Questo lavoro è stato finanziato da un BBSRC concedere a parlamentare europeo (BB / H010297 / 1), una quota borsa di studio BBSRC a SAR, e una borsa di studio MRC in SWF.

<ol>
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D., Moore, W. M., Guiral, E. C., Holme, A., Turnbull, J. E., Pownall, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+regulation+of+developmental+cell+signaling+by+XtSulf1.">Extracellular regulation of developmental cell signaling by XtSulf1.</a> Developmental Biology. 320 (2), 436-445 (2008).</li><li>Tao, Q., 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=Maternal+wnt11+activates+the+canonical+wnt+signaling+pathway+required+for+axis+formation+in+Xenopus+embryos.">Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos.</a> Cell. 120 (6), 857-871 (2005).</li><li>Chamberlain, C. E., Jeong, J., Guo, C., Allen, B. L., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notochord-derived+Shh+concentrates+in+close+association+with+the+apically+positioned+basal+body+in+neural+target+cells+and+forms+a+dynamic+gradient+during+neural+patterning.">Notochord-derived Shh concentrates in close association with the apically positioned basal body in neural target cells and forms a dynamic gradient during neural patterning.</a> Development. 135 (6), 1097-1106 (2008).</li><li>Ramsbottom, S. A., Maguire, R. J., Fellgett, S. W., Pownall, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulf1+influences+the+Shh+morphogen+gradient+during+the+dorsal+ventral+patterning+of+the+neural+tube+in+Xenopus+tropicalis.">Sulf1 influences the Shh morphogen gradient during the dorsal ventral patterning of the neural tube in Xenopus tropicalis.</a> Developmental Biology. 391 (2), 207-218 (2014).</li><li>Fellgett, S. W., Maguire, R. J., Pownall, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulf1+has+ligand+dependent+effects+on+canonical+and+non-canonical+WNT+signalling.">Sulf1 has ligand dependent effects on canonical and non-canonical WNT signalling.</a> Journal of Cell Science. 128 (7), 1408-1421 (2015).</li><li>Dytham, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Choosing and using statistics : A biologist's guide. , (2005).</li><li>Fay, M. P., Proschan, 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=Wilcoxon-Mann-Whitney+or+t-test?+On+assumptions+for+hypothesis+tests+and+multiple+interpretations+of+decision+rules.">Wilcoxon-Mann-Whitney or t-test? 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The authors have no competing financial interests.

Una parte essenziale di questo protocollo genera ligandi biologicamente attivi che vengono normalmente trattati, secreti, e in grado di suscitare una risposta nella cellula ricevente, pur avendo una porzione fluorescente allegata. E &#39;fondamentale per stabilire che il prodotto del gene fluorescente-tag è biologicamente attivo utilizzando un dosaggio adeguato. Per Shh-GFP, la capacità di attivare l&#39;espressione di PTC1 stato confermato 16. Wnt8a ha notevolmente potente capacità di indurre un ...

