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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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><tbody><tr><td>&lt;strong&gt;Chemicals&amp;nbsp;&lt;/strong&gt;</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&amp;nbsp;</td><td>A. Hartenstein GmbH</td><td>211 677</td><td>Peptone Media&amp;nbsp;</td></tr><tr><td>Yeast extract&amp;nbsp;</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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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>K&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth</td><td>T875.2</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth&amp;nbsp;</td><td>3904.2</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR</td><td>25,163,364</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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&amp;nbsp;</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>&lt;strong&gt;Incubators&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Incubator&amp;nbsp;</td><td>New Brunswick Scientific C24</td><td>M1247-0052</td><td>for heat-shock</td></tr><tr><td>Incubator&amp;nbsp;</td><td>Sanyo MIR-5</td><td>5534210</td><td>for maintenance of worm strains at 15 &amp;deg;C or 25 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;Microscopes&lt;/strong&gt;</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>&lt;strong&gt;Bacterial strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Escherichia coli HT115&lt;/em&gt;</td><td></td><td></td><td>F-, mcrA, mcrB, IN(rrnD-rrnE)1, rnc14::Tn10(DE3 lysogen: lavUV5&lt;br/&gt; promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNAse&lt;br/&gt; III minus).</td></tr><tr><td>&lt;em&gt;Escherichia coli OP50&amp;nbsp;&lt;/em&gt;</td><td></td><td></td><td>Uracil auxotroph E. coli strain</td></tr><tr><td>&lt;strong&gt;Worm strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;BAT28:&lt;/strong&gt; &lt;em&gt;otIs305 (hsp16.2prom::che-1::&lt;br/&gt; 3xHA) ntIs1 (gcy-5prom::gfp) V.&lt;/em&gt;</td><td></td><td></td><td>derived from OH9846 by 4x times more back crossing with N2</td></tr><tr><td>&lt;strong&gt;RNAi Clones&lt;/strong&gt;</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&amp;nbsp;</td><td>#1654</td><td></td></tr><tr><td>&lt;strong&gt;Misc.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Worm pick&amp;nbsp;</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level. While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is&#160;presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay. Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Here, standardized protocols are presented to assess induction of the heat shock response (HSR) in Caenorhabditis elegans using RT-qPCR at the molecular level, fluorescent reporters at the cellular level, and thermorecovery at the organismal level.

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, ...

Environment

Epigenetic Inheritance and ChIP in Caenorhabditis elegans

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through factors such as non-coding RNAs and chromatin modifications. The phenomenon of RNA interference (RNAi) inheritance in the nematode Caenorhabditis elegans is an effective model to investigate TEI that takes advantage of this model organism's short life cycle, self-propagation, and transparency. In RNAi inheritance, exposure of animals to RNAi leads to gene silencing and altered chromatin signatures at the target locus that persist for multiple generations in the absence of the initial trigger. This protocol describes the analysis of RNAi inheritance in C. elegans using a germline-expressed nuclear green fluorescent protein (GFP) reporter. Reporter silencing is initiated by feeding the animals bacteria expressing double-stranded RNA targeting GFP. At each generation, animals are passaged to maintain synchronized development, and reporter gene silencing is determined by microscopy. At select generations, populations are collected and processed for chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) to measure histone modification enrichment at the GFP reporter locus. This protocol for studying RNAi inheritance can be easily modified and combined with other analyses to further investigate TEI factors in small RNA and chromatin pathways.

Author Spotlight: RNAi Inheritance and ChIP in C. elegans 

This protocol describes an RNA interference and ChIP assay to study the epigenetic inheritance of RNAi-induced silencing and associated chromatin modifications in C. elegans.

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through ...

Genetics

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network function1. These proteins are found at the presynaptic and postsynaptic membranes 2. Studies in mice indicate that neurexins and neurologins have an essential role in synaptic transmission 1. Recent reports have shown that altered neuronal connections during the development of the human nervous system could constitute the basis of the etiology of numerous cases of autism spectrum disorders 3.
Caenorhabditis elegans could be used as an experimental tool to facilitate the study of the functioning of synaptic components, because of its simplicity for laboratory experimentation, and given that its nervous system and synaptic wiring has been fully characterized. In C. elegans nrx-1 and nlg-1 genes are orthologous to human NRXN1 and NLGN1 genes which encode alpha-neurexin-1 and neuroligin-1 proteins, respectively. In humans and nematodes, the organization of neurexins and neuroligins is similar in respect to functional domains.
The head of the nematode contains the amphid, a sensory organ of the nematode, which mediates responses to different stimuli, including osmotic strength. The amphid is made of 12 sensory bipolar neurons with ciliated dendrites and one presynaptic terminal axon 4. Two of these neurons, named ASHR and ASHL are particularly important in osmotic sensory function, detecting water-soluble repellents with high osmotic strength 5. The dendrites of these two neurons lengthen to the tip of the mouth and the axons extend to the nerve ring, where they make synaptic connections with other neurons determining the behavioral response 6.
To evaluate the implications of neurexin and neuroligin in high osmotic strength avoidance, we show the different response of C. elegans mutants defective in nrx-1 and nlg-1 genes, using a method based on a 4M fructose ring 7. The behavioral phenotypes were confirmed using specific RNAi clones 8. In C. elegans, the dsRNA required to trigger RNAi can be administered by feeding 9. The delivery of dsRNA through food induces the RNAi interference of the gene of interest thus allowing the identification of genetic components and network pathways.

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexins and neuroligins are membrane-neuron adhesion proteins which perform essential roles in synaptic differentiation and transmission. Neuroligin deficient mutants of C. elegans are defective in detecting osmotic strength, but when they also contain a mutation in the gene coding neurexin, they recover the wild type phenotype.

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

Biology

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

Neuroscience

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis. 
	Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.
	We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

We describe a protocol using C. elegans and RNAi feeding libraries that allows automated measurement of multiple parameters such as fluorescence, size and opacity of individual worms in a population. We give one example of a screen to identify genes involved in anti-fungal innate immunity in C. elegans.

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the importance of intercellular signaling processes in the regulation of cell-nonautonomous proteostasis. Recent studies are now beginning to uncover signaling components and pathways that coordinate protein quality control from one tissue to another. It is therefore important to identify mechanisms and components of the cell-nonautonomous proteostasis network (PN) and its relevance for aging, stress responses and protein misfolding diseases. In the laboratory, we use genetic knockdown by tissue-specific RNAi in combination with stress reporters and tissue-specific proteostasis sensors to study this. We describe methodologies to examine and to identify components of the cell-nonautonomous PN that can act in tissues perceiving a stress condition and in responding cells to activate a protective response. We first describe how to generate hairpin RNAi constructs for constitutive genetic knockdown in specific tissues and how to perform tissue-specific genetic knockdown by feeding RNAi at different life stages. Stress reporters&#160;and behavioral assays function as&#160;valuable readouts&#160;that enable&#160;the fast screening of genes and conditions modifying systemic stress signaling processes. Finally, proteostasis sensors expressed in different tissues are utilized to determine changes in the tissue-specific capacity of the PN at different stages of development and aging. Thus, these tools should help clarify and allow monitoring the capacity of PN in specific tissues, while helping to identify components that function in different tissues to mediate cell-nonautonomous PN in an organism.

Maintenance of organismal proteostasis requires the coordination of protein quality control responses such as chaperone expression from one tissue to another. Here, we provide tools used in C. elegans that allow monitoring of proteostasis capacity in specific tissues and determine intercellular signaling responses.

Over the past decade there has been a transformative increase in knowledge surrounding the regulation of protein quality control processes, unveiling the ...

RNAi in C. elegans

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target gene. In the nematode, C. elegans, RNAi is particularly easy and effective because it can be delivered simply by feeding the worms bacteria that express double stranded RNA (dsRNA) that is complementary to a gene of interest. First, this video will introduce the concept of RNA interference and explain how it causes targeted gene knockdown. Then, we will demonstrate a protocol for using RNAi in C. elegans, which includes preparation of the bacteria and RNAi worm plates, culturing of the worms, and how to assess the effects of RNAi on the worms. RNAi is frequently used to perform reverse genetic screens in order to reveal which genes are important to carry out specific biological processes. Furthermore, automated reverse genetic screens allow for the efficient knockdown and analysis of a large collection of genes. Lastly, RNAi is often used to study the development of C. elegans. Since its discovery, scientists have used RNAi to make tremendous progress on the understanding of many biological phenomena.

RNA Interference RNAi in Caenorhabditis elegans

RNA interference (RNAi) is a widely used technique in which double stranded RNA is exogenously introduced into an organism, causing knockdown of a target ...

Education

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Biology I: yeast, Drosophila and C. elegans

RNAi Plating for C. elegans Feeding: A Technique to Induce Target dsRNA Expression in E. coli

Source: Kolundzic, E., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56889/application-rnai-heat-shock-induced-transcription-factor-expression">Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in&#160;C. elegans</a>.&#160;J. Vis. Exp.&#160;(2018).
This video describes the principles behind RNAi treatment in C. elegans and demonstrates a protocol to knockdown lin-35 in a transgenic worm strain.

Source: Kolundzic, E., et al. Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in&#160;C. ...

JoVE Encyclopedia of Experiments

Caenorhabditis elegans (worm)

RNAi Feeding in Liquid Culture: A High-Throughput Method to Knockdown Gene Expression in C. elegans

Source: Groh, N. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/56464/methods-to-study-changes-inherent-protein-aggregation-with-age">Methods to Study Changes in Inherent Protein Aggregation with Age in&#160;Caenorhabditis elegans</a>.&#160;J. Vis. Exp.&#160;(2017).
RNAi is powerful genetic tool available to C. elegans researchers. This video introduces a method to treat more worms with RNAi than plate feeding by utilizing liquid culture conditions.

Source: Groh, N. et al. Methods to Study Changes in Inherent Protein Aggregation with Age in&#160;Caenorhabditis elegans.&#160;J. Vis. Exp.&#160;(2017).
...

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

Summary

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Chapters in this video

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

Tissue-Specific RNAi Tools to Identify Components for Systemic Stress Signaling

RNAi in C. elegans

RNAi Plating for C. elegans Feeding: A Technique to Induce Target dsRNA Expression in E. coli

RNAi Feeding in Liquid Culture: A High-Throughput Method to Knockdown Gene Expression in C. elegans