Name: investigating scarless tissue regeneration in embryonic wounded chick corneas
Uploaded: 2022-05-02T14:00:00
Description: Watch this Scientific Journal Video about Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas at JoVE.com

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 w...

<ol>
	<li>Wilson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+wound+healing.">Corneal wound healing.</a> Experimental Eye Research. 197, 108089 (2020).</li><li>Ljubimov, A. V., Saghizadeh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+corneal+wound+healing.">Progress in corneal wound healing.</a> Progress in Retinal and Eye Research. 49, 17-45 (2015).</li><li>Whitcher, J. P., Srinivasan, M., Upadhyay, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+blindness:+a+global+perspective.">Corneal blindness: a global perspective.</a> Bulletin of the World Health Organization. 79 (3), 214-221 (2001).</li><li>Ritchey, E. R., Code, K., Zelinka, C. P., Scott, M. A., Fischer, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chicken+cornea+as+a+model+of+wound+healing+and+neuronal+re-innervation.">The chicken cornea as a model of wound healing and neuronal re-innervation.</a> Molecular Vision. 17, 2440-2454 (2001).</li><li>Berdahl, J. P., Johnson, C. S., Proia, A. D., Grinstaff, M. W., Kim, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+sutures+and+dendritic+polymer+adhesives+for+corneal+laceration+repair+in+an+in+vivo+chicken+model.">Comparison of sutures and dendritic polymer adhesives for corneal laceration repair in an in vivo chicken model.</a> Archives of Ophthalmology. 127 (4), 442-447 (2009).</li><li>Fowler, W. C., Chang, D. H., Roberts, B. C., Zarovnaya, E. L., Proia, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+paradigm+for+corneal+wound+healing+research:+the+white+leghorn+chicken+(Gallus+gallus+domesticus).">A new paradigm for corneal wound healing research: the white leghorn chicken (Gallus gallus domesticus).</a> Current Eye Research. 28 (4), 241-250 (2004).</li><li>Huh, M. I., Kim, Y. E., Park, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+distribution+of+TGF-&#946;+isoforms+and+signaling+intermediates+in+corneal+fibrotic+wound+repair.">The distribution of TGF-&#946; isoforms and signaling intermediates in corneal fibrotic wound repair.</a> Journal of Cellular Biochemistry. 108 (2), 476-488 (2009).</li><li>Spurlin, J. W., Lwigale, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wounded+embryonic+corneas+exhibit+nonfibrotic+regeneration+and+complete+innervation.">Wounded embryonic corneas exhibit nonfibrotic regeneration and complete innervation.</a> Investigative Ophthalmology &amp; Visual Science. 54 (9), 6334-6344 (2013).</li><li>Koudouna, E., Spurlin, J., Babushkina, A., Quantock, A. J., Jester, J. V., Lwigale, P. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+corneal+innervation:+interactions+between+nerves+and+specialized+apical+corneal+epithelial+cells.">Developmental corneal innervation: interactions between nerves and specialized apical corneal epithelial cells.</a> Investigative Ophthalmology &amp; Visual Science. 51 (2), 782-789 (2010).</li><li>Schwend, T., Deaton, R. J., Zhang, Y., Caterson, B., Conrad, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+sulfated+glycosaminoglycans+and+their+effects+on+trigeminal+nerve+growth+cone+behavior+in+vitro:+roles+for+ECM+in+cornea+innervation.">Corneal sulfated glycosaminoglycans and their effects on trigeminal nerve growth cone behavior in vitro: roles for ECM in cornea innervation.</a> Investigative Ophthalmology &amp; Visual Science. 53 (13), 8118-8137 (2012).</li><li>Lee, M. K., Tuttle, J. B., Rebhun, L. I., Cleveland, D. W., Frankfurter, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+and+posttranslational+modification+of+a+neuron-specific+beta-tubulin+isotype+during+chick+embryogenesis.">The expression and posttranslational modification of a neuron-specific beta-tubulin isotype during chick embryogenesis.</a> Cell Motility and the Cytoskeleton. 17 (2), 118-132 (1990).</li><li>Chen, X., Nadiarynkh, O., Plotnikov, S., Campagnola, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+harmonic+generation+microscopy+for+quantitative+analysis+of+collagen+fibrillar+structure.">Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure.</a> Nature Protocols. 7, 654-669 (2012).</li><li>Campagnola, P. J., Millard, A. C., Terasaki, M., Hoppe, P. E., Malone, C. 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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 ...

Damage to the adult corneal stroma leads to a rapid wound healing response that compromises one's vision due to opaque scar tissue formation. This protocol generates wounds in embryonic chick corneas which unlike adults, can heal without a scar. Given the intrinsic capacity of the injured embryonic cornea to undergo a complete recapitulation of the native corneal structure with no detectable scarring, the embryonic chick serves an important animal model for elucidating the molecular and cellular factors for scarless corneal wound healing.The most challenging aspect of this technique is generating a consistent wound that penetrates both the outermost epithelium and the deeper corneal stroma. As one is learning, it may be helpful to view the wounded corneas in a cross section so that the depth of the wound penetration into the corneal stroma can be assessed. To begin, arrange the eggs horizontally on a tray and mark them at the top of the egg to denote the expected position of the embryo.Incubate these eggs in a 38 degree Celsius humidified incubator with the rocking function activated. Remove the egg from the incubator on the third day of embryonic development and position it horizontally in a secure egg holder. Create a small hole in the top of the egg shell near the pointed end of the egg using the sharp end of dissecting scissors.Insert an 18 gauge beveled hypodermic needle through the hole. With the needle pushed to the bottom of the inner surface of the egg and the bevel side of the needle facing the pointed end of the egg, remove two to three milliliters of the albumin from the chicken egg and discard. 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 albumin with clear tape. With the sharp end of dissecting scissors, make a second window hole in the top of the eggshell. Ensure that the scissors do not extend too far into the egg shell to avoid contacting and damaging the embryo or embryonic vasculature positioned directly below the side of the second hole.Using curved forceps, widen the window hole to span approximately two to three centimeters in diameter to reveal and gain access to the developing embryo beneath the shell. Now, 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 two to three centimeter window that directly overlays the embryo. To limit bacterial contamination, add 100 to 200 microliters of ringer solution containing 50 units per milliliter of penicillin and 50 micrograms per milliliter of streptomycin through the window hole. Seal the window hole using clear adhesive tape.Perform this egg sealing by aligning a corner of the tape on the long axis of the hole and pressing the tape to the shell one to two centimeters away from the edge of the hole. 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 overhanging tape to the shell to finish sealing the egg.Return the windowed eggs to the incubator for further development. Ensure to keep these eggs horizontal and turn off the incubators rocking function. Use a dissecting microscope to ensure the embryo is at the proper developmental stage and locate the positions of the amniochorionic membrane and the chorioallantoic membrane.Then use sterilized micro dissecting scissors to cut a hole in the amniochorionic membrane directly above the forelimb that extends from the membrane, overlying the forelimb to the membrane overlying the head. Use two pairs of fine sterile forceps and gently grab the amnion in two adjacent positions between the amniochorionic membrane ACM and the chorioallantoic membrane. 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.Use sterilized fine forceps to dissect and remove any remaining amnion membrane covering the embryo. Using sterilized forceps, grasp the amnion near the mid cranial region of the embryo and carefully pull the amnion in a coddle direction with respect to the embryo toward the earlier displaced chorioallantoic membrane. Reseal the window hole using clear tape as described in the manuscript and return the egg to the incubator for further development.The extra embryonic membranes may need to be repositioned to assure the right eye is accessible. If the right eye is not accessible for wounding due to the position of the embryo in the egg, it can serve as a non wounded control. Use a micro dissecting knife to make an incision that spans the extent of the cornea of the right eye which is parallel to and inline with the choroid tissue.Use the micro dissecting knife to again, lacerate the cornea in the same spot as the first incision, two times more in such a way that the cut two and cut three occurring along with the same position in the cornea as cut one. To help with viability, use the curved iris forceps to tuck the embryo back under the chorioallantoic membrane after surgery to promote proper growth of the chorioallantoic membrane. Next, add three to four drops of ringer solution containing antibiotics to hydrate the embryo and sterilize the egg.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. After the incubation, euthanize the embryo and use curved iris forceps to harvest the eye in a Petri dish of Ringer'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.Use fine forceps to poke a small hole of three to five centimeters in the back of the whole eye and fix the whole eye in 4%paraformaldehyde at four degrees Celsius overnight with mild agitation. To achieve an ideal wound, it is crucial to use a sharp micro dissection knife and apply the correct amount of pressure during the laceration. Applying too little pressure will result in a shallow wound that tears the corneal epithelium without sufficiently penetrating the anterior stroma.Applying too much pressure results in a full extent wound penetrating the entire stroma and exposing the aqueous humor to the external environment. Carrying out the proper lacerating incisions produces an ideal wound that initially enlarges zero to three days post wounding. After that, re-epithelialization and new tissue formation occurs to ultimately close the wound in a scar free fashion by 11 days, post wounding.Spatiotemporal localization of the extracellular matrix proteins, fibronectin and tenascin within the healing wound was found to be elevated at time points corresponding to corneal re-epithelialization, suggesting their involvement in wound closure, epithelial cell migration and survival. Whole mount immunohistochemistry revealed that the nerves that directly juxtaposed the wounded central cornea are temporarily inhibited from the healing corneal tissue. Despite earlier inhibition, corneal nerves eventually innervate the fully healed corneal tissue 11 days post wounding to similar density levels and in similar patterns to stage matched non-wounded controls embryonic day 18.As evidenced by second generation harmonic imaging, bundles of collagen fibers throughout varying depths of the central cornea wound are seen arranged orthogonally, matching the native macro structure of non wounded central corneal tissue. When combined with classic developmental biology approaches, such as tissue grafting and bead implantation, as well as modern day approaches for gene manipulation such as retroviral infection and DNA electroporation, this animal model promises to reveal molecular factors and cellular mechanisms necessary to promote scarless cornea wound healing. Determining key molecular factors in matrix proteins that regulate fetal scarless wound healing will pave the way for therapies that foster a more restorative healing process with less scarring and better recapitulation of the normal tissue architecture.

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

Research

JoVE Journal

Developmental Biology

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 ...

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 ...

Avian Embryonic Left Atrial Ligation Model for HLHS and Cardiovascular Research

Left Atrial Ligation in the Avian Embryo as a Model for Altered Hemodynamic Loading During Early Vascular Development

Due to its four-chambered mature ventricular configuration, ease of culture, imaging access, and efficiency, the avian embryo is a preferred vertebrate animal model for studying cardiovascular development. Studies aiming to understand the normal development and congenital heart defect prognosis widely adopt this model. Microscopic surgical techniques are introduced to alter the normal mechanical loading patterns at a specific embryonic time point and track the downstream molecular and genetic cascade. The most common mechanical interventions are left vitelline vein ligation, conotruncal banding, and left atrial ligation (LAL), modulating the intramural vascular pressure and wall shear stress due to blood flow. LAL, particularly if performed in ovo, is the most challenging intervention, with very small sample yields due to the extremely fine sequential microsurgical operations. Despite its high risk, in ovo LAL is very valuable scientifically as it mimics hypoplastic left heart syndrome (HLHS) pathogenesis. HLHS is a clinically relevant, complex congenital heart disease observed in human newborns. A detailed protocol for in ovo LAL is documented in this paper. Briefly, fertilized avian embryos were incubated at 37.5 &#176;C and 60% constant humidity typically until they reached Hamburger-Hamilton (HH) stages 20 to 21. The egg shells were cracked open, and the outer and inner membranes were removed. The embryo was gently rotated to expose the left atrial bulb of the common atrium. Pre-assembled micro-knots from 10-0 nylon sutures were gently positioned and tied around the left atrial bud. Finally, the embryo was returned to its original position, and LAL was completed. Normal and LAL-instrumented ventricles demonstrated statistically significant differences in tissue compaction. An efficient LAL model generation pipeline would contribute to studies focusing on synchronized mechanical and genetic manipulation during the embryonic development of cardiovascular components. Likewise, this model will provide a perturbed cell source for tissue culture research and vascular biology.

Author Spotlight: Effect of Left Atrial Ligation on Avian Embryonic Hearts and HLHS Implications 

Here, we present a detailed visual protocol for executing the left atrial ligation (LAL) model in the avian embryo. The LAL model alters the intracardiac flow, which changes wall shear stress loading, mimicking hypoplastic left heart syndrome. An approach to overcome the challenges of this difficult microsurgery model is presented.

Due to its four-chambered mature ventricular configuration, ease of culture, imaging access, and efficiency, the avian embryo is a preferred vertebrate ...

Longitudinal Monitoring of Neural Plate Mechanics in Chick Embryo Development

Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo

Neural tube closure (NTC) is a critical process during embryonic development. Failure in this process can lead to neural tube defects, causing congenital malformations or even mortality. NTC involves a series of mechanisms on genetic, molecular, and mechanical levels. While mechanical regulation has become an increasingly attractive topic in recent years, it remains largely unexplored due to the lack of suitable technology for conducting mechanical testing of 3D embryonic tissue in situ. In response, we have developed a protocol for quantifying the mechanical properties of chicken embryonic tissue in a non-contact and non-invasive manner. This is achieved by integrating a confocal Brillouin microscope with an on-stage incubation system. To probe tissue mechanics, a pre-cultured embryo is collected and transferred to an on-stage incubator for ex ovo culture. Simultaneously, the mechanical images of the neural plate tissue are acquired by the Brillouin microscope at different time points during development. This protocol includes detailed descriptions of sample preparation, the implementation of Brillouin microscopy experiments, and data post-processing and analysis. By following this protocol, researchers can study the mechanical evolution of embryonic tissue during development longitudinally.

Author Spotlight: Non-Contact Measurement of Tissue Mechanics in Live Chick Embryos Using Brillouin Microscopy

This protocol was developed to longitudinally monitor the mechanical properties of neural plate tissue during chick embryo neurulation. It is based on the integration of a Brillouin microscope and an on-stage incubation system, enabling live mechanical imaging of neural plate tissue in ex ovo cultured chick embryos.

Neural tube closure (NTC) is a critical process during embryonic development. Failure in this process can lead to neural tube defects, causing congenital ...

Left Atrial Ligation in Chick Embryos

Start by gently cleaning the eggshells of fertilized white leghorn chicken eggs with lint-free ethanol soaked wipes. Incubate the eggs blunt end up until the desired stage, usually HH20 to 21. Before starting the procedure, prepare the required number of knots by tying a loose overhand knot into a 1.5-centimeter-long 10-zero suture.Ensure that the knot is not tight and is large enough to fit over the atria. Crack open the eggshell gently with the reverse end of the tweezers, supporting the egg firmly to prevent unwanted cracks. Open a window from the blunt end of the egg and remove both the outer and inner membranes.Use micro scissors to remove only the necessary vitelline membrane. Once the embryo is free of the vitelline membrane, place the closed-tip tweezer under the dorsal segment of the embryo and gently flip it to expose the left side. Remove all membranes immediately around the atrial bud by removing the coarse membranes first, and then using finer tweezers for fine membrane removal.Position the pre-prepared knot close to the embryo. Correctly orient the open knot over the left atrial bud to execute the knot tightening. Using micro scissors, cut the excess suture ends as close as possible to the bud and remove the excess suture pieces with tweezers.Finally, using closed tweezers, return the embryo to its original position. After completing the microsurgery, cover the egg with a double-layer of param and return it to the incubator. The left atrial ligation, or LAL models, showed that a more compact myocardial structure was achieved with significant morphological changes compared to normal development.Extracellular matrix deposition was observed around the cardiac interstitium, resembling myocardial fibrosis. Morphometric porosity measurements revealed a smaller left ventricular cavity and trabecular compaction in the LAL models. At HH 25 of the LAL model, an enlarged right ventricular cavity, altered trabecular architecture, and myocardial volume were observed.Optical coherence tomography imaging of LAL models showed a significant reduction in left ventricular size and diameter compared to the control. LAL groups also exhibited an increase in wall thickness compared to control groups at HH 25.