di circa 0,5 centimetri su uno scivolo e posizionare una larva iniettata in PBS sullo scivolo alla fine dell'incubazione di 30 minuti. Usando un paio di forcep, afferrare la larva a metà del corpo larvale e utilizzare una seconda coppia di forcep per separare le metà anteriore e posteriore della larva.Successivamente, utilizzare un paio di pinze per afferrare la regione anteriore ai ganci della bocca e utilizzare la seconda coppia di pinze per invertire la parete del corpo sulla punta della prima coppia di pinze. Il cervello e il cordone nervoso ventrale saranno esposti. Separare il cervello e il cordone nervoso ventrale dalla parete del corpo tagliando i nervi se necessario e rimuovere la parete corporea dallo scivolo.Coprire il campione con 10 microlitri di glicerolo all'80%. Quindi posizionare un foglietto di copertura sopra il campione per l'imaging e immaginare i campioni per determinare la percentuale di campioni con una barriera emato-encefalica compromessa come dimostrato. Quando gli embrioni di tipo selvatico in fase avanzata 17 vengono iniettati con 10 kilodalton dextran coniugati in colorante fluorescente al cloruro acido solforato 101, la grande molecola di dextran è esclusa dal cordone nervoso ventrale, come previsto.Eterozigoti embrioni mutanti grezzi 1 mostrano una barriera ematico-encefalica intatta, simile a quella osservata negli embrioni di controllo del tipo selvatico. Al contrario, gli embrioni mutanti grezzi omozigoti 1 presentano difetti nell'integrità della barriera ematico-encefalica, con il dextran di 10 kilodalton che si inonda nel cordone nervoso ventrale, indicando un fallimento della barriera ematico-encefalica per formarsi. Nei campioni larvali di controllo del tipo selvaggio, 10 kilodalton dextran non riescono a penetrare nella barriera emato-encefalica ed è escluso dal cervello e dal cordone nervoso ventrale.Quando si tenta questa procedura, un corretto invecchiamento degli embrioni è fondamentale per garantire che i campioni siano esaminati ad un'età in cui si prevede che la formazione di barriere ematiche-encefalica sia completa. Seguendo questa procedura, i mutanti che presentano una barriera emato-encefalica compromessa possono essere ulteriormente studiati usando genetica, immunoistochimica e microscopia per comprendere meglio la funzione genica a livello cellulare. Questa tecnica consentirà ai ricercatori di identificare geni aggiuntivi che sono fondamentali per la creazione e il mantenimento della barriera ematico-encefalica.

assay-for-blood-brain-barrier-integrity-in-drosophila-melanogaster

Research

JoVE Journal

Developmental Biology

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

Saggio popolazioni cellulari del sangue<em&gt; Drosophila melanogaster</em&gt; Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Ricerca

Biologia dello sviluppo

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Analisi delle larve Zebrafish muscolo scheletrico Integrità con Blu di Evans Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

Allevamento la mosca della frutta<em&gt; Drosophila melanogaster</em&gt; Sotto axeniche e Gnotobiotic Condizioni

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

Protocolli per la visualizzazione steroidogenica organi e dei loro organi interattivi con Immunocolorazione nella mosca della frutta<em&gt; Drosophila melanogaster</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissezione e macchiatura immunofluorescente del corpo di fungo e del fotoricettore neuroni nel cervello adulto Drosophila melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Un metodo In Vivo per studiare l'integrità della barriera ematotesticolare di Mouse

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

Metodi per testare la disgregazione endocrina nella Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

Generazione di un chip di barriera emato-encefalica basata sull'iPSC umano

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

Imaging di animali interi di Drosophila melanogaster utilizzando la tomografia microincisa

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Imaging di espianti cerebrali vivi da larve di Drosophila melanogaster terzo stadio

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

Autore in primo piano: Indagine sulle dinamiche di divisione cellulare asimmetrica: un protocollo per l'imaging dal vivo di espianti di cervello larvale di Drosophila 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

Imaging delle cellule vive di Drosophila melanogaster Terzo stadio Larvale Cervelli

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...