Name: rapid genetic analysis of epithelial-mesenchymal signaling during hair regeneration
Uploaded: 2013-02-28T12:30:00
Duration: 10 min 9 s
Description: Watch this Scientific Journal Video about Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration at JoVE.com

This work was supported by NIH NRSA 1F32CA14208701 (S.X.A.) and NIH grants AR052785 and AR046786 (A.E.O. and W.-M.W.).

1. Prepare 0 to 2 days Old Newborn Mice for Skin Dissection
<ol>
	<li>Euthanize mouse pups using a CO2 chamber for at least 20 min. Leave pups on ice up to an hour until dissection. P0-P2 mice are highly recommended as cells prepared from older mice have reduced progenitor capacity that results in lower graft yields. Prepare one dish of 70% ethanol (for step 1.3) and three dishes of wash solution (for step 3) when ready to perform skin dissection. Thaw Dispase Solution and leave it at 4 &deg;C. Cervical dislocate pups after CO2 overexposure to ensure euthanasia.</li>
	<li>Place euthanized pups in a culture dish on ice and transfer to a sterile flow hood.</li>
	<li>Wash pups by briefly immersing in 70% ethanol and place onto sterile culture dish.</li>
</ol>
2. Dissect Mouse Skin
<ol>
	<li>Cut off each limb and tail at the base of the torso with sterile scissors.</li>
	<li>Grasp the body firmly between a pair of curved forceps and make an incision along the dorsal skin from head to tail using a scalpel without penetrating the underlying fascia.</li>
	<li>Carefully peel the skin away from the midline of the mouse.</li>
	<li>Grasp the exposed mouse firmly with the long side of the curved forceps and insert another pair of curved forceps underneath the skin at the posterior end of the mouse and gently pull skin over hips toward the ventral half of the mouse.</li>
	<li>Carefully peel skin completely off the mouse in one smooth motion and discard the carcass.</li>
</ol>
3. Wash Skin and Incubate with Dispase Solution
<ol>
	<li>Place the skin in the first dish of wash solution with dermis-side down. Spread skin out and agitate with forceps. Leave skin in the first dish of wash solution while dissecting the next skin.</li>
	<li>After placing the next dissected skin in the first dish of wash solution, transfer the prior skin to the second dish of wash solution. Keep skins in the second wash solution until all skins are collected. Transfer all of the skins to the final wash, agitate briefly.</li>
	<li>Add 10 ml ice cold Dispase to a sterile 10 cm culture dish. Transfer skin to the Dispase Solution and flatten skin dermis-side down.</li>
	<li>Float skin for 8-16 hr at 4 &deg;C (alternative option: 1 hr at 37 &deg;C).</li>
</ol>
4. Separate Dermis and Epidermis
<ol>
	<li>Transfer skin to a new sterile culture dish and flatten skin dermis-side down. Carefully hold down the dermis from one corner with a scalpel.</li>
	<li>Grasp the epidermis with forceps and slowly peel away from the dermis. Epidermis should peel away as one piece. The epidermis will be white and thin and the dermis should be brownish, thicker, and gelatinous.</li>
	<li>Separate the two tissues into separate sterile culture dishes.
	<ol class="lalpha">
		<li>To make a dermal specific gene knockdown/overexpression graft, take dermis and mince into very small pieces with two scalpels. Discard epidermis. On day of infection (step 7), dissect another set of mouse skins to prepare fresh, untreated epidermal cells to combine with lentiviral-infected dermal cells.</li>
		<li>To make an epidermal specific gene knockdown/overexpression graft, slice each epidermis into 6 - 8 smaller pieces. Discard dermis. On day of infection (step 7), dissect another set of mouse skins to prepare fresh, untreated dermal cells to combine with lentiviral-infected epidermal cells.</li>
	</ol>
	</li>
</ol>
5. Dissociate Primary Mouse Dermal Cells (mDCs) from Minced Tissue
<ol>
	<li>Dissociate mDCs by incubating dermal pieces with freshly made Collagenase Solution at 37 &deg;C for 1 hr. Use 10 ml of Collagenase Solution per mouse pup.</li>
	<li>Gently mix solution containing dermis every 10 min. Check the extent of dissociation under a microscope; the digested cell suspension should be mostly single cells. The solution should change from clear to cloudy as dermal cells are dissociated from the dermis.</li>
	<li>Add FBS to Collagenase Solution containing dermis to a 10% final volume to reduce the activity of collagenase.</li>
	<li>Pass the cell suspension through a 70 &mu;m cell strainer into a new 50 ml tube to remove large undigested clumps and whole follicles. Add 10 ml of DMEM/10%FBS per 30 ml of the flow-through to further dilute the collagenase.</li>
