Our work is funded by NIH 1R15GM1131-01 to CFH and NIH PA -12-149 TO CFH.

1. Media Preparation
<ol>
	<li>Prepare grape agar according to manufacturer instructions (see Table of Materials) and pour into a 35 mm Petri dish to half-full (Figure 5).&#160; Allow to solidify for approximately 1 h. After the grape agar solidifies, immediately use or store at 4 &#176;C.</li>
	<li>Gently make scratches across the agar plate using a small plastic knife (flies like to lay on uneven surfaces) leaving the middle of the plate without scratches (Figure 5). Place a small amount of yeast paste (made fresh; see Table of Materials) at the center of plate (Figure 5).</li>
</ol>
2. Embryo Collection Mini Cage Set Up
<ol>
	<li>Set up a cross using two virgin females and one young male inside the embryo collection cage containing the grape agar plate supplemented with yeast paste (Figure 5).</li>
	<li>After 24 h, inspect the cages for laid embryos without opening the cage by looking at the bottom of the agar plate.&#160; If embryos have been laid, remove the agar plate from the chamber and place it inside a humid chamber for microscopic observation (Figure 6).&#160; If several days of laying are scored (e.g., fertility/longevity assays), keep the breeding parents and replace the plate with a fresh one prepared as described. Early collections of less than 24 h can be done but, when using virgin females, be aware that they will not lay until 48 h after emerging from their pupa case.</li>
	<li>Cover the agar plate containing embryos with the Petri dish lid to avoid dehydration and place it immediately inside a humid chamber (Figure 7). Observe under a dissecting microscope and record hatched embryos and L1.&#160; Replace lid and store at room temperature in humid chamber until all embryos had hatched and developed into L1 larvae (Figure 6).</li>
</ol>
3. Counting Embryos and Larvae
<ol>
	<li>After 48 h, observe the plates under the dissecting microscope and record the numbers one last time before transfer of agar disc to food vial.&#160; Fertilized/viable embryos should become L1 by then.&#160; Longer incubation periods prior to transfer are possible but be aware that plates may start losing moisture and the agar may crack compromising a clean transfer to food vials (Figure 7).&#160; To ensure the plates remain hydrated and embryo viability is not compromised while counting, avoid using a direct gooseneck light over the agar surface (Figure 7E shows an appropriate distance).</li>
	<li>After counting, cover the plate and store it in the humid chamber until ready to transfer. Record findings.</li>
</ol>
4. Transfer the Grape Agar Disc to a Food Vial to monitor Viability During Development
<ol>
	<li>Once the numbers are recorded (hatched embryos/L1/L2), use a spatula to carefully transfer the grape agar disc to a vial large enough to accommodate a 35 mm disc containing Drosophila food media prepared according to manufacturer&#8217;s instructions (refer to list of reagents). Place the grape agar disc L1-side down on the food (Figure 7). &#160;After transferring the agar disc with larvae to the food vial, carefully inspect the empty Petri dish for any larvae left behind (Figure 7E).</li>
	<li>Set up a schedule to inspect food vials daily, at roughly the same time each day, to ensure L2/L3 larvae are observed making their way to the food in the vial (Figure 8).</li>
	<li>Record the number of pupae and adult Drosophila (Figure 9). Keep counting until no more adults are observed and avoid counting the following generation (do not count past 9 days after observing the first adults).</li>
	<li>Observe and record the findings. Adjust the time frame of embryo collection, counting, and recording accordingly to the mutant stock being used.</li>
	<li>Perform a chi square analysis. The null hypothesis assumes 100% viability such that the number of adults will be equal to the number of hatched embryos and L1 originally recorded and transferred to the food vials</li>
</ol>

