We thank N. &#214;zel and R. Hiesinger for their advice on the explant culture; M. Wagner for technical help of the two-photon microscopy; D.J. Luginbuhl for generating transgenic flies; D. Friedmann for suggestions of Fiji software analysis; Y. Ge for assistance on fly work; C. McLaughlin and K.K.L. Wong for comments on the manuscript. L.L. is a Howard Hughes Medical Institute investigator. This work was supported by National Institutes of Health grants 1K99DC01883001 (to T.L.) and R01-DC005982 (to L.L.).

1. Preparation of reagents
NOTE: All the steps in this protocol are carried out at room temperature (20-25 &#176;C) unless explained otherwise.
<ol>
	<li>To prepare the culture dish for immobilizing the explant during time lapse imaging, lay 0.5 cm thick Sylgard (thoroughly mix two liquid components at 10:1 ratio before use) on the bottom surface of a 60 mm x 15 mm Petri dish and let it cure for 48 h at room temperature (Figure 2A, referred as Sylgard plate in the following text).</li>
	<li>To prepare micro pins for immobilizing explant on this plate, use a pair of forceps to stick multiple micro pins on a tape with the sharp ends aligned on one side (Figure 2B). Use a pair of scissors to cut ~2 mm from the sharp ends of the micro pins (Figure 2B&#39;). Use forceps to hold the cut micro pins and insert into the Sylgard layer of a pre-made Sylgard plate (Figure 2C). Two micro pins are used to immobilize one explant.</li>
	<li>Use a brush to collect white pupae, which form puparium within 1 h, of hsFLP, pebbled-GAL4/+; UAS-FRT100-stop-FRT100-mCD8-GFP8 genotype and transfer them to new vials. Heat shock in 37 &#176;C water bath for 40 min to induce sparse ORN clones from random types. After heat shock, put the vials at 25 &#176;C for 30 h, resulting in pupae aged at 30 h after puparium formation (APF).</li>
	<li>To prepare the culture medium for explant, add 5 mL of Penicillin-Streptomycin (10,000 U/mL) to 500 mL Schneider&#39;s Drosophila Medium. Filter the medium and make 45 mL aliquots in 50 mL conical tubes. The medium can be stored at 4 &#176;C for 1-2 months.</li>
	<li>On the day of imaging, take one tube of 45 mL of Schneider&#39;s Drosophila medium and add 5 mL of Fetal Bovine Serum (10% v/v), 125 &#181;L of 4 mg/mL human insulin stock solution (10 &#181;g/mL final concentration), 50 &#181;L of 1 mg/mL 20-hydroxyecdysone stock solution dissolved in ethanol (1 &#181;g/mL final concentration). Mix well and transfer 15 mL of full medium into a new 50 mL conical tube. The rest of the full medium can be stored at 4 &#176;C for a week. Fetal Bovine Serum, human insulin stock solution and 20-hydroxyecdysone stock solution are aliquoted and stored at -20 &#176;C.</li>
	<li>Oxygenate the 15 mL full medium by pumping oxygen bubbles from an oxygen cylinder under the liquid surface through a sterile 5 mL pipette tip at the rate of one bubble/s for 20-30 min. Use a paraffin film to cover the opening of the tube during this process.</li>
	<li>Sterilize the dissection well surface and the Sylgard plate (with micro pins inserted on the Sylgard layer, prepared in steps A1 and A2) with 70% ethanol. Let them dry before use.</li>
</ol>
2. Explant dissection
<ol>
	<li>Use a brush to transfer 30 h APF (30 h after puparium formation) pupae to a paper tissue and dry the external surface of the pupae for 5 min.</li>
	<li>Put a piece of double-sided tape on a glass slide. Carefully attach the dried pupae on the sticky surface of the tape with the dorsal side facing upward. Gently press the pupae with a brush to help the ventral side of the pupae attach well to the tape (Figure 3A). Do not damage the pupa.</li>
	<li>Use a pair of forceps to remove brown pupal case covering the dorsal side of the head (Figure 3A,B). Insert one sharp tip of the forceps between the brown pupal case and the pupa from the lateral side and carefully break the brown pupal case through a line to the posterior end of the pupa (Figure 3B,C). Open the brown pupal case. Use a pair of forceps to gently hold the pupa and transfer to the dissection well with 1 mL of oxygenated full medium. Submerge floating pupa on the medium surface to help it sink to the bottom of the well (Figure 3D). 
	NOTE: Do not insert the forceps tip too deeply inside the brown pupal case to prevent injuring the pupa with the forceps.</li>
	<li>To dissect the antennae-brain explant from the pupa, use forceps to gently hold the pupa with one hand and use a pair of microscissors to cut a small hole from the posterior side of the pupa with the other hand (Figure 3E). This small hole releases the high pressure inside the pupa.</li>
	<li>Cut through the ventral midline of the pupa from the hole until the neck (the narrow structure that connects the head and the thorax) with the microscissors (Figure 3F). Then, cut through the circumference of the neck to detach the head from the body of the pupa (Figure 3G). Remove the body and place it in a different well. 
	NOTE: Do not cut the neck directly from the dorsal/ventral side of the pupa, which may squeeze the brain.</li>
	<li>Cut the transparent cuticle that covers the dorsal side of the brain (Figure 3H). This will expose the fat body on top of the brain. Keep some cuticle to which the retina and antennae attach. Repeat the same procedure to the ventral side of the brain. 
	NOTE: Do not insert the blade of the scissors too deeply under the cuticle as this will cause severing of the antennal nerves connecting the antennae and brain (Figure 3H&#39;).</li>
	<li>Use a P10 pipette to gently wash out the fat body that covers the brain and antennae by pipetting the medium toward the open regions on the dorsal and ventral sides of the head (Figure 3I). 
	NOTE: Be very gentle when pipetting the medium as the brain can easily be detached from the cuticle. Make sure all fat body is removed during this step. Arrested development of ORN axons were observed when fat body was not cleaned well, probably due to poor oxygen access from the medium.</li>
	<li>To study the interaction of bilateral ORN axons or ORN axons to PN dendrite targeting, sever one or two antennal nerves with the microscissors during this stage8 (Figure 3J). Carefully place the blades of the scissors between the cuticle and the brain and sever interested antennal nerves.</li>
	<li>To transfer the dissected explant to the Sylgard plate, place a droplet of oxygenated full medium (~200 &#181;L) on the Sylgard surface. Coat the inner surface of a 200 &#181;L wide tip pipette tip with the fat body from the dissected trunk (step 1.6) by pipetting the fat body several times, which prevents the explants from sticking the pipette tip during transfer. Then, use this wide tip pipette tip to transfer the explant from the dissection well to the medium droplet on the culture plate (Figure 3K).</li>
	<li>Use forceps to pin the explant on the Sylgard layer in the two optic lobes (Figure 3L). Carefully position the Sylgard plate on the imaging station and immobilize the plate with tapes. Slowly add 10 mL of oxygenated full medium to the Sylgard plate using P1000 pipette. 
	&#8203;NOTE: Avoid disrupting the explant when adding the medium to the Sylgard plate.</li>
</ol>
3. Two-photon microscopy-based live imaging
<ol>
	<li>To perform time-lapse imaging, use a two-photon microscope, a Ti:Sapphire laser, a 20x water-immersion objective (1.0 NA) and an imaging software. Use the excitation wavelength at 920 nm for imaging GFP proteins. Adjust the pixel dwell time to 10 &#181;s.</li>
	<li>Adjust the imaging station position so that the explants are roughly under the objective. Use 70% ethanol to sterilize the lens before imaging. Slowly lower the objective under the medium close to the explants. Check whether there is any bubble on the lens of objective.
	<ol>
		<li>If so, lift the objective above the medium and repeat this until the bubble is gone. Find the explants using the eyepiece and center one explant in the field.</li>
	</ol>
	</li>
	<li>To ensure an explant with a few ORNs sparsely labeled for time lapse imaging, dissect ~10 explants each time and align on y axis on the culture plate. Screen all explants by moving the objective along y axis and choose an explant in which a few single ORN axons have just reached the antennal lobe for imaging (Figure 4A).
	<ol>
		<li>Recognize the antennal lobe by its oval shape and the ORN axons that are beginning to circumnavigate it. Image a ~150 &#181;m x 150 &#181;m area in the xy plane (3x zoom with the 20x objective). Estimate the boundary of the two antennal lobes and center them in the imaging area.</li>
	</ol>
	</li>
	<li>Select an initial imaging region along the z axis by defining the bottom section and top section of scanning. Set up imaging area along the z axis. Set the deepest section with ORN axon signals as the first imaging session and the session 100 &#181;m above (more superficial side) as the last imaging session (Figure 4B). 
	NOTE: This leaves some sections on top (superficial side) of the ORN axons and avoids shifting of ORN axons upward outside the imaging area due to the growth of the brain during culture.
	<ol>
		<li>Image at 2 &#181;m intervals. Set automatic imaging scanning at the frequency of every 20 min using imaging software.</li>
	</ol>
	</li>
	<li>Shift the imaging region 20 &#181;m upward along the z axis after the first 4 h imaging and another 20 &#181;m upward along the z axis after 16 h imaging. This can be achieved by setting a script and different z stacks in the imaging software.</li>
	<li>Culture the explant for an additional period post imaging (up to 24 h ex vivo) before fixation and staining with N-cadherin, a neuropil marker, to reveal the genetic identity of each single ORN by the glomerulus it targets to.</li>
</ol>
4. Image processing
<ol>
	<li>To process z stack images from section series taken at each time point using the Fiji software, open image section series, click on Image | Stacks | Z project [/].</li>
	<li>To correct lateral drift of the sample during culture, install TurboReg Plugin in Fiji.
	<ol>
		<li>Open a z stack image series and a single z stack image from the series. Open Plugins | Registration | TurboReg.</li>
		<li>Select the z stack image series in Source and the single z stack image in Target | Translation. Click on the Batch button to register all the images from the opened z stack image series.</li>
	</ol>
	</li>
	<li>To maximize the utility of imaged samples, separate sparsely labeled single axons in the vicinity to each other from the z stack images following 3D image sections.
	<ol>
		<li>To extract single ORN axons from a few axons in the same image, open the image section series, click on Plugins | Segmentation | Segmentation Editor [/]. Select the brush tool and mask interested ORN axon in the Segmentation Editor working window using Select &#34;+&#34; or &#34;-&#34; buttons on each image section.</li>
		<li>Click on Process | Image Calculator [/]. Select &#34;Image X&#34; in Image 1, &#34;Multiply&#34; in Operation, &#34;Image X. labels&#34; in Image 2, [/]. This generates a new image series file with the interested axon only. Perform step 4.1 to process the z stack image. Repeat this step for all time points to generate a time series image file.</li>
	</ol>
	</li>
	<li>To pseudocolor different axons from the same image, first perform step 4.3 to generate the time series image file of each axon separately. Open the time series image files for different single axons from the same raw data image. Click on Image | Color | Merge Channels. Select different time series image files in different color channels and click OK.</li>
</ol>

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The authors have nothing to disclose.

The Drosophila antennae-brain explant retains normal targeting of the olfactory circuit. We did notice that the development is 2 times slower ex vivo compared to in vivo. It is noted that the explant system does not retain maxillary palp, which hosts six types of ORNs. To ensure normal development is recapitulated ex vivo, stretching of the antennal nerves needs to be avoided during explant dissection. During ex vivo culture bacteria growth usually causes arrested development ...

Neurons are precisely interconnected to form circuits essential for the proper function of the brain. For over 100 years, neuroscientists have been trying to understand how neurites extend toward their intermediate and final targets with extreme precision. As a result, they have identified important genes that encode guidance cues for developing neuronal processes1. The Drosophila olfactory system provides an excellent model to investigate this process since olfactory receptor neurons (ORNs, the primary sensory neurons) project to 50 identifiable glomeruli with stereotypical size, shape, and relative position, where they form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs), each of which send dendrites to one of the 50 glomeruli2 (Figure 1A). Therefore, it is relatively easy to identify mutant phenotypes at synaptic (glomerular) resolution in the fly olfactory system. This led to discoveries of important genes that regulate olfactory circuit assembly3.
The assembly of the fly olfactory circuit relies on temporally and spatially coordinated developmental processes3. ORNs and PNs acquire distinct cell fates, which set up the program for their wiring specificities. Next, PN dendrites prepattern the antennal lobe (Figure 1B). The axons of ORNs then circumnavigate the ipsilateral antennal lobe and cross the midline of the brain to reach the contralateral antennal lobe. Subsequently, ORN axons invade both ipsi- and contralateral antennal lobes and form synapses with dendrites of their partner PNs in specific glomeruli. This coarse model for olfactory circuit assembly was proposed based on the characterization of fixed samples from intermediate time points during the development. The poor temporal resolution and inability to follow the same neuronal processes across development from fixed tissue limit the mechanistic understanding of the circuit assembly process.
It is technically challenging to live image ORN and PN processes in vivo since the wiring process occurs in the first half of the pupal stage when the antennal lobe is surrounded by opaque fat body inside the pupal case. It is, therefore, impossible to directly image the developing olfactory circuit from intact pupae. Dissected tissues cultured ex vivo can circumvent tissue opacity and have been successfully used to study neural development4,5,6. The challenge of using a similar ex vivo explant culture strategy to study neuronal wiring in the pupal brain is whether it recapitulates the precise neuron targeting in a culture condition. Based on a previously reported ex vivo culture condition for the fly eye-brain complex7, an explant that contains the whole pupal brain, antennae, and the connecting antennal nerves intact has been recently developed, which retains precise targeting of the olfactory circuit and can be subjected to two-photon microscopy-based live imaging for up to 24 h at the frequency of every 20 min8. Here, a detailed protocol of the explant culture and imaging is described. The explant system provides a powerful method to study the assembly of olfactory circuit and potentially other circuits in the central brain.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20-hydroxyecdysone</td>
			<td>Sigma</td>
			<td>H5142</td>
			<td></td>
		</tr>
		<tr>
			<td>Chameleon Ti:Sapphire laser</td>
			<td>Coherent</td>
			<td>Coherent MRU X1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Human insulin</td>
			<td>Thermo Fisher Scientific</td>
			<td>12585014</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Prairie</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors</td>
			<td>World Precision Instruments</td>
			<td>501778</td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen cylinder</td>
			<td>Praxair</td>
			<td>OX M-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#8217;s Drosophila Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21720024</td>
			<td></td>
		</tr>
		<tr>
			<td>SYLGARD 184 Silicone Elastomer</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC0162601</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-photon microscopy</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>water immerse objective (20X)</td>
			<td>Zeiss</td>
			<td>421452-9800-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

an explant system for time-lapse imaging studies of olfactory circuit assembly in drosophila

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an excellent model to investigate this process since 50 types of olfactory receptor neurons (ORNs) from the antennae and maxillary palps project their axons to 50 identifiable glomeruli in the antennal lobe and form synaptic connections with dendrites from 50 types of second-order projection neurons (PNs). Previous studies mainly focused on identifying important molecules that regulate the precise targeting in the olfactory circuit using fixed tissues. Here, an antennae-brain explant system that recapitulates key developmental milestones of olfactory circuit assembly in culture is described. Through dissecting the external cuticle and cleaning opaque fat bodies covering the developing pupal brain, high quality images of single neurons from live brains can be collected using two-photon microscopy. This allows time-lapse imaging of single ORN axon targeting from live tissue. This approach will help reveal important cell biological contexts and functions of previously identified important genes and identify mechanisms underpinning the dynamic process of circuit assembly.

ORN axons arrive at the antennal lobe between 18 h and 36 h APF. They then navigate the antennal lobe, cross the midline, and innervate the glomeruli. Video 1 is a representative video showing the entire process for several individually identifiable axons, taken at the frequency of every 20 min for 24 h. Before registration using TurboReg, the axons exhibit some lateral drifting as the brain develops (first half of the video). After registration, the drifting is corrected (second half of the video).
...

Watch this Scientific Journal Video about An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila at JoVE.com

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

~Neurons are precisely interconnected to form circuits essential for the proper function of the brain. The Drosophila olfactory system provides an ...

an-explant-system-for-time-lapse-imaging-studies-olfactory-circuit

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Developmental Biology

2.1K Views.  Stanford University. This protocol describes the dissection procedure, culture condition, and live imaging of an antennae-brain explant system for the study of the olfactory circuit assembly.

Video: An Explant System for Time-Lapse Imaging Studies of Olfactory Circuit Assembly in Drosophila

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...