This research was funded by the Max Planck Society. C.B.H. was supported by a fellowship from the International Max Planck Research School &#8211; Molecular Biomedicine. We thank Shelby Blythe and Eric Wieschaus for kindly providing the eGFP-PCNA Drosophila melanogaster line.

<ol>
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The authors have nothing to disclose.

The protocol presented here is very effective at generating high-quality maps of the chromatin architecture in early Drosophila embryos. Compared to an earlier protocol34, the approach described here uses an up-to-date in situ Hi-C procedure5, resulting in quicker processing, higher resolution, and less reagent usage. The overall procedure including the in situ Hi-C protocol is expected to work on a wide range of stages and experimental systems besides Drosophila. Since the protocol has a low input requirement, it could also be used on isolated cell populations. In Drosophila, when using the protocol for embryos outside the range described here, some parameters, in particular the fixation of the material, might need to be adjusted. Since older embryos develop a highly impermeable cuticle, raising the concentration of formaldehyde and prolonging fixation may be appropriate. For collection of embryos at stages other than nuclear cycle 14, the incubation times of embryos at 25 &#176;C in step 1.4 need to be adjusted as follows: nuclear cycle 12, 70 min; nuclear cycle 13, 90 min; 3&#8211;4 hpf, 3:30 h.
During the 13 cleavage divisions (stage 1-4), the nuclei density roughly doubles with each division. The nuclei can easily be identified by their bright GFP fluorescence. During mitosis, eGFP-PCNA is not located in the nucleus, and its signal is dispersed throughout the embryo. This feature makes identifying embryos that are undergoing a synchronous cleavage division possible. For studying chromatin conformation, these mitotic embryos are usually not desirable, since the mitotic organization of chromatin is drastically different than the interphase organization35. It is possible to adapt the protocol to specifically select embryos undergoing a synchronous mitotic division. In this case, only embryos with dispersed, non-nuclear distribution of eGFP-PCNA should be kept, and all other embryos should be discarded. Since the nuclear density cannot be determined, alternative methods to stage embryos by their morphology viewed in transmitted light microscopy must be employed. Presence of pole cells and nuclei at the embryo periphery indicate that the embryo has completed at least nuclear cycle 9, whereas visible cellularization at the periphery indicates nuclear cycle 1412.
Hi-C experiments can be successfully performed using a wide selection of restriction enzymes5. Current approaches typically use enzymes that recognize either a 4-base sequence, such as MboI, or a 6-base recognition site, such as HindIII. The advantage of 4-base cutters over 6-base cutters is that they offer higher potential resolution, given enough sequencing depth, and a more even coverage of restriction sites across the genome. There is no clear advantage in choosing one 4-base cutter over another5,23,36,37. The two most commonly used enzymes, MboI and DpnII, both recognize the same GATC recognition site. DpnII is less sensitive to CpG methylation, which is of no concern in Drosophila. The protocol presented here can also be successfully completed using DpnII as a restriction enzyme. In section 4.2. restriction enzyme and buffer have to be adjusted for DpnII compatibility, according to the manufacturer&#39;s recommendations.
If the fragment size of the sequencing library deviates significantly from the range shown in Figure 2A, cluster formation during sequencing may be less efficient or fail completely. In this case, the size distribution after shearing should be checked and shearing parameters adjusted accordingly. Peaks in the distribution of DNA fragments of very small (&#60;100 bp) or very large (&#62;1,000 bp) sizes indicates problems with size selection, such as carry over of beads or supernatant that are supposed to be discarded. Often these libraries with small peaks at these undesirable sizes, such as the one pictured, are still sequenced successfully with only a minor decrease in clustering efficiency.
High rates of PCR duplication should be avoided because this drastically reduces the number of usable sequence reads. The rate of PCR duplicates is directly related to the amount of input material. Using more input therefore usually alleviates problems with PCR duplication.
Higher numbers of reads filtered due to read orientation (Figure 2B) indicate insufficient digestion, which can be the result of using too little enzyme, too much input material, or incomplete homogenization of the embryos.

Chromatin conformation capture (3C) has emerged as an exceptionally useful method to study the topology of chromatin in the nucleus1. The 3C variant Hi-C allows measuring the contact frequencies of all chromatin interactions that occur in the nucleus in a single experiment2. Application of Hi-C has played an important role in the discovery and characterization of many fundamental principles of chromatin organization, such as TADs, compartments, and loops3,4,5.
Studies of chromatin architecture in the context of developmental transitions and cell differentiation are increasingly used to unravel the mechanisms of gene regulation during these processes6,7,8,9. One of the model organisms of great interest is Drosophila melanogaster, whose development and genome are well characterized. However, few studies that investigate chromatin architecture in Drosophila outside of in vitro tissue culture settings have been conducted10,11. In embryos 16&#8211;18 h post fertilization, TADs and compartments reminiscent of similar structures in mammals were identified10, which raises the question of which role they are playing in gene regulation during Drosophila embryo development. Especially in the early stages of development, prior to gastrulation, such studies are technically challenging. Before gastrulation, Drosophila embryos undergo 13 synchronous nuclear divisions that proceed at an extremely rapid pace of 8&#8211;60 min per cycle12,13. In addition to this, the lack of visual features to distinguish the different stages make it difficult to obtain tightly staged embryo material in sufficient quantities.
In order to develop a protocol that allows studying chromatin architecture in early Drosophila development at nuclear cycle resolution, we combined two existing techniques: in situ Hi-C, which allows the generation of high resolution whole genome contact maps5, and embryo staging using a transgenic Drosophila line expressing a eGFP-PCNA transgene13,14. This transgene localizes to the nucleus during interphase and disperses throughout the syncytial blastoderm during mitosis. Using this property, it is possible to easily distinguish different stages by their nuclear density and mitotic embryos by the dispersion of the GFP signal.
Together, these techniques enable studying the three-dimensional structure of chromatin in high resolution from as few as 20 Drosophila embryos. This protocol includes the instructions for harvesting and sorting Drosophila embryos to obtain populations of embryos from a single nuclear division cycle. It further describes how the obtained embryos are used to perform in situ Hi-C. The end result is a nucleotide library suitable for sequencing on next generation sequencing machines. The resulting sequencing reads can then be processed into detailed chromatin interaction maps covering the entire Drosophila genome.

<table><tbody><tr><td>Biotin-14-dATP</td><td>Life Technologies</td><td>19524016</td><td></td></tr><tr><td>MboI</td><td>New England Biolabs</td><td>R0147L</td><td></td></tr><tr><td>DNA Polymerase I Klenow Fragment</td><td>New England Biolabs</td><td>M0210L</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>Thermo Fisher</td><td>EL0012</td><td>T4 DNA Ligase Buffer included</td></tr><tr><td>T4 DNA Polymerase</td><td>New England Biolabs</td><td>M0203L</td><td></td></tr><tr><td>Proteinase K</td><td>AppliChem</td><td>A4392</td><td></td></tr><tr><td>GlycoBlue</td><td>Life Technologies</td><td>AM9516</td><td></td></tr><tr><td>Complete Ultra EDTA-free protease inhibitors</td><td>Roche</td><td>5892791001</td><td></td></tr><tr><td>NEBNext Multiplex Oligos for Illumina&amp;nbsp;(Index Primers Set 1)</td><td>New England Biolabs</td><td>E7335</td><td>Sequencing Adaptor, Forward (unindexed) PCR primer and Reverse (indexed) PCR primer and USER enzyme used in the Library preparation section are components of this kit</td></tr><tr><td>NEBNext Ultra II DNA Library Prep Kit</td><td>New England Biolabs</td><td>E7645</td><td>End Prep Enzyme Mix, End Prep Reaction Buffer, Ligation Enhancer, Ligation Master Mix and Polymerase Master Mix used in the Library preparation section are components of this kit</td></tr><tr><td>Covaris S2 AFA System</td><td>Covaris</td><td></td><td></td></tr><tr><td>DNA LoBind Tubes, 1.5 mL</td><td>Eppendorf</td><td>0030108051</td><td></td></tr><tr><td>Falcon cell strainer 100 &amp;micro;m</td><td>Corning</td><td>352360</td><td>Embryo collection baskets</td></tr><tr><td>37% formaldehyde</td><td>VWR</td><td>437536C</td><td></td></tr><tr><td>Heptane</td><td>AppliChem</td><td>122062.1612</td><td></td></tr><tr><td>M165 FC fluorescent stereo microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>M165 FC DFC camera</td><td>Leica</td><td></td><td></td></tr><tr><td>Metal micro pestle</td><td>Carl Roth</td><td>P985.1</td><td>Used to lyse embryos in step 4.1.4</td></tr><tr><td>RNase A</td><td>AppliChem</td><td>A3832,0050</td><td></td></tr><tr><td>Dynabeads MyOne Streptavidin C1</td><td>Life Technologies</td><td>65002</td><td>Streptavidin coated magnetic beads</td></tr><tr><td>Ampure XP beads</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>Qubit 3.0 Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33216</td><td></td></tr><tr><td>Qubit assay tubes</td><td>Thermo Fisher Scientific</td><td>Q32856</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher Scientific</td><td>Q32854</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P4417</td><td></td></tr><tr><td>eGFP-PCNA flies</td><td>Gift from S. Blythe and E. Wieschaus</td><td></td><td></td></tr><tr><td>Sodium hypochlorite 13%</td><td>Thermo Fisher</td><td>AC219255000</td><td></td></tr><tr><td>Triton X-100</td><td>AppliChem</td><td>A4975</td><td></td></tr><tr><td>Tris buffer pH 8.0 (1 M) for molecular biology</td><td>AppliChem</td><td>A4577</td><td></td></tr><tr><td>NaCl</td><td>AppliChem</td><td>A2942</td><td></td></tr><tr><td>IGEPAL CA-630</td><td>Sigma-Aldrich</td><td>I8896</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>Greiner Bio-One</td><td>616201</td><td></td></tr><tr><td>SDS for molecular biology</td><td>AppliChem</td><td>A2263</td><td></td></tr><tr><td>10x CutSmart buffer</td><td>New England Biolabs</td><td>B7204S</td><td>Restriction enzyme buffer</td></tr><tr><td>PCR Nucleotide Mix</td><td>Sigma-Aldrich</td><td>11814362001</td><td>Unmodified dCTP, dGTP, dTTP</td></tr><tr><td>BSA, Molecular Biology Grade</td><td>New England Biolabs</td><td>B9000S</td><td></td></tr><tr><td>EDTA 0.5 M solution for molecular biology</td><td>AppliChem</td><td>A4892</td><td></td></tr><tr><td>Sodium acetate 3 M pH 5.2</td><td>Sigma-Aldrich</td><td>S7899</td><td></td></tr><tr><td>DynaMag-2 Magnet</td><td>Life Technologies</td><td>12321D</td><td>Magnetic stand</td></tr><tr><td>Intelli-Mixer RM-2L</td><td>Omnilab</td><td>5729802</td><td>Rotator</td></tr><tr><td>ThermoMixer F1.5</td><td>Eppendorf</td><td>5384000012</td><td>Mixer</td></tr><tr><td>Small Embryo Collection Cages</td><td>Flystuff.com</td><td>59-100</td><td>Egg collection cage</td></tr><tr><td>Centrifuge 5424&amp;nbsp;R</td><td>Eppendorf</td><td>5404000413</td><td></td></tr><tr><td>C1000 Touch Thermal Cycler</td><td>Bio-Rad</td><td>1851148</td><td></td></tr><tr><td>PCR tube strips</td><td>Greiner Bio-One</td><td>673275</td><td></td></tr><tr><td>NEBuffer 2.1</td><td>New England Biolabs</td><td>B7202S</td><td>T4 DNA Polymerase buffer</td></tr></tbody></table>

generation of genome-wide chromatin conformation capture libraries from tightly staged early drosophila embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Sorted embryo populations at nuclear cycle 12, 13, and 14 (corresponding to 1:30, 1:45, and 2:10 hours post fertilization, respectively12) and 3&#8211;4 h post fertilization (hpf) were obtained according to the procedures described in the protocol. By taking pictures of the eGFP-PCNA signal of each sorted embryo batch, it is possible to document the precise stage and cell cycle status of every single embryo that is used in downstream experiments. Example pictures of embryos from sorted populations are shown in Figure 1B-E. The output of the in situ Hi-C protocol is a nucleotide library ready to be sequenced on next generation sequencing machines. For this purpose, a final library concentration of at least 2&#8211;4 nM is usually required. Using the recommended amounts of input material, this concentration is reliably achieved (Table 1).
The expected size distribution of DNA fragments after size selection is between 300&#8211;600 bp, with a maximum at around 500 bp (Figure 2A), depending on the exact shearing and size selection parameters. For sequencing, we recommend paired-end reads of at least 75 bp length to minimize the number of unmappable restriction fragments in the genome. High-resolution maps with 1&#8211;2 kb bin size can be obtained from 400 million reads. We recommend sequencing multiple biological replicates at a lower depth of ~150 million reads each, instead of sequencing a single replicate at very high depth. This allows assessment of the biological variation and leads to a lower number of discarded reads due to PCR duplication. For visual representation, the replicates can be combined. Before committing to sequencing a sample at high depth, we recommend running samples using shallow sequencing (a few million reads per sample) to determine basic library quality parameters as in Figure 2B.
Analysis of Hi-C data requires significant computational resources and bioinformatics expertise. As a rough overview, the paired reads are mapped independently to the reference genome, the resulting alignments are filtered for quality and orientation, then a matrix of contacts at a given bin resolution or fragment level can be generated from the filtered alignments. The contact matrix is the basis for all further downstream analysis exploring TADs, loops, and compartments. For the initial analysis of the sequencing reads, several bioinformatics pipelines are available that enable processing of raw reads into contact matrices without much specialized bioinformatics knowledge18,19,20,21,22,23. How further analysis is carried out depends largely on the exact biological question under study and might require significant experience in programming and scripting in R or Python. However, several tools and algorithms to call TADs are available5,24,25,26,27,28, as well as software to analyze and explore Hi-C data in the web browser and as stand-alone desktop applications29,30,31,32.
Once processed, the quality of the library can be determined using different metrics (Figure 2B). First, the rate of PCR duplicates, which is the number of sequenced read pairs arising from the same original molecule, should be as low as possible to limit the amount of wasted sequence reads. However, even libraries with &#62;40% PCR duplication can be processed into high-quality contact maps if the duplicates are filtered. Second, the rate of filtered reads due to their orientation, as described in4, should consistently be lower than 10% of aligned read pairs.
During pre-gastrular development of Drosophila between nuclear cycle 12 and 14, the nuclear architecture is drastically remodeled33 (Figure 3). At nuclear cycle 12, few TADs are detected, and the overall distribution of contacts is very smooth without many discernable features. This is dramatically changed at nuclear cycle 13 and 14, when TADs are increasingly prominent and unspecific long-range contacts are depleted.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57001/57001fig1.jpg" /> 
Figure 1: Representative pictures of eGFP-PCNA embryos during sorting. (A) eGFP-PCNA signal from an unsorted population of embryos after 60 min collection and 2 h incubation at 25 &#176;C (B-E) Examples of embryos from sorted populations at nuclear cycle 12 (B), nuclear cycle 13 (C), nuclear cycle 14 (D), and from embryos undergoing synchronous mitosis (E). Scale bars = 200 &#181;m. <a href="https://cloudflare.jove.com/files/ftp_upload/57001/57001fig1large.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/57001/57001fig2.jpg" /> 
Figure 2: Examples of in situ Hi-C library quality metrics. (A) Bioanalyzer traces showing the distribution of DNA fragment sizes from a successful Hi-C library (Library 1, top) and from a library that displays a peak of fragments that are too large for sequencing (Library 2, bottom). Library 2 was successfully sequenced, but even larger amounts of undesired DNA fragments may lead to decreased sequencing yields. (B) Filtering statistics of two Hi-C libraries: displayed is the number of aligned read pairs that are excluded from further analysis due to read orientation and distance (inward, outward)4 or PCR duplication (duplicate). In each bar, the number of reads passing the filter (remaining) and failing (filtered) are plotted. The percentage of reads passing the filter is additionally shown as text. <a href="https://cloudflare.jove.com/files/ftp_upload/57001/57001fig2large.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/57001/57001fig3.jpg" /> 
Figure 3: Hi-C interaction maps from staged embryos. Hi-C interaction maps are binned at 10 kb resolution and balanced as described before33. Shown is a region on chromosome 2L. <a href="https://cloudflare.jove.com/files/ftp_upload/57001/57001fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Library</td>
			<td>Stage</td>
			<td>Number of embryos</td>
			<td>Amount DNA before shearing (ng)</td>
			<td>PCR cycles</td>
			<td>Final library concentration (nM)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>nuclear cycle 12</td>
			<td>71</td>
			<td>46</td>
			<td>12</td>
			<td>28.2</td>
		</tr>
		<tr>
			<td>2</td>
			<td>nuclear cycle 12</td>
			<td>46</td>
			<td>40</td>
			<td>12</td>
			<td>22.2</td>
		</tr>
		<tr>
			<td>3</td>
			<td>nuclear cycle 12</td>
			<td>60</td>
			<td>13</td>
			<td>13</td>
			<td>12.3</td>
		</tr>
		<tr>
			<td>4</td>
			<td>nuclear cycle 13</td>
			<td>36</td>
			<td>39</td>
			<td>12</td>
			<td>22.2</td>
		</tr>
		<tr>
			<td>5</td>
			<td>nuclear cycle 13</td>
			<td>35</td>
			<td>10</td>
			<td>12</td>
			<td>5.0</td>
		</tr>
		<tr>
			<td>6</td>
			<td>nuclear cycle 13</td>
			<td>48</td>
			<td>18</td>
			<td>12</td>
			<td>8.7</td>
		</tr>
		<tr>
			<td>7</td>
			<td>nuclear cycle 14</td>
			<td>33</td>
			<td>30</td>
			<td>12</td>
			<td>39.8</td>
		</tr>
		<tr>
			<td>8</td>
			<td>nuclear cycle 14</td>
			<td>24</td>
			<td>36</td>
			<td>12</td>
			<td>20.4</td>
		</tr>
		<tr>
			<td>9</td>
			<td>nuclear cycle 14</td>
			<td>14</td>
			<td>8</td>
			<td>12</td>
			<td>4.2</td>
		</tr>
		<tr>
			<td>10</td>
			<td>3-4 hpf</td>
			<td>17</td>
			<td>30</td>
			<td>12</td>
			<td>24.0</td>
		</tr>
		<tr>
			<td>11</td>
			<td>3-4 hpf</td>
			<td>18</td>
			<td>42</td>
			<td>11</td>
			<td>19.1</td>
		</tr>
		<tr>
			<td>12</td>
			<td>3-4 hpf</td>
			<td>22</td>
			<td>63</td>
			<td>11</td>
			<td>48.4</td>
		</tr>
	</tbody>
</table>
Table 1: List of representative sequencing library statistics. For each library in the list, the number of embryos that were used for its generation, the amount of total DNA before biotin pulldown and shearing measured by Qubit, the number of PCR cycles used for amplification, and the final concentration of the sequencing library after purification and size selection are indicated.

Watch this Scientific Journal Video about Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos at JoVE.com

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

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JoVE Journal

Genetics

19.8K Views.  Max Planck Institute for Molecular Biomedicine. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het gebied van chromatinearchitectuur, zoals hoe de 3D-structuur van chromatine de genexpressie en -ontwikkeling kan beïnvloeden. Het belangrijkste voordeel van deze techniek is dat het een hoge resolutie weergave van chromatine architectuur en precies geënsceneerde vliegen embryo's biedt. Hoewel deze methode inzicht kan geven in chromatineorganisatie en -ontwikkeling, kan men ook bestaande lijnen uit transgene vliegbibl...

Video: Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq&#8212;the most popular approach to investigate chromatin features at a global level&#8212;is outlined here for Xenopus embryos.

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

Developmental Biology

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Biology

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

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