Durante lo sviluppo embrionale precoce, le cellule sono progressivamente impegnati a seguire linee specifiche di differenziazione: questo significa un gruppo di totipotente (o pluripotenti) cellule diventano gradualmente limitarsi a verificare popolazioni di cellule progenitrici decisi a dare origine a un tipo di cellula. Segnalazione cellula-cellula è centrale per la regolamentazione specifica stirpe durante lo sviluppo embrionale. La manipolazione di questi segnali dovrà dirigere le cellule staminali verso particolari sorti per sostenere cure mediche innovative. Un numero relativamente piccolo di vie di segnalazione sono ribadito durante lo sviluppo, tra cui percorsi di rispondere alla superfamiglia TGF (nodals e BMP) 1-2, FGFs 3, 4 Wnt, e Hedgehogs 5. Queste proteine ​​secrete legano recettori presenti sulla membrana cellulare per attivare trasduzione del segnale modificando così l&#39;espressione genica e / o il comportamento cellulare. La stretta regolazione di segno cellulareAlling è essenziale per la specifica linea cellulare e lo sviluppo normale. Mentre il cross-talk tra questi percorsi è importante per determinare il destino delle cellule, un singolo ligando può sé suscitare risposte diverse a diverse concentrazioni. Gradienti morfogeno sono stati descritti più di 100 anni fa come una teoria per spiegare come i diversi tipi di cellule possono derivare da un campo di celle 6. Segnalazione molecole prodotte da un gruppo di cellule si diffonda in un determinato intervallo, diminuendo in concentrazione con una maggiore distanza dalla sorgente. Cellule esposte al segnale risponderà alla concentrazione locale nella loro posizione nel campo delle celle, con le cellule nelle posizioni distinte risposta diverso a diversi livelli di segnale. La prova dell&#39;esistenza di morphogens deriva da studi dei primi Drosophila embrione 7 e il disco dell&#39;ala 8, così come l&#39;arto vertebrato 9 e 10 del tubo neurale. I metodi sono necessari alvestigate come gradienti morfogeno sono stabilite e di individuare altre molecole importanti nella regolazione questi gradienti. Eleganti esperimenti che utilizzano immunoistochimica di visualizzare le proteine ​​endogene in vivo nel contesto di differenti background genetici sono stati utilizzati per indagare gradienti morphogen 11-12. Tuttavia, i buoni anticorpi e mutanti specifici non sono sempre disponibili, così abbiamo descritto qui un protocollo con sovraespressione di ligandi fluorescenti Xenopus, per fornire un&#39;alternativa, metodo semplice per analizzare come i prodotti genici esogeni possono influenzare la distribuzione di ligandi attraverso un campo di celle. Xenopus laevis fornisce un ottimo sistema di intraprendere questo tipo di esperimenti come i loro embrioni si sviluppano esternamente in modo che siano accessibili nelle prime fasi. Loro grandi dimensioni (1-1.5mm di diametro) semplifica microiniezione e manipolazione chirurgica e blastula stadi cellule sono facili da immagine in quanto sono ancora relativamente grande (circa 2081; m di diametro). Studi iperespressione di Xenopus sono semplici da fare: mRNA iniettate nel embrione precoce possono essere mirati a particolari cellule ed è efficace tradotti. I costrutti Wnt8a / Wnt11b-HA-eGFP fluorescente con tag sono stati generati utilizzando pCS2 Wnt8a-HA 13, pCS2 Wnt11b-HA 14 e eGFP. Il peptide HA è importante includere non solo per fornire un ulteriore indicatore molecolare, ma anche perché è pensato per fungere da distanziatore che separa le proteine ​​Wnt e eGFP permettendo entrambi i prodotti genici di funzionare. Il costrutto utilizzato per la visualizzazione di Shh è stato precedentemente utilizzato per generare un topo transgenico che esprime una proteina di fusione shh-eGFP 15; questo è stato gentilmente fornito da Andy McMahon. È importante sottolineare che il tag GFP per tutti i costrutti è clonato 3 &#39;alla sequenza del segnale in modo tale che viene mantenuta dopo l&#39;elaborazione. È anche essenziale per garantire che la proteina finale comprende sequenze necessarie modifiche, come la addition di lipidi, come è il caso di Shh e Wnt ligands.The cDNAs stati subclonato nel vettore pCS2 + di espressione che è ottimizzato per il prodution di mRNA sintetico; esso include un promotore SP6 e il segnale di poliadenilazione (http://sitemaker.umich.edu/dlturner.vectors). Il lavoro qui descritto fornisce un protocollo semplice per confrontare la secrezione e la diffusione di fluorescente tag Wnt e Shh contrassegnati leganti. Iniettando quantità definite di mRNA sintetico, il protocollo circumnaviga eventuali problemi connessi con l&#39;espressione variabile da vettori diversi utilizzando diversi promotori. Questi metodi sono stati recentemente applicato per studiare gli effetti del solfato endosulfatase eparan Sulf1 su Shh-eGFP e Wnt8a / secrezione Wnt11b-HA-eGFP e diffusione in Xenopus 16-17.

