We thank Alina El-Khalili, and Martina Hajduskova for technical assistance. We thank members of the Tursun group for comments on the manuscript. This work was sponsored by the ERC-StG-2014-637530 and ERC CIG PCIG12-GA-2012-333922 and is supported by the Max Delbrueck Center for Molecular Medicine in the Helmholtz Association.

All methods described here with regards to animal care have been approved by the LaGeSo Berlin, Germany
1. Solution Preparation
<ol>
	<li>NGM-Agar plates (1 L)
	<ol>
		<li>Add 3 g of NaCl, 2.5 g of peptone media (e.g., Bacto-Peptone), and 20 g of agar. After autoclaving, add 1 mL of cholesterol (5 mg/mL in 95% EtOH stock), 1 mL of 1 M MgSO4, 1 mL of 1 M CaCl2, 25 mL of 1 M K2PO4, and 1 mL of amphotericin B (2.5 mg/mL stock).</li>
	</ol>
	</li>
	<li>NGM-Agar RNAi plates (1 L)
	<ol>
		<li>Add 3 g of NaCl, 2.5 g of peptone media, and 20 g of agar. After autoclaving add, 1 mL of cholesterol (5 mg/mL in 95% EtOH stock), 1 mL of 1 M MgSO4, 1 mL of 1 M CaCl2, 25 mL of 1 M K2PO4, 1 mL of amphotericin B (2.5 mg/mL stock), 1 mL of 1 M IPTG, and 1 mL carbenicillin (50 mg/mL).</li>
	</ol>
	</li>
	<li>LB-Agar (1 L)
	<ol>
		<li>Add 10 g of peptone media, 5 g of yeast extract, 5 g of NaCl, 20 g of agar, 10 mL of 1 M Tris pH 8.0. Add H2O to 1 L. After autoclaving, add 1 mL of 50 mg/mL carbenicillin and 2.5 mL of 5 mg/mL tetracycline.</li>
	</ol>
	</li>
	<li>LB Medium (1 L)
	<ol>
		<li>Add 10 g of peptone media, 5 g of yeast extract, 5 g of NaCl, and 10 mL of 1 M Tris pH 8.0. Add H2O to 1 L. After autoclaving,&#160; add 1 mL of 50 mg/mL carbenicillin.</li>
	</ol>
	</li>
	<li>M9-buffer (1 L)
	<ol>
		<li>Add 6.0 g of Na2HPO4, 3 g of KH2PO4, 5 g of NaCl, and 50 mg of gelatin. Before usage, add 1 mL of 1 M MgSO4.</li>
	</ol>
	</li>
	<li>Bleaching solution (10 mL)
	<ol>
		<li>Add 1 mL of NaClO and 2 mL of 5 N NaOH. Add H2O to 10 mL.</li>
	</ol>
	</li>
</ol>
2. Preparation of RNAi Plates
Note: C. elegans is typically cultured in the laboratory on 6 cm Petri plates containing 7.5 mL of Nematode Growth Medium Agar (NGM-Agar)10,11. This protocol is optimized for worms kept at 15 &#176;C. To prepare plates with NGM, use standard sterile techniques to prevent fungal and bacterial contamination. The following is the protocol to prepare NGM plates supplemented with reagents for RNAi.
<ol>
	<li>Prepare 6-cm NGM-Agar RNAi plates containing 1 mM Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) and 50 &#181;g/mL carbenicillin. Let them dry for 24 &#8211; 48 h at room temperature in the dark12.
	<ol>
		<li>Keep RNAi plates at 4 &#176;C in the dark. Do not use if older than 14 days.</li>
	</ol>
	</li>
	<li>Select the RNAi bacteria (Escherichia coli HT115) clone containing the L4440 plasmid with the lin-53 gene DNA sequence from the frozen glycerol stock from the available RNAi library13 on LB-agar plates containing 50 &#181;g/mL carbenicillin and 12.5 &#181;g/mL tetracycline by using the three-phase streaking pattern. Grow the bacteria at 37 &#176;C overnight12. This clone allows IPTG-dependent production of lin-53 dsRNA in the bacteria.
	<ol>
		<li>Additionally, grow RNAi bacteria that contain the L4440 plasmid without any gene sequence as the empty vector control14.</li>
	</ol>
	</li>
	<li>The next day, pick a single colony from lin-53 or empty vector LB-agar plates using a 200 &#181;L pipette tip and inoculate each of them into a separate culture tube containing 2 mL of liquid LB medium14 supplemented with 50 &#181;g/mL carbenicillin. Grow cultures overnight at 37 &#176;C until they reach an optical density (OD) at 600 nm of 0.6 &#8211; 0.8. Measure the OD using a spectrophotometer15. 
	CAUTION: The liquid LB media should not contain tetracycline in contrast to the LB-agar plate used for streaking the bacteria from the glycerol stock, since inclusion of tetracycline during feeding leads to a decreased RNAi efficiency 12</li>