	<li>Centrifuge tubes containing digested dermis at 30 x g on a tabletop centrifuge for 5 min to further remove whole follicles.</li>
	<li>Carefully transfer the supernatant to a new 50 ml tube. Pellet cells by centrifuging at 200 x g on a tabletop centrifuge for 5 min.</li>
	<li>Resuspend cells with DMEM/10%FBS (1 ml per pup) and count cells. Plate cells with Amniomax C-100 media for infection (step 7), or to combine with lentivirus-infected KC for grafting (step 9). Typical yield is 20 x 106 cells per pup.</li>
</ol>
6. Dissociate Primary Keratinocytes (KC) from Minced Tissue
<ol>
	<li>Dissociate KC by incubating epidermal pieces with freshly thawed 0.05% Trypsin-EDTA at 37 &deg;C for 15 min. Use 7 ml of Trypsin-EDTA per mouse pup.</li>
	<li>Gently swirl solution containing epidermis every 5 min.</li>
	<li>Add equal volume of Neutralizing Solution to Trypsin-EDTA containing epidermis to reduce the activity of trypsin.</li>
	<li>Pellet cells by centrifuging at 200 x g on a tabletop centrifuge for 5 min. Remaining undigested tissue should float on top.</li>
	<li>Carefully pour off most of the supernatant with undigested tissue. Remove remaining supernatant with a 10 ml pipette without disturbing cell pellet. Resuspend cell pellet with PBS. In case KCs are not pelleted completely, pour off most of the supernatant and repeat centrifugation until most of the cells are pelleted.</li>
	<li>Strain cell suspension through 70 &mu;m cell strainer into a new tube to remove remaining tissue or clumps.</li>
	<li>Pellet cells by centrifuging at 200 x g on a tabletop centrifuge for 5 min.</li>
	<li>Resuspend cells in CnT-07 media or PBS (1 ml per pup) and count cells. Use CnT-07 for plating cells for infection (step 7), use PBS to combine with lentivirus-infected mDCs for grafting (step 9). Typical yield is 3-10 x 106 cells per pup.</li>
</ol>
7. Infect Primary Cells with Lentivirus Containing shRNA or cDNA
On day of infection dissect another set of mouse skins to prepare fresh, untreated primary KCs or mDCs to combine with virus-infected cells on day of grafting (step 9).
<ol>
	<li>
	<ol class="lalpha">
		<li>For mDCs, plate 4 x 106 mDCs per 10-cm plate with AmnioMAX-C100 media and incubate at 37 &deg;C + 5% CO2 for next day infection at 50% cell density. Usually 3-4 plates of mDCs are used to generate one graft, and a total of 10-12 plates are needed to generate three grafts in one experiment. Alternatively, let mDCs attach and spread for a minimum of 2 hr at 37 &deg;C + 5% CO2 and perform infection the same day of cell plating. mDCs will have fibroblast-like morphology.</li>
		<li>For KC, plate 12 x 106 KC per 10-cm plate with CnT-07 media and incubate at 37 &deg;C + 5% CO2 for next day infection. Alternatively, let KC attach and spread for a minimum of 2 hr at 37 &deg;C + 5% CO2 and perform infection on the same day. Usually 3-4 plates of KC are used to generate one graft. A total of 10-12 plates are needed to generate three grafts for one experiment. KCs will have cobblestone morphology.</li>
	</ol>
	</li>
	<li>Preincubate cells for 5-10 min with 8 &mu;g/ml Polybrene Solution at 37 &deg;C + 5% CO2.</li>
	<li>Exchange media and add lentivirus + 8 &mu;g/ml Polybrene Solution. Lentiviral titer will vary depending on the construct used and should be determined before infection.</li>
	<li>Centrifuge the plates at 200 x g on a tabletop centrifuge for 1 hr at 32 &deg;C to infect cells with lentivirus.</li>
	<li>Remove lentivirus-containing media and add fresh media. For KC, wash cells once with PBS before adding back fresh media. Usually infected cells recover overnight at 37 &deg;C + 5% CO2. Alternatively, allow at least two hours of recovery at 37 &deg;C + 5% CO2 before trypsinizing the cells for grafts. Continue to culture a small portion of infected cells to check for infection efficiency as described in the Representative Results section.</li>
</ol>
8. Prepare Infected Cells for Grafting
<ol>
	<li>Isolate fresh primary mDCs or KCs from skin using steps 4-6. Resuspend cells in ice cold sterile PBS. Count cells.</li>
	<li>
	<ol class="lalpha">
		<li>For dermal specific gene knockdown/overexpression graft, dissociate infected mDCs from plates using 0.05% Trypsin-EDTA at 37 &deg;C for 8 min.</li>
		<li>For epidermal specific gene knockdown/overexpression graft, dissociate infected KC from plates using 0.125% Trypsin-EDTA at 37 &deg;C for 15 min.</li>
	</ol>
	</li>
	<li>Neutralize trypsin activity using DMEM + 10%FBS (mDCs) or Neutralizing Solution (KC). Transfer the trypsinized cells to a 50 ml tube.</li>
	<li>Pellet cells by centrifuging at 200 x g on a tabletop centrifuge for 5 min.</li>
	<li>Resuspend cells in ice cold sterile PBS. Count cells.</li>
	<li>Combine the virus-infected cells with the freshly prepared untreated counterpart cell type. For one graft, combine 7-10 x 106 mDCs with 7-10 x 106 KCs in ice cold sterile PBS and pellet the cell slurry in a tabletop centrifuge at 4 &deg;C. Resuspend cells in 50 -100 &mu;l ice cold sterile PBS. Keep cells on ice until nu/nu mouse chambers are ready.</li>
</ol>
9. Create Wound Bed and Fix Chamber onto Back of nu/nu Mouse
A day before grafting, sterilize surgical tools with autoclave and sterile silicon chambers by soaking in 70% ethanol. Replace the 70% ethanol with sterile PBS before using the chambers.
<ol>
	<li>Anesthetize nu/nu mouse (7-12 week old females) using ketamine (8 mg/ml)/xylazine (1.6 mg/ml) via intraperitoneal injection (100 &mu;l/10 g body weight) or other preferred method. Apply eye lubricant. Place heating pad under mouse.</li>
	<li>Disinfect nude mouse back skin with iodine and clean with alcohol swabs.</li>
	<li>Add 1-3 drops 0.25% Bupivicaine to incision site topically. Pinch the nude mouse back skin at base of neck with forceps.</li>
	<li>Cut a small circular hole (~1 cm in diameter) in pinched skin with sterile scissors.</li>
	<li>Place sterile chamber onto wound bed and cover chamber edges with surrounding skin.</li>
	<li>Secure chamber into place with sutures along chamber edges (6 stitches per chamber) as shown in Figure 2A.</li>
	<li>When chambers are ready, gently swirl cell slurry and draw up into 23 gauge needle attached to 1 ml syringe. If cell slurry has condensed at the bottom, gently resuspend with 1 ml pipette tip before drawing into the needle.</li>
	<li>Puncture chamber with needle and slowly drip cells onto wound.</li>
	<li>Place mice into clean, autoclaved cages. House 2 mice per cage and add antibiotics and Tylenol (3 mg/ml) to drinking water. In our experience, two female mice per cage have not resulted in adverse trauma to the chamber, graft, or animals.</li>
	<li>Place warming pad under half the cage and observe mice until anesthesia wears off.</li>
</ol>
10. Remove Chamber After 7-9 Days
<ol>
	<li>Anesthetize chambered nu/nu mouse as in step 9.1. Apply eye lubricant.</li>
	<li>Disinfect skin around chamber with iodine and clean with alcohol swabs.</li>
	<li>Carefully cut stitches and remove from chamber.</li>
	<li>Gently remove chamber from mouse. If new skin graft sticks to the chamber, lift part of the chamber and gently separate graft from chamber using forceps. Hold graft down during removal of the chamber. Graft should look dull and opaque as in Figure 2B.</li>
	<li>Apply a non-adherent pad with a thin film of antibiotics on the open wound and suture into place (2 stitches).</li>
	<li>Wrap bandage around mouse to secure the pad and protect the wound. Suture bandage into place.</li>
	<li>The silicon chambers are cleaned by scrubbing with mild soap and thoroughly rinsed with deionized water. Soak the chambers in 70% ethanol overnight, air dry, and store.</li>
</ol>
11. Examine Mouse for Hair Growth
<ol>
	<li>Remove bandages and pad after 3 days of chamber removal. Graft should be dry and opaque to brown in color.</li>
	<li>Check mice periodically for hair growth. Hair should begin to show at 16 days after graft.</li>
</ol>
Procedure Outline
Day 1 (2-3 hr): Sacrifice newborn pups, remove skin, and leave on dispase at 4 &deg;C overnight.
Day 2 (2-3 hr): Isolate primary cells from skin and plate at recommended cell density.
Day 3 (3-4 hr): Infect cells with lentivirus. Remove skin from another set of newborn pups and leave on dispase at 4 &deg;C overnight.
Day 4 (6-8 hr): Isolate primary cells from skin, count, and leave on ice. Recover lentivirus-infected cells by trypsinization and centrifugation, count, and combine 7-10 x 106 dermal cells and keratinocytes into 50-100 &mu;l of PBS per graft. Create wound bed on mouse, attach chamber, and apply cell mixture to wound bed.
Alternative Procedure Outlines
Alternative procedure outlines will shorten the procedure from four days to three.
<ol>
	<li>Combine Day 1 and Day 2 by treating mouse skins in dispase at 37 &deg;C for one hour (estimated time 5-7 hr).</li>
	<li>Combine Day 2 and Day 3 by infecting cells with lentivirus after plating cells for two hours (estimated time 5-7 hr).</li>
</ol>