<table>
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Melford</td>
			<td>MB 1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>Ambion</td>
			<td>From Megascript SP6 Kit AM 1330</td>
			<td></td>
		</tr>
		<tr>
			<td>Bicarbonate</td>
			<td>VWR International</td>
			<td>RC-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium nitrate</td>
			<td>Sigma</td>
			<td>C13961</td>
			<td></td>
		</tr>
		<tr>
			<td>Cap analog (m7G(5&#39;))</td>
			<td>Applied Biosystems</td>
			<td>AM 8050</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C 2432</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;L-Cysteine hydrochloride monohydrate</td>
			<td>Sigma</td>
			<td>C7880</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Invitrogen</td>
			<td>18427-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>O3690</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR International</td>
			<td>20821.33</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Sigma</td>
			<td>F 4375</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji image J software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Free download http://fiji.sc/Fiji</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Melford</td>
			<td>G 0124</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Scientific</td>
			<td>A/0400/PB17</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cover slips, No.1.5&#160;</td>
			<td>Scientific Laboratory Supplies</td>
			<td>22X22-SGJ3015. 22X50-SGJ3030&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass needle puller</td>
			<td>Narishige</td>
			<td>Narishige PC -10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pull needles</td>
			<td>Drummond Scientific</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>Human chronic gonadotropin (HCG)</td>
			<td>Intervet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>P/7500/PB17</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium chloride (LiCl)</td>
			<td>Sigma</td>
			<td>L-7026</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM710 and Zen software (2008-2010)</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab software</td>
			<td>Mathworks</td>
			<td></td>
			<td>http://uk.mathworks.com/</td>
		</tr>
		<tr>
			<td>Molecular grade water&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP 2819-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail varnish&#160;</td>
			<td>Boots</td>
			<td>Bar code 3600530 373048</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Lab.tech International</td>
			<td>ND-1000 / ND8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (55mm)</td>
			<td>VWR International</td>
			<td>391-0865</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol-chloroform</td>
			<td>Sigma</td>
			<td>P3803</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop software</td>
			<td>Adobe</td>
			<td>N/A</td>
			<td>http://www.photoshop.com/products</td>
		</tr>
		<tr>
			<td>High fidelity DNA polymerase and buffers&#160;</td>
			<td>Biolabs</td>
			<td>M0530S&#160; Buffer - M0531S</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P/4280/53</td>
			<td></td>
		</tr>
		<tr>
			<td>PVC insulation tape</td>
			<td>Onecall</td>
			<td>SH5006MPK</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel extraction kit&#160;</td>
			<td>Qiagen</td>
			<td>S28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction enzymes buffers</td>
			<td>Roche</td>
			<td colspan="2">SuRE/CUT Buffer Set 11082 035 001</td>
		</tr>
		<tr>
			<td>RNAse-free DNAse</td>
			<td>Promega</td>
			<td>ME10A</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel back single edge blades</td>
			<td>Personna</td>
			<td>66-0403-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>27810.364</td>
			<td></td>
		</tr>
		<tr>
			<td>SP6 transcription kit</td>
			<td>Ambion</td>
			<td>AM1330</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Thermo Fisher</td>
			<td>SHE 2505</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Invitrogen</td>
			<td>15504-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten needles</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>homemade</td>
		</tr>
		<tr>
			<td>Zen lite software</td>
			<td>Carl Zeiss</td>
			<td>N/A</td>
			<td>Free download&#160; http://www.zeiss.co.uk/microscopy/en_gb/downloads/zen.html</td>
		</tr>
	</tbody>
</table>

using confocal analysis of xenopus laevis to investigate modulators of wnt and shh morphogen gradients