	<li>Add 500 &#181;L of each bacterial culture (lin-53 or empty vector) to 6 cm NGM-Agar RNAi plates using a multipipette. Incubate plates with a closed lid overnight at room temperature in the dark to dry. During this time, the IPTG in the NGM plates will induce production of dsRNA in the bacteria.
	<ol>
		<li>Use at least 3 plates per bacterial culture per experiment. This will provide 3 technical replicates for each experiment.</li>
	</ol>
	</li>
	<li>Store dried NGM-agar RNAi plates with bacteria at 4 &#176;C in the dark for up to two weeks.</li>
</ol>
3. Preparation of C. elegans Strain BAT28
<ol>
	<li>Maintain the worm strain BAT288 containing the transgenes otIs305 [hsp-16.2prom::che-1, rol-6(su1006)] and ntIs1 [gcy-5prom::gfp] on OP50 bacteria using standard NGM-Agar plates at 15 &#176;C. 
	Note: For detailed protocol on Nematode culture, see10. The rol-6(su1006) is a dominant allele and commonly used phenotypic injection marker16 that causes the typical rolling movement. It is used in the otIs305 transgene to track hsp-16.2prom::che-1.
	<ol>
		<li>Keep the BAT28 strain at 15 &#176;C at all times in order to prevent precautious activation of the hsp-16.2prom::che-1 transgene. Reduce exposure to temperatures higher than 15 &#176;C while handling the strain as much as possible.</li>
	</ol>
	</li>
	<li>To age-synchronize worms, use the bleaching technique17.
	<ol>
		<li>Wash off 6 cm NGM-Agar plates containing adults and eggs of BAT28 using 900 &#181;L M9 buffer. Pellet worms by centrifuging at 900 x g for 1 min. Remove the supernatant.</li>
		<li>Add 0.5 &#8211; 1 mL of bleaching solution and shake the tube for approximately 1 min until adult worms start to burst open. Monitor using a standard stereomicroscope.</li>
		<li>Pellet worms by centrifuging at 900 x g for 1 min. Remove the supernatant.</li>
		<li>Wash the worm pellet 3 times by adding 800 &#181;L of M9 buffer and centrifuging at 900 x g.</li>
		<li>Place the cleaned eggs on fresh NGM-plates seeded with OP50 bacteria. Grow a bleached population on OP50 bacteria at 15 &#176;C until they reach L4 stage (approximately 4 days). L4 larvae can be recognized by a white patch approximately halfway along the ventral side of the worm18 using a standard stereomicroscope. 
		Note: To achieve the germ cell to neuron conversion phenotype upon depletion of lin-53, it needs to be depleted in the parental generation (P0). The scoring for the conversion phenotype is undertaken in the following generation (filial generation F1). To achieve the deplete lin-53 already in the P0, L4 animals are subjected to RNAi.</li>
	</ol>
	</li>
	<li>Manually transfer 50 L4 stage worms per replicate using a platinum wire to an NGM plate that does not contain any bacteria and let worms move away from any transferred OP50 bacteria. Let worms move on the plate for around 5 minutes. Use bacteria from the NGM-Agar RNAi plates previously seeded with lin-53 RNAi bacteria (or empty vector control) to transfer to the respective RNAi plates.
	<ol>
		<li>Avoid transferring OP50 bacteria to the actual RNAi plates. Work fast to minimize exposure time of the strain to temperatures higher than 15 &#176;C.</li>
	</ol>
	</li>
	<li>Incubate worms on NGM-Agar RNAi plates at 15 &#176;C for approximately 7 days until F1 progeny of the worms reaches L3 &#8211; L4 stage. They can be clearly separated from the P0 animals since they are bigger and thicker than the F1 progeny.
	<ol>
		<li>Visually check under a standard stereomicroscope whether F1 progeny worms show the protruding vulva (pvul) phenotype as shown in Figure 1. RNAi against lin-53 has pleiotropic effects and causes the pvul phenotype which can be used to assess whether RNAi against lin-53 has been successful. 
		Note:&#160; RNAi against lin-53 can also cause lethality of F1 embryos. This effect increases if plates are exposed to higher degrees than 15 &#176;C before animals reached the L3 &#8211; L4 stage. Dead embryos can be recognized by arrested development and lack of hatching.</li>
		<li>Incubate plates in the dark since IPTG is light sensitive.</li>
	</ol>
	</li>
</ol>
4. Induction of Germ Cell Reprogramming
<ol>
	<li>Incubate examined RNAi plates containing lin-53 and empty vector RNAi-treated F1 progeny for 30 min at 37 &#176;C in the dark to activate CHE-1. Use a vented incubator since it allows for a more efficient heat-shock.</li>
	<li>Allow heat-shocked animals to recover for 30 min at room temperature in the dark and then incubate plates at 25 &#176;C overnight in the dark.</li>
	<li>Using a standard stereomicroscope coupled to a fluorescence light source, examine animals under the GFP filter for gcy-5prom::gfp-derived signals in the mid-body area of the worms as shown in Figure 2. Set microscope for GFP signals: excitation maximum at 488 nm with emission maximum at 509 nm.</li>
	<li>Evaluate the degree of germ cell to neuron conversion by mounting animals with GFP signals in the mid-body area on agar pad-containing microscopy slides19.
	<ol>
		<li>Count the number of animals showing the germ cell to neuron conversion vs. the dark animals to assess the phenotype penetrance. Typically, around 30% of the animals show discrete GFP signals in the germline upon lin-53 depletion, whereas not more than 5% of the animals show diffuse GFP signals in the germline upon empty vector RNAi. &#160;</li>
	</ol>
	</li>
</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=H3K9me3-Dependent+Heterochromatin:+Barrier+to+Cell+Fate+Changes.">H3K9me3-Dependent Heterochromatin: Barrier to Cell Fate Changes.</a> Trends Gent. 32 (1), 29-41 (2016).</li><li>Tursun, B., Patel, T., Kratsios, P., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Conversion+of+C.+elegans+Germ+Cells+into+Specific+Neuron+Types.">Direct Conversion of C. elegans Germ Cells into Specific Neuron Types.</a> Science. 331 (6015), 304-308 (2011).</li><li>Patel, T., Tursun, B., Rahe, D. P., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+Polycomb+Repressive+Complex+2+Makes+C.elegans+Germ+Cells+Susceptible+to+Direct+Conversion+into+Specific+Somatic+Cell+Types.">Removal of Polycomb Repressive Complex 2 Makes C.elegans Germ Cells Susceptible to Direct Conversion into Specific Somatic Cell Types.</a> Cell Rep. , 1-9 (2012).</li><li>Etchberger, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+signature+and+cis-regulatory+architecture+of+a+C.+elegans+gustatory+neuron.">The molecular signature and cis-regulatory architecture of a C. elegans gustatory neuron.</a> Genes Dev. 21 (13), 1653 (2007).</li><li>Yu, S., Avery, L., Baude, E., Garbers, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guanylyl+cyclase+expression+in+specific+sensory+neurons:+a+new+family+of+chemosensory+receptors.">Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors.</a> Proc Nat Acad Sci. USA. 94 (7), 3384-3387 (1997).</li><li>Seelk, 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=Increasing+Notch+signaling+antagonizes+PRC2-mediated+silencing+to+promote+reprograming+of+germ+cells+into+neurons.">Increasing Notch signaling antagonizes PRC2-mediated silencing to promote reprograming of germ cells into neurons.</a> eLife. 5, (2016).</li><li>Patel, T., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+control+of+terminal+differentiation+and+restriction+of+cellular+plasticity.">Coordinated control of terminal differentiation and restriction of cellular plasticity.</a> eLife. 6, e24100 (2017).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Genetics+of+Caenorhabditis+Elegans.">The Genetics of Caenorhabditis Elegans.</a> Genetics. 77 (1), 71 (1974).</li><li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Introduction+to+Worm+Lab:+from+Culturing+Worms+to+Mutagenesis.">An Introduction to Worm Lab: from Culturing Worms to Mutagenesis.</a> J Vis Exp. 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The authors have declared that no competing interests exist.