<ol>
	<li>Hartung, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+observations+on+the+larval+growth+rate+and+viability+of+two+tumor+strains+of+Drosophila+melanogaster.">Some observations on the larval growth rate and viability of two tumor strains of Drosophila melanogaster.</a> Science. 107 (2777), 296-297 (1948).</li><li>Moree, R., King, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Studies+on+Relative+Viability+in+Drosophila+Melanogaster.">Experimental Studies on Relative Viability in Drosophila Melanogaster.</a> Genetics. 46 (12), 1732-1752 (1961).</li><li>Da Costa, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viability+of+F1+and+F2+generations+of+crossed+Drosophila+and+Oregon+previously+adapted+to+2+different+nutrient+media.">Viability of F1 and F2 generations of crossed Drosophila and Oregon previously adapted to 2 different nutrient media.</a> Comptes rendus de l'Acad&#233;mie des Sciences. 242 (1), 177-180 (1956).</li><li>Garcia-Dorado, A., Caballero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mutational+rate+of+Drosophila+viability+decline:+tinkering+with+old+data.">The mutational rate of Drosophila viability decline: tinkering with old data.</a> Genome Research. 8 (2), 99-105 (2002).</li><li>Lence, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=m6A+modulates+neuronal+functions+and+sex+determination+in+Drosophila.">m6A modulates neuronal functions and sex determination in Drosophila.</a> Nature. 540 (7632), 304-318 (2016).</li><li>Haussmann, I. U., 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=m(6)A+potentiates+Sxl+alternative+pre-mRNA+splicing+for+robust+Drosophila+sex+determination.">m(6)A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination.</a> Nature. 54 (7632), 301-304 (2016).</li><li>Stockwer, H., Gallant, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+started:+An+overview+on+raising+and+handling+Drosophila.">Getting started: An overview on raising and handling Drosophila.</a> Methods in Molecular Biology. 420, 27-44 (2008).</li><li>Hales, K. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+on+the+Fly:+A+Primer+on+the+Drosophila+Model+System.">Genetics on the Fly: A Primer on the Drosophila Model System.</a> Genetics. 201 (3), 815-842 (2015).</li><li>Gardner, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Variation+for+Preadult+Viability+in+Drosophila+melanogaster.">Genetic Variation for Preadult Viability in Drosophila melanogaster.</a> Evolution. 55 (8), 1609-1620 (2001).</li><li>Hongay, C. F., Orr-Weaver, T. 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There are no conflicts of interest.

In summary, this method provides an accurate and simple assessment of viability in Drosophila. The entire protocol takes approximately 14 days to complete. The procedure does not require expert technical skills; however, proper timing, a schedule of daily observations, and careful agar transferring is important for accuracy and reproducibility.
In addition to the placement of the grape-agar disc embryo-side down in the food vial, another crucial step in the procedure is transferring the agar disc to a food vial no later than 48 h after removing the grape agar plate from the embryo collection mini cages. Transferring the agar after 48 h resulted in embryo and larvae loss, likely due to dehydration of the agar disc. To count L2/L3 transitions, the plate needs to incubate for 72 h. If this stage is crucial, grape agar plates should be poured thicker and a humid chamber must be used to prevent desiccation. The crosses were set up in embryo collection cages that accommodate 35 mm plates; however, this procedure can be performed using larger embryo collection cages as well, such as one that accommodates 60 mm or 100 mm Petri plates. &#160;Food bottles must then be big enough to accommodate those sizes.&#160;
There are steps that can be added to this protocol. As mentioned above, L2/L3 transitions are challenging to quantify on plates due to the time required and the potential dehydration of the agar. Additionally, larvae become highly mobile on the surface of the agar, posing a challenge to accurately count them. Placing the plates in the refrigerator for 30 min or adding a few drops of a mild anesthetic (lidocaine solution) on the surface of the agar prior to counting L2/L3 larvae can help slow down their movements to count them more accurately. &#160;A caveat to these modifications is that they can introduce variables (cold sensitivity, anesthetic sensitivity) and confound the viability results.&#160; Even without these steps, by using colored food, researchers can quantify wandering L3s/prepupae as they settle on the side of the food vials to initiate pupation. A limitation to this method is that it requires the adults used in the crosses to survive being anesthetized with CO2 for phenotyping to set up the crosses in the embryo collection cages. Dm ime4 homozygous mutant males do not recover well and die a few hours after waking up from CO2 treatment. &#160;Other methods to immobilize adults for sorting can be explored, such as placing vials in the refrigerator for a few minutes and then transfer vials to crushed ice to slow movement and sort quickly and efficiently.&#160;
Apart from comparing allelic strengths and their effects on viability, this method can be used to screen sensitivity or resistance to defined pharmaceutical compounds. Unlike other methods that screen compound toxicity in cell culture15,16,17,18, this method uses whole organisms, making assessment of developmental effects easier to analyze. In sum, using this protocol and modifications therein, will allow to measure allelic strengths as well as the effects of environmental factors and chemical compounds on Drosophila viability, fecundity, fertility, lifespan, and duration of developmental cycle.