Questo protocollo descrive un metodo per visualizzare ligandi distribuiti attraverso un campo di cellule. La facilità di esprimere proteine ​​esogene, insieme con le grandi dimensioni delle loro cellule in embrioni precoci, fare Xenopus laevis un modello utile per la visualizzazione di ligandi GFP-tag. MRNA sintetici sono efficacemente tradotte dopo l&#39;iniezione in fase iniziale di Xenopus embrioni, e le iniezioni possono essere mirati a una singola cella. Se combinato con un tracciante lignaggio come membrana legato RFP, la cella iniettato (ei suoi discendenti) che stanno producendo la proteina overexpressed possono facilmente essere seguite. Questo protocollo descrive un metodo per la produzione di fluorescenza etichettato Wnt e Shh ligandi da iniettare mRNA. I metodi coinvolgono la micro dissezione di espianti ectodermici (tappi di animali) e l&#39;analisi del ligando diffusione in più campioni. Utilizzando confocale, informazioni sulla secrezione ligando e diffusione su un campo di cellule può essere ottenuta. Le analisi statistiche delle immagini confocale forniscono dati quantitativi sulla forma di gradienti ligando. Questi metodi possono essere utili per i ricercatori che vogliono testare gli effetti dei fattori che possono regolare la forma di gradienti morphogen.

Analisi confocale di espianti protezione animale che esprimono proteine ​​fluorescenti tag fornisce un sistema efficace per la visualizzazione di distribuzione ligando in diverse condizioni sperimentali. In un esempio, la distribuzione di GFP etichettato Shh è mostrato (Figura 1). Nella fase 2 cellule, embrioni di Xenopus vengono iniettate in entrambe le cellule con una o con mRNA di controllo o con mRNA codificante per Sulf1, un enzima che modifica superficie cellulare eparan solfato e in...

Watch this Scientific Journal Video about Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients at JoVE.com

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Il manoscritto qui fornisce un semplice insieme di metodi per analizzare la secrezione e la diffusione di fluorescenza ligandi marcati nel Xenopus. Ciò fornisce un contesto per valutare la capacità di altre proteine ​​di modificare la distribuzione ligando e permettendo esperimenti che possono dare comprensione dei meccanismi che regolano gradienti morphogen.

Utilizzando Analisi confocale di<em&gt; Xenopus laevis</em&gt; Per Indagare modulatori di Wnt e Shh morphogen gradienti

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

using-confocal-analysis-xenopus-laevis-to-investigate-modulators-wnt

Research

JoVE Journal

Developmental Biology

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

11.0K Views.  The Bateson Centre, University of Sheffield.  Il manoscritto qui fornisce un semplice insieme di metodi per analizzare la secrezione e la diffusione di fluorescenza ligandi marcati nel Xenopus. Ciò fornisce un contesto per valutare la capacità di altre proteine ​​di modificare la distribuzione ligando e permettendo esperimenti che possono dare comprensione dei meccanismi che regolano gradienti morphogen. 

Video: Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Capire in anticipo Organogenesis Utilizzando una semplificata<em&gt; In Situ</em&gt; Protocollo di ibridazione in<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Ricerca

Biologia dello sviluppo

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Manipolazione e<em&gt; In Vitro</em&gt; Maturazione di<em&gt; Xenopus laevis</em&gt; ovociti, seguito da Iniezione intracitoplasmatica di spermatozoi, per studiare lo sviluppo embrionale

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Clonazione funzionale utilizzando un<em&gt; Xenopus</em&gt; Ovociti Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

Loss e approccio guadagno-di-funzione per indagare cellulare precoce Fate Determinanti in preimpianto embrioni di topo

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Tecnica a Target microiniezione per lo sviluppo<em&gt; Xenopus</em&gt; Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

Studiare Wnt segnalazione Durante Patterning di condurre Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

Costruire modelli ad elementi finiti per Indagare Zebrafish Jaw Biomeccanica

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Visualizzazione e analisi quantitativa di angiogenesi embrionale in<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Microiniezione di DNA in boccioli di occhiali in embrioni di Xenopus lavis e imaging di GFP Expressing Optic Aboral Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Imaging fluorescente del calcio e successiva ibridazione in situ per la caratterizzazione neuronale del precursore in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...