While the described protocol using the transgenic C. elegans strain BAT28 and RNAi against lin-53 is straightforward, a number of steps are critical in order to ensure the expected result of germ cell to neuron reprogramming. It is important that the strain BAT28 is kept at 15 &#176;C at all times prior to the experiments. During handling and maintenance of the strain, the time at temperatures above 15 &#176;C needs to be minimized as much as possible. Proper heat-shock conditions are important since insufficient induction of che-1 overexpression will not result in germ cell to ASE neuron conversion. This can be detected by measuring the phenotype penetrance as described in the protocol. Extensive heat-shock can lead to lethality of the animals. For instance, plates that have been placed near the inner walls or on the bottom ground of a static air incubator often experience extensive heat treatment. Therefore, it is recommended to use a vented heat incubator. Additionally, timing of the heat-shock induction is critical since over-expression of che-1 during larval stages earlier then L3 can lead to ectopic gcy-5prom::gfp induction in different body regions such as the vulva (Figure 3)9. Furthermore, extensive heat-shock can be detected by the phenotype penetrance in empty vector RNAi animals which should not extend 5% and increased auto-fluorescence of other tissues, namely the intestine.
Sporadically, RNAi bacteria can lose their activity to efficiently produce dsRNA of the target gene. Unfortunately, this can not be detected prior to the RNAi experiment. In such cases a fresh streak from the glycerol should be grown as described in Section 2 of the protocol. If there is still no RNAi-caused pleiotropic phenotype visible such as the pvul phenotype caused by successful RNAi against lin-53, then a plasmid DNA isolation from the respective bacteria should be performed and fresh HT115 bacteria need to be transformed with the L4400 plasmid containing the lin-53 sequence.
Importantly, the BAT28 strain has a the roller phenotype caused by the injection marker pRF4, which was used during transgenesis of the animals with the hsp-16.2prom::che-1 DNA construct. Occasionally, animals lose the rolling phenotype, which can indicate silencing of the hsp-16.2prom::che-1 transgene. To properly maintain the BAT28 strain, pick 10 roller animals to a fresh plate and propagate selecting for rolling animals.
The application of the protocol is limited to the F1 RNAi procedure, which means that animals whose parents (P0 generation) have not been exposed to lin-53 RNAi will not show germ cell to neuron conversion due to maternal rescue4. An alternative procedure to convert germ cells to neurons has been described by Ciosk and colleagues15 using RNAi knock-down of gld-1 and mex-3 without over-expression of a transcription factor. However, the obtained neurons do not belong to a specific type of neurons and germ cells also convert to muscle-like cells upon RNAi-mediated depletion of gld-1 and mex-315. Previously, it has been demonstrated the RNAi against lin-53 allowed germ cell conversion into GABAergic neuron-like cells upon over-expression of the Pitx-type homoedomain transcription factor UNC-3016 instead of CHE-14. For future directions, different fate-inducing transcription factors can be tested whether lin-53 depleted germ cells can be converted to other cell types than specific neurons. In this context, overexpression of the myogenic bHLH transcription factor HLH-1, homolog of the mammalian MyoD17, induces conversion of germ cell to muscles upon lin-53 RNAi5. The established reprogramming of germ cells to a specific somatic cell type upon lin-53 RNAi and over-expression of an appropriate fate-inducing transcription factor can be used for a number of different genetic screens. Such suppressor or enhancer screens can help dissecting regulatory pathways that play a role during cell fate conversion.