Lifespan is affected by genetic and non-genetic factors. In standard lab growth conditions at room temperature, our lab has observed significant variation of fitness and viability among different Dm ime4 alleles grown under identical conditions (Figure 1 and Supplemental Figures). Viability studies are frequently done to investigate the effects of a certain allele combination or growth condition in population genetic studies1,2,3,4. However, detailed analyses of viability within a non-complementary group of mutations are hard to find in the scientific literature. An allele is usually labeled &#34;non-essential&#34;&#160;if the researcher finds a few individuals homozygous for that allele within the food vial that houses the balanced stock5,6. However, accurate Chi-square analyses to assess whether these homozygotes arise at the expected Mendelian ratios are not reported5,6. The most permissive temperature for any Drosophila stock is room temperature (22-23 &#176;C) and, with appropriate nutrients, the life cycle of wild-type flies takes approximately twelve days to complete7,8. As the duration of each developmental stage of wild-type Drosophila is known7,8, the method described in this report can be used to examine whether the Drosophila strain under study is fit at each stage in comparison to a control appropriate for the genetic background tested. In contrast to studies that focus on one specific aspect of development9, this protocol provides a practical way to assess viability at different developmental stages.
In our lab, this protocol is used to assess the viability of stocks that are deficient for Drosophila Inducer of Meiosis 4 (Dm ime4). Dm ime4 is an essential gene10 that encodes an RNA methyltransferase with critical roles in RNA metabolism in Drosophila and other multicellular organisms5,6,10,11,12,13,14. To quickly evaluate novel alleles of Dm ime4 generated via CRISPR/Cas9 (Supplemental Figures), an end-point viability assay was performed that only counted adult progeny produced within vials of balanced stocks (Figure 1). Some of the stocks used were described in previous Dm ime4 reports5,6. Homozygous mutants emerged at sub-Mendelian levels, as determined by chi-square analyses (Figure 1 and Supplemental Materials). To assess whether these lower-than-expected numbers were due to fewer embryos being laid, or fewer hatched embryos, or loss of viability in L1/L2 or pupae, we expanded the tracking to include counts at each of these developmental stages (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, and Figure 9).
Here, we describe the method using wild-type (OreR) flies. To empirically test this method for use with other genetic backgrounds or Drosophila species, we recommend using OreR as a reference and adjust timepoints according to the experimental organism.&#160; The protocol was further evaluated to assess viability of progeny generated by crossing virgin wild-type females with males from a heterozygous Dm ime4 mutant stock5,6 (Figure 4).

<table border="0" cellpadding="0" cellspacing="0" style="width:665px;" width="665">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antimold additive</td>
			<td style="width:121px;">Carolina</td>
			<td style="width:161px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Beaker</td>
			<td style="width:121px;">Fisher Scentific</td>
			<td style="width:161px;">S76100J</td>
			<td>Beaker</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Benchmark scientific digital hotplate</td>
			<td style="width:121px;">The Lab Depot</td>
			<td style="width:161px;">H3760H</td>
			<td>Hot plate</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Britta-filtered tap water to prepare grape agar and instant fly food</td>
			<td style="width:121px;">Any tap water filtration system</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CO2 pistol or FlyNap</td>
			<td style="width:121px;">Carolina</td>
			<td style="width:161px;"></td>
			<td>Anesthetic to sort/count adult flies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscope/stereoscope</td>
			<td style="width:121px;">Amscope</td>
			<td style="width:161px;">SM-1TSZ-V203</td>
			<td>Dissecting microscope</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Drosophila culture vials and stoppers</td>
			<td style="width:121px;">Carolina</td>
			<td align="right" style="width:161px;">173120</td>
			<td>Food vials for growing Drosophila</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Embryo collection mini cage</td>
			<td style="width:121px;">Genesee Scientific</td>
			<td style="width:161px;">59-105</td>
			<td>chamber used to gather embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Erlenmeyer flask</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td align="right" style="width:161px;">70980</td>
			<td>Erlenmeyer flask</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Formula 4-24 Instant Drosophila Medium, Blue</td>
			<td style="width:121px;">Carolina</td>
			<td style="width:161px;"></td>
			<td>https://www.carolina.com/drosophila-fruit-fly-genetics/formula-4-24-drosophila-medium-blue/FAM_173210.pr</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Grape agar</td>
			<td style="width:121px;">Genesee Scientific</td>
			<td style="width:161px;">47-102</td>
			<td>Media used for agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Instant fly flood</td>
			<td style="width:121px;">Carolina</td>
			<td align="right" style="width:161px;">173210</td>
			<td>Blue media for food vials</td>
		</tr>
		<tr height="21">
			<td></td>
			<td></td>
			<td></td>
			<td height="21" style="height:21px;">Large Petri dish or any clear container to be used as a humid chamber by placing wet paper towels to keep the grape-agar plates moist during incubation/pre and post microscopic observation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Metal spatula (small)</td>
			<td style="width:121px;">Carolina</td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microwave</td>
			<td style="width:121px;">Any</td>
			<td style="width:161px;"></td>
			<td>To cook grape agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:121px;">Kord-Valmark</td>
			<td align="right" style="width:161px;">2901</td>
			<td>Petri dish for grape agar</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stir bar</td>
			<td style="width:121px;">The Lab Depot</td>
			<td style="width:161px;">58948-981-EA</td>
			<td>Magnetic stir bar</td>
		</tr>
	</tbody>
</table>

