This work was supported by an Artistic and Scholarly Development grant through Illinois Wesleyan University to TS and funded in part by NIH-R01EY022158 (PL).

The strain of eggs used in this protocol was White Leghorn, and all animal procedures were approved by the Institutional Animal Care and Use Committee at Illinois Wesleyan University.
1. Incubation of chick eggs
<ol>
	<li>Keep the eggs at ~10 &#176;C for up to 1 week after they are laid to halt development. When ready to initiate chick embryo development, wipe the entire eggshell with lint-free wipes (see Table of Materials) saturated with room temperature water to remove dirt and debris.</li>
	<li>Ensure the eggshell is sanitized. Wipe down the entire egg surface with lint-free wipes dampened with 70% ethanol. Quickly wipe off the ethanol to dry the egg and avoid ethanol absorption through the eggshell to the embryo.</li>
	<li>Arrange the eggs horizontally on a tray. Mark the top of the egg to denote the expected position of the embryo. Incubate the eggs horizontally, with the rocking function activated, in a 38 &#176;C humidified incubator.</li>
</ol>
2. Windowing the eggs to prepare for membrane dissection
<ol>
	<li>Remove eggs from the incubator on the third day of embryonic development (E3). Sterilize the top of the eggs with lint-free wipes dampened with 70% ethanol. Dry the ethanol from the eggshell surfaces. 
	NOTE: To ensure that development was not delayed during the windowing procedure, 6-12 eggs were removed, and the procedure was quickly carried out on these while the remaining eggs were left in the incubator.</li>
	<li>Position an egg horizontally in a secure egg holder (see Table of Materials). Using the sharp end of dissecting scissors, create a small hole in the top of the eggshell near the pointed end of the egg. 
	NOTE: This hole will facilitate the removal of albumen, which is necessary to drop the yolk and embryo away from the inner eggshell surface. For egg holders, paper-pulp egg filler flats, within which the eggs come shipped, were used.</li>
	<li>Through the hole (Step 2.2.), insert an 18 G beveled hypodermic needle. With the needle pushed to the bottom inner surface of the egg and the bevel-side of the needle facing the pointed end of the egg (e.g., away from the expected location of the yolk and the embryo near the middle of the egg), remove 2-3 mL of albumen from the chicken egg and discard. 
	NOTE: If one&#39;s needle nicks the embryo or its associated vasculature during this step, it will result in blood being aspirated with the albumen. This will result in embryo death. Further, if the yolk is inadvertently aspirated along with the albumen during this step, the embryo will not be viable. In either case, the egg should be discarded if the embryo cannot be immediately used for other purposes.</li>
	<li>Clean the eggshell surface surrounding the hole with lint-free wipes lightly dampened with 70% ethanol and wipe dry. Seal the hole made for removing albumen with clear tape.</li>
	<li>With the sharp end of dissecting scissors, make a second &#34;window&#34; hole in the top of the eggshell at the marking site (Step 1.3.). Ensure that the scissors do not extend too far into the eggshell to avoid contacting and damaging the embryo or embryonic vasculature, which will often be positioned within the egg directly below the site of the second hole.</li>
	<li>Using curved iris forceps, widen the &#34;window&#34; hole to span ~2-3 cm in diameter and serve as a &#34;window&#34; to the developing embryo beneath the shell.
	<ol>
		<li>Insert one end of the forceps into the hole, keeping it parallel to and closely juxtaposed with the eggshell. With the other forceps end positioned outside the eggshell, carefully pinch the two forceps ends together, allowing them to break and remove small pieces of the eggshell. Continue breaking and removing eggshell fragments until there remains a 2-3 cm window that directly overlays the embryo. 
		NOTE: Eggs in which embryos do not lay directly beneath the hole made in Step 2.6. must not be used as the forthcoming membrane dissections would be challenging to complete. Even with rocking the eggs horizontally, about 10% of the eggs are unusable due to the poor positioning of the embryo. These embryos can be used for other purposes.</li>
	</ol>
	</li>
	<li>To limit bacterial contamination, add through the window hole (e.g., into the egg) ~100-200 &#181;L of Ringer&#39;s solution (8 g of NaCl, 0.37 g of KCl, and 0.23 g of CaCl2.2H20 per L of distilled H20) containing Penicillin/Streptomycin antibiotics (50 U/mL of Penicillin and 50 &#181;g/mL of Streptomycin, see Table of Materials).</li>