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No conflicts of interest declared.

Hair reconstitution assays provide a unique organ regeneration model for examining the mechanism of de novo hair follicle formation. Here, we describe a modified chamber hair assay useful for determining gene function in hair follicle formation. Our assay is based on the reported chamber hair reconstitution assay 5, 6,11, which we modified to introduce a lentiviral expression technique to achieve gene overexpression or knockdown in either the dermal or epidermal cell types. Our assay provides a rapid alternative to generating tissue-specific genetic knockout. This assay allows us to demonstrate gene function in hair follicle formation in as little as three weeks from infecting primary cells with lentivirus to graft collection. Our assay can be extended to address genetic interactions using combination shRNA/cDNA delivery by lentivirus into one cell type 12, as well as allows examination of signaling between the dermal and epidermal cell types.
Our hair assay is best suited for detecting strong gain- or loss-of-function phenotypes in hair follicle regeneration, while subtle defects are harder to observe. We have successfully demonstrated dermal specific gene function of a few genes using our hair regeneration assay 1,12. Based on the GFP marker co-expressed from lentivirus vector expressing shRNA 10, we demonstrated that the shRNA expressing donor dermal cells persisted in the regenerated hair grafts. GFP-positive cells were present in the dermal papilla (DP) of the regenerated hair follicles (Figure 3). The DP cells likely consisted of different levels of gene knockdown as cells expressed varying levels of GFP, thus the observed hair follicle phenotype was a range from genetic hypomorph to null.
One improvement for this assay will be to establish a quick and efficient selection for the high shRNA expressing cells before grafting such as using FACS to purify strong GFP-expressing cells. An alternative is to substitute mutant or conditional knockout cells in this assay 9. On the other hand, a culture system that can preserve DP cell function is very useful as DP potency is gradually lost once the primary dermal cells are passaged. Potential culture systems include the use of biomaterials derived from artificial niche or aggregation cultures2,7,8,13. Another improvement will be to generate a reliable epidermal cell freezing method with a high cell recovery rate as this will reduce the use of the second set of mouse pups (step 7) to generate fresh keratinocytes in dermal-specific loss- or gain- of-function studies.
This chamber hair assay produces hair follicle density and quality similar to normal mouse skin, although hair growth is slightly less synchronized and follicle orientation is more random. A few limitations of the hair regeneration genetic assay include the requirement of large amount of lentivirus and cell numbers, and it is very labor intensive in terms of surgical methods when compared to other forms of hair reconstitution assays such as the patch and flap assays 3,4,14. However, the quality of hair follicles and timeline to detect hair growth is superior to other assays 4. Our targeted hair regeneration assay provides a fast and convenient companion to existing genetic models.

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog #</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Invitrogen</td>
			<td>14190-144</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14170-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11995-065</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200-056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cnt-07</td>
			<td>Cell N Tec</td>
			<td>CnT-07</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AminoMAX-C100</td>
			<td>Invitrogen</td>
			<td>17001-074</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>AminoMAX-C100 Supplement</td>
			<td>Invitrogen</td>
			<td>12556-015</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Invitrogen</td>
			<td>26140-079</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Invitrogen</td>
			<td>15290-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Invitrogen</td>
			<td>15710-064</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Roche</td>
			<td>4942078001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Collagenase, Type 1</td>
			<td>Sigma</td>
			<td>C-0130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>107689-10G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue culture dish</td>
			<td>Fisher Scientific</td>
			<td>08-772E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>11-999</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>10-275</td>
			<td>curved</td>
		</tr>
		<tr>
			<td>Scalpal</td>
			<td>Medex Supply</td>
			<td>GRF-2975#10</td>
			<td>Size no. 10</td>
		</tr>
		<tr>
			<td>70 &mu;m cell stainer</td>
			<td>VWR</td>
			<td>21008-952</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>15 ml conical centrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>14-959-49D</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alcohol swabs</td>
			<td>Fisher Scientific</td>
			<td>1368063</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>23 gauge needle</td>
			<td>Fisher Scientific</td>
			<td>14-821-13F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Eye lubricant</td>
			<td>CVS</td>
			<td>8883660</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Providone-lodine swabs</td>
			<td>Fisher Scientific</td>
			<td>NC0116841</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Silicon Chambers</td>
			<td>Renner GmbH</td>
			<td>F2U, 30268</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sutures</td>
			<td>Acuderm</td>
			<td>SUP3524</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Flexible bandage</td>
			<td>Fisher Scientific</td>
			<td>22-363-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Non-adherent dressing</td>
			<td>TELFA</td>
			<td>1050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>
			Wash solution
			PBS 
			500 &mu;g/ml Penicillin/Streptomycin 
			12.5 &mu;g/ml Fungizone 
			100 &mu;g/ml Gentamycin
			Dispase solution
			HBSS 
			2.5 mg/ml final Dispase 
			100 &mu;g/ml Gentamycin
			Neutralizing media
			HBSS 
			15% (v/v) FBS
			Collagenase solution 
			0.25% (w/v) Collagenase, Type 1
			100 &mu;g/ml Penicillin/Streptomycin
			2.5 &mu;g/ml Fungizone
			50 &mu;g/ml Gentamycin
			Polybrene solution
			HBSS 
			8 &mu;g/mL (w/v) Polybrene</td>
		</tr>
	</tbody>
</table>

rapid genetic analysis of epithelial-mesenchymal signaling during hair regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

In Figure 1 we show the result of dermal-specific lentiviral shRNA knockdown of Smoothened (Smo), a critical Shh signaling component, in a 3-week old regenerative hair graft. Control grafts using lentiviral vector alone show robust hair growth (Figure 1A). By contrast, dermal-specific knockdown of Smo results in loss of hair growth (Figure 1B). Hematoxylin and eosin stain depicts the stage of hair follicles in control and experimental conditions (Figure 1C, D). The hair growth phenotype from similar treatments can be variable among experiments, including the positive control with lentiviral vector alone infection. Therefore positive control grafts should always be included in every experiment as a reference as 30% of grafts can fail as a result of scarring or incomplete attachment of grafts. In the case of testing an interaction between two factors such as overexpressing cDNA of one factor to rescue shRNA-mediated knockdown of another factor, one should always include knockdown alone grafts to manifest the loss-of-function result in each experiment. This will provide a necessary range to determine how a particular gene perturbs the hair follicle regeneration pathway and how two genes interact.
Removal of the chamber (Figure 2A) must be done with care. The newly formed graft should be slightly opaque with a dull surface (Figure 2B). The graft may stick to the chamber, so gently lifting one side is important to make sure the graft remains attached to the wound bed. The graft is quite stable after three days and should present little difficulty when removing the dressing. Robust hair growth should be observed after three weeks (Figure 2C). Omitting either mDCs or KCs will result in a recovered wound site without any hair (Figure 2D)11. Cell death or cell loss in one of the cell types also results in no hair, in contrast to reduced hair regeneration in dermal-Smo knockdown as shown in Figure 1B.
To ensure a successful assay, it is critical to achieve greater than 90% viral infection of cells with appropriate overexpression or knockdown efficiency before grafting. Due to the time constraints of the grafting procedure, we recommend trouble shooting viral infection efficiencies before starting a grafting experiment, and always save a small portion of cells on day of grafting procedure to verify loss- or gain-of-expression. Lentiviral titer will vary depending on the construct used and should be determined before infection to achieve 90-100% efficiency. We monitor viral infection efficiency with GFP coexpressed from the lentiviral vector. We routinely perform quantitative PCR and/or Western blot to determine the extent of knockdown or overexpression. Utilizing dual-expression vectors with fluorescent tags to label virally infected cells provides a way to report perdurance of signal and number of cells expressing our constructs in mature grafts. We use GFP driven by a CMV promoter from the lentiviral vector pSicoR-CMV-GFP 10 in combination with Smo shRNA. In Figure 3, we show a 3-week old dermal-specific graft where GFP is lentivirally-expressed in dermal papilla of a representative hair follicle.
<img alt="Figure 1" src="/files/ftp_upload/4344/4344fig1.jpg" /> 
Figure 1. Histological analysis of hair follicle growth. (A) View of control hair regeneration graft with dermal cells infected with empty lentiviral vector and untreated keratinocytes. (B) Smoothened knockdown using lentiviral shRNA in dermal cells combined with untreated keratinocytes results in loss of hair follicles. (C) Hematoxylin and eosin staining shows anagen hair follicles in control grafts extend down into the dermis and (D) Smoothened knockdown hair follicles remain stunted and largely absent. All images are three weeks after grafting. Scale bar denotes 100 &mu;m.
<img alt="Figure 2" src="/files/ftp_upload/4344/4344fig2.jpg" /> 
Figure 2. Grafting primary cells. (A) nu/nu mouse with sutured chamber housing primary dermal cells and keratinocytes. (B) Removal of chamber exposes newly formed skin that is dull and opaque in character. (C) Fully grown hairs on graft using wild-type primary dermal cells and keratinocytes at three weeks after grafting. (D) Addition of primary dermal cells without keratinocytes leads to no hair after three weeks. Similar results are seen with addition of primary keratinocytes without dermal cells.
<img alt="Figure 3" src="/files/ftp_upload/4344/4344fig3.jpg" /> 
Figure 3. Maintenance of GFP expression in dermal papilla. Confocal images of dermal papilla from a 3-week old regenerated graft. GFP was expressed in the dermal cell population from a lentiviral vector and detected in the dermal papilla (green). Versican (red) demarcates the dermal papilla and nuclei were label with Hoechst (blue). Note GFP is expressed specifically in dermal cells but not in the surrounding keratinocytes. Scale bar denotes 20 &mu;m.

Watch this Scientific Journal Video about Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration at JoVE.com

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

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Research

JoVE Journal

Biology

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Extraction and Analysis of Cortisol from Human and Monkey Hair

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT &#34;point samples&#34; obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.
In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair&#39;s protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings.

Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...