a direct and simple method to assess drosophila melanogaster's viability from embryo to adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or complete loss of viability at different stages of development. Our lab has developed a method to assess viability in Drosophila from embryo to fully mature adult. The method relies on quantifying the number of progeny present at different stages during development, starting with hatched embryos. After embryos have been quantified, additional stages are counted, including L1/L2, pupae, and mature adults. After all stages have been examined, a statistical analysis such as the chi-square test is used to determine if there is a significant difference between the starting number of progeny (hatched embryos) and later stages culminating in the observed number of adults, thus rejecting or accepting the null hypothesis (that the number of hatched embryos will be equal to the number of larvae, pupae, and adults recorded throughout the stages of development). The primary advantage of this assay is its simplicity and accuracy, as it does not require an embryo rinse to transfer them to the food vial, avoiding losses from technical errors. Although the protocol described here does not directly examine L2/L3 larvae, additional steps can be added to account for these. Comparing the number of hatched embryos, L1, pupae, and adults can help determine if viability was compromised during the L2/L3 stages for further studies (the use of colored food helps with visual identification of larvae). Overall, this method can help Drosophila researchers and educators determine when viability is compromised during the fly life cycle. Routine assessment of stocks using this assay can prevent accumulation of secondary mutations that may affect the phenotype of the originally isolated mutant, especially if the original mutations affect fitness. For this reason, our lab maintains multiple copies of each of our Dm ime4 alleles and routinely checks the purity of each stock with this method in addition to other molecular analyses.&#160;

This method accurately and reproducibly allows one to gauge viability from embryos to emerged adults when coupled to chi square analyses.
In initial studies, after counting embryos and larvae, the grape agar was placed inside the vial upright on the side of the vial. Unfortunately, when the agar was placed on the side of the vial many of the embryos and larvae did not mature to adults. This was likely due to the grape agar disc drying out (Figure 3). This placement introduced an environmental variable (hydration of agar disc) that could confound the results. Approximately 39% of the progeny was lost between embryos to adult as only 61% of adults emerged. The biggest losses occurred between the L1 larvae (counted on surface of grape agar plates) and pupae (counted on walls of food vials) counts. However, when the grape agar was placed faced down inside the vial in direct contact with the food surface, less than 6% of the progeny was lost (Figure 2). The agar remained hydrated as the entire grape agar disc remained in contact with the moisture of the instant food inside the vial (Figure 7, Figure 8). These results indicate that the position of the agar inside the vial is important for obtaining reliable data and minimize contribution of environmental variables. Further data to support the effectiveness of the method was collected by comparing wild-type progeny used in initial method validation and progeny produced by crossing virgin wild-type females to males from a balanced Dm ime4 mutant stock (Supplemental Figure 2, ime4&#916;null/TM3sb). Approximately 91% of the progeny matured to adulthood compared to 94% of the progeny from wildtype (Figure 4).&#160; This difference was not statistically significant.
Homozygous Dm ime4 mutant males5 were also used to provide further validation of the method. However, the homozygous mutants were too sick to reproduce and died before embryos were deposited. Therefore, one limitation of the experiment is that males need to be healthy enough to reproduce to generate a starting embryo population to track.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59996/59996fig1.jpg" /> 
Figure 1: Dm ime4 mutant stocks emerge at sub-Mendelian levels. Stocks in which portions of the catalytic domain or Ado-Met binding domain were mutated are shown (refer to Supplemental Figure 1 for details). Some stocks had portions of either domain replaced with Ala (alanine-scanning mutagenesis), while other stocks had either or both domains deleted. The ratio of observed number of homozygotes to the expected numbers is represented in percentages (compared to heterozygous sibling controls, refer to Supplemental Figure 3 for sorting scheme and chi square analysis). Chi square analyses were performed to determine if the difference between observed and expected was statistically significant and not due to chance alone (p-values &#60; 0.01). Error bars represent standard error of the mean (minimum of three trials per cross indicated).&#160; A spreadsheet with all the data can be found in Supplemental Materials. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig1large.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/59996/59996fig2.jpg" /> 
Figure 2: Agar position inside the vial can introduce unintended environmental variables. Histogram of the number of individuals at different stages of development counted from grape agar disc embryo side down in food vial or agar on the side of the food vial. Placing the agar disc embryo-side down in the vial resulted in 94% (SD &#177; 0.02) of initial embryos surviving until adulthood. Placing the agar on the side of the food vial resulted in only 61% (SD &#177; 0.07) of the originally counted embryos reaching adulthood. A spreadsheet with all the data can be found in the Supplemental Materials. The expected numbers at each stage is set as the initial number of embryos/L1 counted on the grape agar disc prior to transfer to food vial (Null hypothesis: 100% of hatched embryos develop). The observed numbers are the actual number of individuals counted for the developmental stage indicated. Thus, each stage is compared to the initial number of hatched embryos counted on the grape agar plate. Chi square analyses were performed to determine if the difference between observed and expected was statistically significant and not due to chance alone using a p-value threshold of 0.05. The difference was significant for embryos grown with agar on the side of the food vial, but not for those grown in a disc embryo side down. Errors bars represent standard error (minimum of three trials per cross indicated). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig2large.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/59996/59996fig3.jpg" /> 
Figure 3: Cartoon showing two ways grape agar discs carrying embryos/L1s can be placed inside the food vial. &#160;There was a significant difference between the total number of embryos and adult progeny based on the position of the agar in the food vial. The difference stems from an environmental variable (hydration) created by the difference in disc placement inside the food vial:&#160; there is a statistically significant difference between expected and observed numbers in the larvae and pupae counts when the agar disc was placed on the side of the vial versus no significant difference in expected versus observed numbers when the agar disc was in contact with the food. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59996/59996fig4.jpg" /> 
Figure 4: Histogram showing the number of individuals at different stages of development from wild-type males versus males from a Dm ime4 mutant balanced stock. Balanced males crossed to virgin wild-type females as described in 2.1.&#160; Agar was transferred embryo-side down into food vials.&#160; The average ratio of adults emerging from the original embryos counted in grape agar discs from wild-type X wild-type was 94%, SD &#177; 0.02, whilst that ratio for Dm ime4 mutant/+ X wild-type was 91%, SD &#177; 0.01. Statistical analysis was performed as described for Figure 2; differences were not significant for either group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59996/59996fig5.jpg" /> 
Figure 5: Experimental setup. (A) Embryo collection mini-cages and grape-agar mini-Petri dishes. Plates are placed inside a humid chamber (standard Petri dish with absorbent paper disc saturated with water) to prevent dehydration.&#160; A small amount of yeast paste was placed at the center of each grape-agar plate.&#160; (B) Detach the embryo collection plate holder lid (red) and place a prepared plate, yeast side up.&#160; (C) Transfer the cross to the embryo cage and immediately place a Petri dish lid to hold flies inside.&#160; (D) Quickly remove cover and flip the embryo cage so opening contacts grape-agar plate.&#160; Incubate for 24-48 h, inspecting daily. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59996/59996fig6.jpg" /> 
Figure 6: Embryo development on grape-agar plates. (A) Wildtype embryos deposited on the grape-agar mini-Petri dishes are counted under a dissecting microscope. Keep plates inside humid chamber when not visualizing and avoid direct light to prevent dehydration.&#160; (B) Grape-agar plates are kept in a humid chamber and hatched wildtype embryos are recorded.&#160; This photo shows three wildtype L1 larvae that developed from embryos shown in A.&#160; (C) Example of recorded numbers of embryos (day 1) and L1 larvae (day 2) for the two plates shown in Figure 5.&#160; The &#34;expected number&#34;&#160;for the null hypothesis is determined by the number of embryos that hatched and became L1 larvae at the time of transfer to the food vial (table below).&#160; For wildtype flies, almost all embryos hatch and develop into L1 larvae.&#160; This may not be the case for other genetic backgrounds and this method is used to determine these differences in viability. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59996/59996fig7.jpg" /> 
Figure 7: Transferring larvae.&#160; (A) After recording L1 counts, the grape-agar discs are carefully removed from the Petri dish using a clean (ethanol-wiped) spatula (B,C) and transferred L1 side-down to contact the food in the vials as shown in D.&#160; (E)&#160; The empty Petri dish is carefully inspected to detect any larvae left behind.&#160; If larvaee left behind are alive and can be transferred to the food vial, do so carefully and immediately (F).&#160; If larvae left behind are dead or cannot be transferred, record this fact to correct the &#34;expected number&#34;&#160;for subsequent calculations. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/59996/59996fig8.jpg" /> 
Figure 8: L2 larvae make their way into the food. See crevices and grooves between the grape-agar disc and the blue food.&#160; Set up a schedule to observe the vials daily, preferably at the same time of day. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/59996/59996fig9.jpg" /> 
Figure 9: Adult emergence. (A)&#160; L3 larvae make their way out of the food to become pupae. &#160;(B) Adults start to emerge on day 11.&#160; Set up a schedule to observe the vials daily, preferably at the same time of day and carefully record your observations.&#160; Stop counting when all the pupae have emerged (count empty pupae cases) and avoid counting the next generation of flies by stopping the experiment on day 15 and count dead pupae if any (black/dried pupae).&#160; Mutants with lengthier life cycles may need longer periods of time, which need to be determined empirically. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/59996fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figures.&#160;&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/Supplemental_Figures.zip" target="_blank">Please click here to download these files.</a>
Supplemental Materials.&#160;&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59996/Supplemental_Materials.zip" target="_blank">Please click here to download these files.</a>

Watch this Scientific Journal Video about A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult at JoVE.com

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

In Drosophila melanogaster, viability assays are used to determine the fitness of certain genetic backgrounds. Allelic variations can result in partial or ...

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Nanyang Technological University

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JoVE Journal

Developmental Biology

10.2K Views.  Clarkson University. This protocol is designed to assess the viability of Drosophila at every developmental stage, from embryo to adult. The method can be used to determine and compare the viability of different genotypes or growth conditions.

Video: A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological problems after stroke and is shown to impair neurogenic processes. Interestingly, the adult zebrafish has recently emerged as a relevant and useful model to mimic hyperglycemia/diabetes and to investigate constitutive and regenerative neurogenesis. This work provides methods to develop zebrafish models of hyperglycemia to explore the impact of hyperglycemia on brain cell proliferation under homeostatic and brain repair conditions. Acute hyperglycemia is established using the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) into adult zebrafish. Chronic hyperglycemia is induced by immersing adult zebrafish in D-glucose (111 mM) containing water for 14 days. Blood-glucose-level measurements are described for these different approaches. Methods to investigate the impact of hyperglycemia on constitutive and regenerative neurogenesis, by describing the mechanical injury of the telencephalon, dissecting the brain, paraffin embedding and sectioning with a microtome, and performing immunohistochemistry procedures, are demonstrated. Finally, the method of using zebrafish as a relevant model for studying the biodistribution of radiolabeled molecules (here,[18F]-FDG) using PET/CT is also described.

This work describes methods to establish acute and chronic hyperglycemia models in zebrafish. The aim is to investigate the impact of hyperglycemia on physiological processes, such as constitutive and injury-induced neurogenesis. The work also highlights the use of zebrafish to follow radiolabeled molecules (here, [18F]-FDG) using PET/CT.

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been described, termed &#34;na&#239;ve&#34; and &#34;primed&#34; pluripotency. The mechanisms that control na&#239;ve-primed transition are poorly understood. In particular, we remain poorly informed about protein kinases that specify na&#239;ve and primed pluripotent states, despite increasing availability of high-quality tool compounds to probe kinase function. Here, we describe a scalable platform to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition in mouse ESCs. This approach utilizes simple cell culture conditions and standard reagents, materials and equipment to uncover and validate kinase inhibitors with hitherto unappreciated effects on pluripotency. We discuss potential applications for this technology, including screening of other small molecule collections such as increasingly sophisticated kinase inhibitors and emerging libraries of epigenetic tool compounds.

Here, we present a quantitative and scalable protocol to perform targeted small molecule screens for kinase regulators of the na&#239;ve-primed pluripotent transition.

Embryonic stem cells (ESCs) can self-renew or differentiate into all cell types, a phenomenon known as pluripotency. Distinct pluripotent states have been ...

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

A Direct and Simple Method to Assess Drosophila melanogaster's Viability from Embryo to Adult

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Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

A Simple Method to Identify Kinases That Regulate Embryonic Stem Cell Pluripotency by High-throughput Inhibitor Screening

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines