Name: isolation of preadipocytes from broiler chick embryos
Uploaded: 2022-08-04T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Preadipocytes from Broiler Chick Embryos at JoVE.com

The authors thank UT AgResearch and the Department of Animal Science for supporting and optimizing this protocol. This work was funded by USDA grant.

All animal procedures were approved by the University of Tennessee Institutional Animal Care and Use Committee. Freshly fertilized commercial broiler eggs (Cobb 500) were obtained from a local hatchery. Eggs were incubated at 38 &#176;C with 60% relative humidity until dissections at embryonic days 16-18 (E16-E18). Adipose tissue was collected from the subcutaneous (femoral) depot.
1. Preparation for isolation and culture
<ol>
	<li>Prepare the culture hood and the instrument...

<ol>
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Although several well-described protocols have reported the isolation of preadipocytes14,15,16,17, isolation for embryonic preadipocytes has been optimized, which can be used for functional analyses of early life fat growth and development in broiler chicks. This protocol yields high viability embryonic adipocyte progenitors with high differentiation potential. Moreover, the presented procedure...

Obesity is a global health threat to both adults and children. Children who are overweight or obese are approximately five times more likely to be obese as adults, placing them at significantly increased risk for cardiovascular disease, diabetes, and many other comorbidities. About 13.4% of US children aged 2-5 have obesity1, illustrating that the tendency to accumulate excess body fat can be set in motion very early in life. For very different reasons, the accumulation of excess adipose tissue is a concern for broiler (meat-type) chickens. Modern broilers are incredibly efficient but still accumulate more lipid than is physiologically necessary2,3. This tendency begins soon after hatch and effectively wastes feed, the most expensive production component, by allocating it away from muscle growth. Therefore, for both children and broiler chickens, albeit for very different reasons, there is a need to understand factors that influence adipose tissue development and identify ways to limit adipose expansion early in life.
Adipocytes form from preadipocytes, adipose tissue-derived stem cells that undergo differentiation to develop mature, lipid-storing fat cells. Accordingly, preadipocytes in vitro are a valuable experimental model for obesity studies. These cells, isolated from the stromal vascular fraction of adipose depots, can provide a fundamental understanding of molecular pathways controlling adipocyte differentiation and metabolism4,5. Chick embryos are a favorable experimental model in developmental studies because culturing eggs on the desired schedule makes experimental manipulation easier, as it enables obtaining embryos without the mother's sacrifice to observe a series of developmental stages of embryos. Moreover, complicated surgical procedures and lengthy periods of time are not required to obtain embryos relative to larger animal models. Therefore, the chick embryo presents an opportunity to obtain preadipocytes from the earliest stages of adipose tissue development. Subcutaneous adipose tissue becomes visible in the chick around embryonic day 12 (E12) as a clearly defined depot located around the thigh. This depot is enriched in highly proliferative preadipocytes that actively undergo differentiation under developmental cues to form mature adipocytes6,7. The process of adipogenic differentiation is comparable between chickens and humans. Therefore, preadipocytes isolated from chick embryos can be used as a dual-purpose model for studies relevant to humans and poultry. However, the yield of preadipocytes declines with aging as cells grows into mature adipocytes5.
The present protocol optimizes the isolation of preadipocytes from adipose tissue during the stage (E16-E18) at which adipogenic differentiation and adipocyte hypertrophy are at their peak in broiler chick embryos8. This procedure can assess the effects of factors to which the developing embryo is exposed in ovo, such as the hen diet, on adipocyte development and adipogenic potential ex vivo. It can also test the impact of various manipulations (e.g., hypoxia, nutrient additions, pharmacological agonists, and antagonists) on adipogenesis or the various 'omes (e.g., transcriptome, metabolome, methylome) of adipocyte progenitors. As a representation of the earliest stage of adipose formation, cells obtained using this protocol are valuable models for studies relevant to poultry and humans.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Pipette</td>
			<td>Eppendorf</td>
			<td>Z683825</td>
			<td>Single Channel Pipette, 100 - 1000 &#181;L</td>
		</tr>
		<tr>
			<td>1 mL Pipette Tip</td>
			<td>Fisher Scientific</td>
			<td>02-707-402</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>A426P4</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL Flask</td>
			<td>Pyrex</td>
			<td>4980-25</td>
			<td></td>
		</tr>
		<tr>
			<td>37% Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>F75P-1GAL</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Well Plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td>Tissue Culture-treated</td>
		</tr>
		<tr>
			<td>96-Well Assay Plate</td>
			<td>Costar</td>
			<td>3632</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Plate, Black Bottom</td>
			<td>Costar</td>
			<td>3603</td>
			<td>Tissue Culture-treated</td>
		</tr>
		<tr>
			<td>AdipoRed</td>
			<td>Lonza</td>
			<td>PT-7009</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gibco</td>
			<td>15290026</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench Top Wiper (Kimtechwiper)</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Up &#38; Up</td>
			<td>NDC 1167300334</td>
			<td>20% Working Solution</td>
		</tr>
		<tr>
			<td>Cell Counter</td>
			<td>Corning</td>
			<td>6749</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer, 40 &#181;m</td>
			<td>SPL</td>
			<td>93040</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifugaton</td>
			<td>Eppendorf</td>
			<td>5702</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken Serum</td>
			<td>Gibco</td>
			<td>16110082</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Centrifuge Tubes, 15 mL</td>
			<td>VWR</td>
			<td>10025-690</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Centrifuge Tubes, 50 mL</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovial</td>
			<td>Nunc</td>
			<td>343958</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Forceps, 100 mm</td>
			<td>Roboz Surgical</td>
			<td>RS-5137</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Surgical Scissors, 115 mm</td>
			<td>Roboz Surgical</td>
			<td>RS-6839</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>Millipore</td>
			<td>SYNSV0000</td>
			<td>Despensed as needed</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>HyClone</td>
			<td>SH30023.01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2701</td>
			<td>70% Working Solution</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Evos</td>
			<td>M7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Plate Reader</td>
			<td>Biotek</td>
			<td>Synergy H1</td>
			<td></td>
		</tr>
		<tr>
			<td>Foil</td>
			<td>Reynolds</td>
			<td></td>
			<td>Reynolds Wrap Heavy Duty Aluminum Foil, 125 SQ. FT.</td>
		</tr>
		<tr>
			<td>Freezing Container</td>
			<td>Thermo Scientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Millipore</td>
			<td>4055</td>
			<td>2% Working Solution</td>
		</tr>
		<tr>
			<td>Hematocytometer (Counting Chamber)</td>
			<td>Corning</td>
			<td>480200</td>
			<td>0.1 mm deep</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Fisher Scientific</td>
			<td>6845</td>
			<td></td>
		</tr>
		<tr>
			<td>Instrument Sterilizer</td>
			<td>VWR</td>
			<td>B1205</td>
			<td></td>
		</tr>
		<tr>
			<td>Linoleic Acid-Oleic Acid-Albumin</td>
			<td>Sigma</td>
			<td>L9655</td>
			<td>1x Working Solution</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Evos</td>
			<td>AMEX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Channel Pipette</td>
			<td>Thermo Scientific</td>
			<td>4661070</td>
			<td>12-Channel Pipetters, 30 - 300 &#181;L</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma</td>
			<td>S-7907</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma</td>
			<td>S-3139</td>
			<td></td>
		</tr>
		<tr>
			<td>NucBlue</td>
			<td>Invitrogen</td>
			<td>R37605</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma</td>
			<td>O-0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>IKA</td>
			<td>KS130BS1</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper Towel</td>
			<td>Tork</td>
			<td>RK8002</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Parafilm M</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Steptomycin (P/S)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>1x Working Solution</td>
		</tr>
		<tr>
			<td>Petri dishes, 100 mm</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, 60 mm</td>
			<td>Falcon</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate Shaker</td>
			<td>VWR</td>
			<td>200</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC Lysis Buffer</td>
			<td>Roche</td>
			<td>11814389001</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent Reservior</td>
			<td>VWR</td>
			<td>89094-680</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Beaker, 100 mL</td>
			<td>Pyrex</td>
			<td>1000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer Plate Reader</td>
			<td>Biotek</td>
			<td>Synergy H1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Gauze</td>
			<td>McKesson</td>
			<td>762703</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Forceps, 120 mm</td>
			<td>Roboz Surgical</td>
			<td>RS-4960</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight Scissors, 140 mm</td>
			<td>Roboz Surgical</td>
			<td>RS-6762</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 Flask</td>
			<td>Corning</td>
			<td>430639</td>
			<td>Tissue Culture-treated</td>
		</tr>
		<tr>
			<td>Tissue Culture Incubator</td>
			<td>Thermo Scientific</td>
			<td>50144906</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Strainer, 250 &#181;m</td>
			<td>Pierce</td>
			<td>87791</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>15400054</td>
			<td>0.1% Working Solution</td>
		</tr>
		<tr>
			<td>Tweezers, 110 mm</td>
			<td>Roboz Surgical</td>
			<td>RS-5035</td>
			<td></td>
		</tr>
		<tr>
			<td>Type 1 Collagenase</td>
			<td>Gibco</td>
			<td>17100017</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Fisher Scientific</td>
			<td>15-462-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Grade 1 Filter Paper</td>
			<td>Whatman</td>
			<td>1001-110</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of preadipocytes from broiler chick embryos

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipose development. This primary cell can be used to identify factors influencing preadipocyte proliferation and adipogenic differentiation, making them a valuable model for studies related to childhood obesity and control of excess fat deposition in poultry. The rapid growth of postnatal adipose tissue effectively wastes feed by allocating it away from muscle growth in broiler chickens. Therefore, methods to understand the earliest stages of adipose tissue development may provide clues to regulate this tendency and identify ways to limit adipose expansion early in life. The present study was designed to develop an efficient method for isolation, primary culture, and adipogenic differentiation of preadipocytes isolated from developing adipose tissue of commercial broiler (meat-type) chick embryos. The procedure has been optimized to yield cells with high viability (~98%) and increased capacity to differentiate into mature adipocytes. This simple method of embryonic preadipocyte isolation, culture, and differentiation supports functional analyses of fat growth and development in early life.

Primary preadipocytes are morphologically similar to fibroblasts, with irregular, star-like shapes and a central nucleus (Figure 2A-C). The cells readily adhere to tissue culture plastic and begin to proliferate soon after attachment. They rapidly differentiate and accumulate lipid droplets (Figure 3D) when provided with fatty acids in the media. The viability (98%, based on dye exclusion) reported in the isolations represented ...

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Isolation of Preadipocytes from Broiler Chick Embryos

The present protocol describes a simple method for isolating preadipocytes from adipose tissue in broiler embryos. This method enables isolation with high yield, primary culture, and adipogenic differentiation of preadipocytes. Oil Red O staining and lipid/DNA stain measured the adipogenic ability of isolated cells induced with differentiation media.

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte differentiation and metabolism. ...

Primary preadipocytes are a valuable experimental system for understanding the molecular pathways that control adipocyte metabolism. Chicken embryos provide the opportunity to isolate preadipocytes from the earliest stage of adipocyte development. This method has been optimized to yield cells with high viability and increased capacity to differentiate into mature adipocyte, which can be used for functional analysis of early life fat growth and development embryo in chicks.The process of adipogenic differentiation is highly comparable between chickens and humans. Therefore, isolated preadipocytes can be used as a dual-proposed model for studies relevant to humans and poultry. To begin swab the embryo body with 70%ethanol.Then to prevent interference during filtration at later steps after digestion gently scrubbed the skin surface with sterile gauze to remove feathers. Then cut off the skin between the legs and abdominal region to reveal the pair of femoral adipose depots. Use curved forceps with one hand to hold the skin around the leg and gently remove the femoral subcutaneous fat.with the other hand. Later, use curved forceps to gently pull the depot away from the leg. Transfer the tissues into a 15 milliliter tube containing five milliliters of collection media and repeat the dissection for the other side of the fat pad.Now, transfer the collected fat pads to a 60 millimeter Petri dish containing sterilization solution and rinse briefly by swirling the dish a few times. Then rinse the sterilization solution by transferring the fat pads to a 60 millimeter Petri dish containing PBS. Gently swirl the plate and once again, wash with PBS.For digestion of the adipose tissues, transfer them to a 15 millimeter tube containing pre-warm enzymatic solution. Then immerse a pair of long straight scissors in the tube and finally mince the adipose tissues in the solution into pieces as small as possible. Transfer the minced tissue and enzymatic solution to a 25 milliliter autoclaved flask.Wrap the flask with paraffin film and place the flask in an orbital shaker incubator at 37 degrees Celsius for digestion. After digestion, gently pipette up and down to mix well. Then remove any bits of undigested tissue and debris by filtering through a 250 micrometer tissue strainer into a 15 milliliter tube by pipetting.Now, remove the cells adhered to the flask by rinsing the flask with four milliliters of growth media and filter the cell suspension into the same 15 milliliter tube. Next rinse the strainer by pipetting the growth media again to remove any cells trapped in the filter. Centrifuge at 300 times G for five minutes at room temperature to pellet the cell fraction and aspirate the supernatant without disturbing the cell pellet.Gently resuspend the pellet in one milliliter of red blood cell lysis buffer, mix well by pipetting and incubate for five minutes at room temperature. Then dilute the cells by adding five milliliters of growth media to the tube containing the cells and mix them gently by pipetting. Now, pipette the cells onto a 40 micrometer cell strainer and filter them into a new 50 milliliter tube.Then rinse the strainer with an additional five milliliters of growth media and pellet the cells by centrifuging at 300 times G for seven minutes at room temperature. Carefully aspirate the supernatant. Resuspend the remaining cell pellet in one milliliter of growth media by pipetting To determine the cell density and viability, mix 10 microliters of each sample in trypan blue by pipetting.Load 10 microliters of the mixture onto the hemocytometer and count the cells. Analysis of preadipocytes after 24, 48, and 72 hours showed normal cell morphology and number. Adipogenic induction of preadipocytes showed rapid differentiation and accumulation of lipid droplets.Aggressive handling of preadipocytes during isolation resulted in cell damage and low cell number. Microbial contamination can be observed despite taking preventative measures. Lipid staining with oil red O indicated the preadipocytes quickly begin to develop lipid droplets under adipogenic inducement and accumulation progresses over time as observed in 24, 48, and 72 hours.The lipid accumulation relative to cell number was quantified using lipid and DNA stains. Both the lipid accumulation and cell proliferation increased over time. Low cell number or damaged cells can be observed when the tissue fractions are digested too long.Therefore, it is recommended that one and a half hour of digestion is not exceeded. Isolated preadipocytes can be used for gene expression studies. Sufficient RNA can be obtained for use in cDNA synthesis and follow-on qPCR or RNAseq.

isolation-of-preadipocytes-from-broiler-chick-embryos

Research

JoVE Journal

Developmental Biology

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#8212; A High-throughput Screen in ES Cells

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

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 ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively na&#239;ve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise na&#239;ve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Generation of Na&amp;#239;ve Blastoderm Explants from Zebrafish Embryos

Zebrafish blastoderm explants are generated by isolating embryonic cells from endogenous signaling centers within the early embryo, producing relatively na&#239;ve cell clusters easily manipulated and cultured ex vivo. This article provides instructions for making such explants and demonstrates their utility by interrogating roles for Nodal signaling during gastrulation.

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.&#160;