Cell fate reprogramming by ectopic TF expression such as the myogenic TF MyoD, which, when overexpressed, converts fibroblasts directly into muscle-like cells1, has been a research focus for decades. However, differentiated cells are often refractory to this process, due to mechanisms that safeguard their cellular identity. Germ cells show a similar degree of protection and there are underlying mechanisms that often act as a barrier to prevent germ cell conversion into other cell types upon over-expression of a fate-inducing TF. Typically, epigenetic regulation such as histone modifications and chromatin structure imposes an impediment for the conversion of cell fates2,3. We have previously applied reverse genetics using RNAi knock-downs in C. elegans and identified factors that safeguard cellular identities and thereby counteract reprogramming of germ cells into neurons4. Specifically, the polycomb repressive complex 2 (PRC2) and the highly conserved histone chaperone LIN-53 (CAF-1/RBBP/4/7 in humans) prevent the direct conversion of germ cells into specific somatic cell types such as glutamatergic taste neurons in C. elegans4,5. To identify these germ cell reprogramming barrier factors, we used transgenic animals carrying the heat-shock inducible Zn-finger TF CHE-1. CHE-1 is normally expressed in two head neurons of C. elegans, where it specifies the fate of glutamatergic taste neurons termed ASE6. The ASE neuron fate can be visualized by expression of the ASE-specific fluorescent reporter gcy-5prom::gfp. GCY-5 is a chemoreceptor only expressed in ASE right neuron7. Upon lin-53 depletion by RNAi and induction of broad ectopic expression of CHE-1 by heat-shock, germ cells can be converted into neurons in vivo4,5. Importantly, timing of RNAi treatment is crucial as only adult worms (P0 generation) exposed to lin-53 RNAi give rise to F1 progeny animals with a germline that is permissive for reprogramming into neurons4.
The above-described germ cell to neuron conversion procedure in C. elegans can be used to study a variety of biological questions such as the implication of signaling pathways in cell fate reprogramming. For instance, we used the CHE-1-induced germ cell reprogramming into ASE neurons upon RNAi against lin-53 in order to study the implication of the Notch signaling pathway in the process of germ cell reprogramming8. By performing double RNAi to co-deplete lin-53 and the histone demethylase-encoding gene utx-1 we showed that the Notch signaling pathway counteracts PRC2-mediated gene silencing by activating expression of UTX-1 in the germline8. This finding demonstrates that the germ cell conversion to neurons described in this protocol could be used to study a complex biological process such as cellular reprogramming in an intact organism and in the context of different developmental and environmental conditions. Furthermore, it provides the perspective to challenge germ cells by over-expressing different fate-inducing TFs to study their role in restriction of cellular plasticity9, or to assess their potential for inducing reprogramming of germ cells to other cell types in vivo.
While mammalian tissue cultures also allow studying different aspects of cell fate reprogramming, cells grown in culture have limited capacity to mimic signaling pathway conditions compared to an intact organism. Therefore, C. elegans is a versatile model for examination of the roles of various biological processes such as Notch signaling during reprogramming in vivo. Additionally, it is a powerful model for genetic screens that is relatively easy to maintain and genetically modify for low costs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar-Agar, Kobe I</td>
			<td>Roth</td>
			<td>5210.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Bactopeptone&#160;</td>
			<td>A. Hartenstein GmbH</td>
			<td>211 677</td>
			<td>Peptone Media&#160;</td>
		</tr>
		<tr>
			<td>Yeast extract&#160;</td>
			<td>AppliChem</td>
			<td>A1552,0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>USBiological</td>
			<td>A2220</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Merck Biosciences</td>
			<td>208290</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Roth</td>
			<td>8866.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatine</td>
			<td>Roth</td>
			<td>4275.3</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma</td>
			<td>15502-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>K2PO4</td>
			<td>Roth</td>
			<td>T875.2</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Roth&#160;</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>VWR</td>
			<td>25,163,364</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Roth</td>
			<td>P030.1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Roth</td>
			<td>9265.1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaClO</td>
			<td>Roth</td>
			<td>9062.3</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH (5 N)</td>
			<td>Roth&#160;</td>
			<td>KK71.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Roth</td>
			<td>AE15.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Roth</td>
			<td>634412</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Roth</td>
			<td>2371.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubators</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td>New Brunswick Scientific C24</td>
			<td>M1247-0052</td>
			<td>for heat-shock</td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td>Sanyo MIR-5</td>
			<td>5534210</td>
			<td>for maintenance of worm strains at 15 &#176;C or 25 &#176;C</td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>M205 FA</td>
			<td>Leica</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (Imaging) + camera</td>
			<td>Axio Imager 2</td>
			<td>Zeiss</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope (Imaging) + camera</td>
			<td>Sensicam</td>
			<td>PCO</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>SMZ745</td>
			<td>Nikon</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacterial strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli HT115</td>
			<td></td>
			<td></td>
			<td>F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10(DE3 lysogen: lavUV5 
			promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNAse 
			III minus).</td>
		</tr>
		<tr>
			<td>Escherichia coli OP50&#160;</td>
			<td></td>
			<td></td>
			<td>Uracil auxotroph E. coli strain</td>
		</tr>
		<tr>
			<td>Worm strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BAT28: otIs305 (hsp16.2prom::che-1:: 
			3xHA) ntIs1 (gcy-5prom::gfp) V.</td>
			<td></td>
			<td></td>
			<td>derived from OH9846 by 4x times more back crossing with N2</td>
		</tr>
		<tr>
			<td>RNAi Clones</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>lin-53</td>
			<td>SourceBioscience</td>
			<td>ID I-4D14</td>
			<td>Ahringer library 16B07 ChromI, K07A1.12</td>
		</tr>
		<tr>
			<td>empty vetor: L4440</td>
			<td>Addgene&#160;</td>
			<td>#1654</td>
			<td></td>
		</tr>
		<tr>
			<td>Misc.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Worm pick&#160;</td>
			<td></td>
			<td></td>
			<td>self-made using a pasteur pipette and a platinum wire</td>
		</tr>
	</tbody>
</table>

application of rnai and heat-shock-induced transcription factor expression to reprogram germ cells to neurons in c. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

As shown in Figure 1, F1 animals that exhibit the pvul phenotype illustrate successful application of lin-53 RNAi. These animals are prone to allow conversion of their germ cells into ASE neuron-like cells upon heat-shock induction of che-1 over-expression as illustrated in Figure 2. In contrast to worms that grew on the control empty vector RNAi lin-53 RNAi treatment will yield animals that display ectopic GFP-signals in the mid-body area after heat-shock and 25 &#176;C incubation overnight as described earlier4,8. Closer examination under an epifluorescence microscope of these worms reveals that the cells showing GFP signals also display neuron-like projections (Figure 2B, white arrows) that are typical for neuronal cells. If RNAi against lin-53 was not successful or heat-shock conditions were inappropriate GFP signals will resemble to those in control RNAi treated animals (Figure 2). Importantly, avoid inducing over-expression of che-1 by heat induction before animals reach mid L3 stage. Animals that were too young at the time of che-1 overexpression induction can display ectopic gcy-5prom::gfp expression in other regions of the body such as the developing vulva as shown in Figure 313.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56889/56889fig1.jpg" /> 
Figure 1: Pvul phenotype caused by RNAi against lin-53. (Top) DIC picture of L4/young adult stage F1 progeny worms derived from control or lin-53 RNAi treated mothers. Scale bars = 20 &#181;m. (Bottom) Animals treated with lin-53 RNAi display the protruding vulva (pvul) phenotype (black arrow-heads). The pvul phenotype confirms that RNAi against lin-53 was successful. Scale bars = 20 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56889/56889fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56889/56889fig2.jpg" /> 
Figure 2: Worms with successfully reprogrammed germ cell into neurons. (A) BAT28 strain animals grown on the empty vector do not show GFP signals in the germline after heat-shock induction of che-1 overexpression. Dashed white line outlines worm. Scale bars = 20 &#181;m. (B) In contrast to BAT28 strain animals grown on the empty vector control RNAi animals grown on lin-53 RNAi display gcy-5prom::gfp signals in the mid-body area. Pictures of the mid-body section with GFP signals taken at 63X magnification reveal GFP-positive cells showing protrusions (white arrow-heads). This morphological feature indicates that germ cells underwent conversion into ASE neuron-like cells. White asterisks mark intestine-derived auto-fluorescence. All pictures were acquired using an epifluorescence microscope with 20X magnification and 63X magnification. Dashed white line outlines worms. Scale bars = 20 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56889/56889fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56889/56889fig3.jpg" /> 
Figure 3: Early stage worm with ectopic GFP. BAT28 strain animals grown on the empty vector RNAi bacteria that were heat-shock induced for che-1 overexpression at early larval stages such as L2 show GFP signals in the mid-body area (white asterisks). These signals are not due to germ cell reprogramming. Dashed white line outlines worm. Scale bars = 20 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/56889/56889fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans at JoVE.com

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

application-rnai-heat-shock-induced-transcription-factor-expression

Research

JoVE Journal

Developmental Biology

7.6K Views.  Max Delbrück Center for Molecular Medicine in the Helmholtz Association. This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo. 

Video: Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...