	<li>Seal the window hole using clear adhesive tape. Perform the egg sealing by aligning a corner of the tape on the long axis of the hole and pressing the tape to the shell ~1-2 cm away from the edge of the hole.
	<ol>
		<li>Continue sealing around the opening until a hanging flap of tape is left on one side. Press the two pieces of tape together, creating a domed shape over the hole, and press the flap of over-hanging tape to the shell to finish sealing the egg. 
		NOTE: Eggs need to be windowed at either E2 or E3. As per experience, windowing prior to E2 results in low embryo viability. Moreover, by E4, the embryo and extraembryonic membranes become attached to the eggshell10, and any attempts to window at E4 or later often result in embryo damage or tearing of extraembryonic blood vessels, with either event leading to the outcome of embryo death.</li>
	</ol>
	</li>
	<li>Return the &#34;windowed&#34; eggs to the incubator for further development. Ensure to keep the eggs horizontal and turn off the incubator&#39;s rocking function.</li>
	<li>Repeat Steps 2.2.-2.9. for each egg.</li>
</ol>
3. Microdissections of the extraembryonic membranes
<ol>
	<li>Remove an E5.5 windowed egg from the incubator. Expose the embryo by cutting the tape away from the window with sterilized dissecting scissors.</li>
	<li>Use a dissecting microscope to observe the embryo and its extraembryonic membranes through the window. If necessary, use scissors or curved iris forceps to widen the window so that the embryo is well-positioned beneath the window, taking care not to damage the embryonic vasculature.
	<ol>
		<li>Add two drops of Ringer&#39;s solution containing Penicillin/Streptomycin antibiotics to hydrate the embryo and sterilize the egg.</li>
	</ol>
	</li>
	<li>Use a dissecting microscope to ensure the embryo is at the proper developmental stage (Hamburger Hamilton stage 27, ~E5.5)11,12 and locate the positions of the amniochorionic membrane (ACM) and the chorioallantoic membrane (CAM). 
	NOTE: At this stage, the embryo is surrounded by the ACM, which comprises the amniotic membrane and overlying chorionic membrane fused and partially covered by the highly-vascularized allantois, which extends from the embryo&#39;s gut region and fuses with the overlaying chorion to form the CAM11,12. The ACM is not heavily vascularized, which enables one to dissect these membranes to expose the embryo without damaging blood vessels and harming the embryo.</li>
	<li>Perform extraembryonic membrane dissections at this stage (E5.5). 
	NOTE: E5.5 is the ideal time to carry out the extraembryonic membrane dissections. Dissecting the membranes earlier (e.g., at E4) before CAM formation reduces embryo accessibility at later stages11. Moreover, at E5.5, the embryo is only partially covered by the highly-vascularized CAM, yet over the next 1-2 days, the CAM quickly envelops and precludes further access to the embryo11,12. For this reason, membrane dissection at E6 or later is challenging as the risk of tearing blood vessels increases.
	<ol>
		<li>Use a pair of sterilized fine forceps to gently grasp the ACM and pull it away from the embryo. Then use sterilized micro-dissecting scissors to cut a hole in the ACM directly above the forelimb that extends from the membrane overlying the forelimb to the membrane overlying the head. 
		NOTE: This step relaxes the chorion and amnion membranes, thus making them easier to grab and further dissect with forceps in the next steps. See Figure 1 for a helpful schematic of how membranes are dissected through the eggshell window.</li>
	</ol>
	</li>
	<li>Use two pairs of fine, sterile forceps to gently grab the amnion in two adjacent positions between the ACM and the CAM (e.g., an area between the cut made above the forelimb and the nearest edge of the CAM).
	<ol>
		<li>Carefully move each pair of forceps, both firmly gripping the amniotic membrane, away from one another, with one pair moving dorsally to the embryo and the other ventrally. 
		NOTE: This motion serves to tear the amnion further while also separating the ACM (which gets pulled in the dorsal direction with respect to the embryo by one pair of forceps) and the CAM (which gets pulled in the ventral direction with respect to the embryo by the other pair of forceps).</li>
		<li>Ensure that the membranes are separated when the CAM no longer covers the embryo and the allantoic artery and vein, which emanate from the embryonic gut to the CAM, are readily apparent.</li>
	</ol>
	</li>
	<li>Use sterilized fine forceps to dissect and remove any remaining amnion membrane covering the embryo. It is most commonly observed that the remaining amnion will partially cover the caudal half of the embryo.
	<ol>
		<li>Using sterilized forceps, grasp the amnion near the mid-cranial region of the embryo and carefully pull the amnion in a caudal direction with respect to the embryo toward the earlier displaced CAM. The embryo will now be fully exposed, and further growth of the CAM will mainly occur away from the developing embryo. 
		NOTE: See Figure 1 for a helpful schematic on how the exposed embryo will appear following membrane dissection. Also, refer to a previously published report11 for helpful schematic diagrams of Steps 3.3.-3.6.</li>
	</ol>
	</li>
	<li>Add a few drops of Ringer&#39;s solution containing Penicillin/Streptomycin antibiotics to hydrate the embryo and sterilize the egg.</li>
	<li>Reseal the window hole using clear tape, as described in Step 2.8. Return the egg to the incubator for further development, keeping the egg horizontal and keeping the incubator&#39;s rocking function inactivated.</li>
	<li>Repeat Steps 3.1.-3.8. for each egg. 
	NOTE: The extraembryonic membrane dissections at E5.5 described above will enable access to the embryo through E7, which is when wounding can be carried out8,9. By E8, the continued growth of the CAM tissue begins to cover the cranial region of the embryo, thus precluding further access to the cornea. If one desires to carry out wounding in older E8-E9 corneas, it is possible to reposition the growing CAM ventrally with respect to the embryo (Step 3.10.). If one wishes to wound at E7, Step 3.10. is not necessary to perform and one may proceed to Step 4, corneal wounding.</li>
	<li>Remove an E7 egg from the incubator whose extraembryonic membranes were previously dissected at E5.5 (Steps 3.1.-3.8.). Grasp with sterilized forceps any available amnion membrane tissues fused to the CAM and gently pull the amnion membrane away from the cranial region of the embryo in a ventral direction with respect to the embryo. 
	&#8203;NOTE: Since the amnionic membrane being displaced away from the embryo is fused to the CAM, the growing and highly vascularized CAM will follow the amnion-grasping forceps and move away from the cranial region. Repeat this step daily to continually displace the CAM away from the embryo until the embryo is at the desired age for wounding.</li>
</ol>
4. Corneal wounding
<ol>
	<li>Obtain an egg from the incubator for wounding at the desired embryonic age, E7-E9. Expose the embryo by cutting the tape away from the window with sterilized dissecting scissors. Add a few drops of Ringer&#39;s solution containing Penicillin/Streptomycin antibiotics to hydrate the embryo and sterilize the egg.</li>
	<li>Use a micro-dissecting knife to make an incision that spans the extent of the cornea of the right eye (due to how the embryo lays in the egg, the left eye is not accessible but can serve as a non-wounded control), which is parallel to and in line with the choroid fissure (Figure 1). The first cut will traverse the corneal epithelium.
	<ol>
		<li>Use the micro-dissecting knife to again lacerate the cornea in the same spot as the first incision 2x more (e.g., three cuts total, with cut 2 and cut 3 occurring along with the same position in the cornea as cut 1)11. The second laceration will traverse the basement membrane, and the third will penetrate the anterior stroma. 
		NOTE: If the embryo has settled under the dissected CAM, one can use sterilized curved iris forceps to carefully move the head out from under the CAM. Position the curved iris forceps beneath the head, making contact with the left side of the head. Cradle the entire head on top of closed curved iris forceps and gently raise the head around and above the CAM. To help with viability, use a similar technique with the curved iris forceps to tuck the embryo back under the CAM after surgery to promote proper growth of the CAM.</li>
	</ol>
	</li>
	<li>Add 3-4 drops of Ringer&#39;s solution containing Penicillin/Streptomycin antibiotics to hydrate the embryo and sterilize the egg.</li>
	<li>Reseal the window hole with clear tape and return to the incubator, leaving the egg horizontal. Allow the embryo to develop and the corneal wound to heal for a desired period (e.g., 0.5-11 days), and then humanely euthanize the embryo by decapitation.</li>
	<li>Use curved iris forceps to harvest the eye from a euthanized embryo floating in a Petri dish of Ringer&#39;s saline solution by gently grasping the eye on its posterior side, where the eye and facial tissue meet, and carefully lifting the whole eye away and free from the facial tissue.
	<ol>
		<li>Use fine forceps to poke a small hole (3-5 cm) in the back of the whole eye and fix the whole eye in 4% paraformaldehyde at 4 &#176;C overnight with mild agitation.</li>
	</ol>
	</li>
</ol>

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The authors have no competing financial interests concerning the information presented in this manuscript.

The chick is an ideal model system for studying fetal, scarless cornea wound repair. Unlike mammals, the chick is easily accessible throughout development using in ovo8&#160;or ex ovo strategies24. The embryonic chick cornea is much larger than rodent corneas, with nearly 50% of the cranial volume dedicated to the eye25, making it highly amenable to physical manipulations such as wounding. Moreover, chicken eggs are readily availabl...

The cornea is the transparent, outer-most tissue of the eye that transmits and refracts light conducive to visual acuity. In the adult cornea, damage or infection to the corneal stroma leads to a rapid and robust wound healing response characterized by keratocyte proliferation, fibrosis, increased inflammation leading to cytokine-induced apoptosis, generation of repair myofibroblasts, and overall remodeling of the extracellular matrix (ECM)1,2. Following injury, such corneal tissue repair results in opaque scar tissue that reduces corneal transparency and occludes the passage of light, thus distorting vision and, in the most severe cases, leading to corneal blindness3. Thus, there is a clear need to develop reliable animal models to address the complexities of wound healing and to identify the cellular and molecular factors responsible for wound closure and tissue regeneration.
To date, most studies examining corneal wound healing have utilized post-natal4 or adult animal models1,2,5,6,7. While these studies have led to a significant advancement in the understanding of the corneal wound healing response and the mechanisms underlying scar formation, the damaged corneal tissues in these healing models fail to fully regenerate, thus limiting their utility for identifying the molecular factors and cellular mechanisms responsible for fully recapitulating corneal morphology and structure post-injury. By contrast, fetal wounds generated with a knife in the embryonic chick cornea possess an intrinsic capacity to heal fully in a scarless fashion8. Specifically, the embryonic chick cornea exhibits nonfibrotic regeneration with the complete recapitulation of the extracellular matrix structure and innervation patterns8,9.
The present protocol describes a sequence of steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to facilitate access to the embryo. 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 and wounding is desired. Third, linear central cornea incisions penetrating through the corneal epithelium and into 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18 G hypodermic needle</td>
			<td>Fisher Scientific</td>
			<td>14-826-5D</td>
			<td></td>
		</tr>
		<tr>
			<td>30 degree angled microdissecting knife</td>
			<td>Fine Science Tools</td>
			<td>10056-12</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8242;,6-diamidino-2-phenylindole (DAPI)</td>
			<td>Molecular Probes</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>14-829-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor labelled secondary antibodies</td>
			<td>Molecular Probes</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2-H20)</td>
			<td>Sigma</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken egg trays</td>
			<td>GQF</td>
			<td>O246</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Forceps, Fine Tip, Serrated</td>
			<td>VWR</td>
			<td>82027-408</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors, sharp tip</td>
			<td>VWR</td>
			<td>82027-578</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris 1 x 2 Teeth Tissue Forceps, Full Curved</td>
			<td>VWR</td>
			<td>100494-908</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Sigma</td>
			<td>Z188956</td>
			<td></td>
		</tr>
		<tr>
			<td>Microdissecting Scissors</td>
			<td>VWR</td>
			<td>470315-228</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-fibronectin (IgG1)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>B3/D6</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-laminin (IgG1)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>3H11</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse antineuron-specific &#946;-tubulin (Tuj1, IgG2a)</td>
			<td>Biolegend</td>
			<td>801213</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-tenascin (IgG1)</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>M1-B4</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>Sportsman 1502 egg incubator</td>
			<td>GQF</td>
			<td>1502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tear by hand packaging (1.88 inch width)</td>
			<td>Scotch</td>
			<td>n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Following the earlier dissection of the ACM and CAM at E5.5 to expose the cranial region of the developing embryo, a series of lacerations that spanned the E7 central cornea was made in ovo (Figure 1). An ideal wound to study cornea regeneration occurs following three lacerations, each made in the same location of the cornea. The first laceration traverses the corneal epithelium, while the second and third lacerations penetrate the underlying basement membrane and anterior stroma, r...

Watch this Scientific Journal Video about Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas at JoVE.com

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

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;

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

investigating-scarless-tissue-regeneration-embryonic-wounded-chick

Research

JoVE Journal

Developmental Biology

1.8K Views.  Illinois Wesleyan University. 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. 

Video: Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

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.

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 ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

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

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

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

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

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue's (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland's stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells' phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary gland is the key regulator of the body's endocrine system. This article describes the development of organoids from the mouse pituitary as a novel 3D in vitro model to study the gland's stem cell population of which the biology and function remain poorly understood.

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, ...

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

Fixation of Embryonic Mouse Tissue for Cytoneme Analysis

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (&#8804;200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.

Here, an optimized step-by-step protocol is provided for fixation, immunostaining, and sectioning of embryos to detect specialized signaling filopodia called cytonemes in developing mouse tissues.

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens ...