The authors would like to acknowledge funding provided by the UCSF Resource Allocation Program.

All animal work was performed under strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Francisco.
1. Mouse Embryonic Lacrimal Glands (LG): Harvesting and Microdissection
<ol>
	<li>Following procedures approved by the Institutional Animals for Scientific Research Ethics committee, euthanize timed pregnant CD-1 (or transgenic) females with rising CO2 inhalation, followed by confirmation by cervical dislocation to harvest embryos on the appropriate embryonic day. Designate the day of vaginal plug discovery e0. 
	Note: Lacrimal glands (LGs) can be harvested from the e14 single bud stage onwards. However, e16 (5 - 10 buds) embryos give the most consistent results for ex vivo culture.</li>
	<li>Sterilize the ventral side of the mouse using 70% ethanol. Pinch the skin and make an incision at the midline with sterile surgical scissors; cut through the skin and peritoneum to expose the abdominal cavity. Remove the 2 uterine horns by cutting along the mesometrium at the top of each uterine horn and place in cold PBS (Phosphate Buffered Saline) or DMEM F12 media supplemented with 100&#160;U/ml penicillin and 100&#160;&#956;g/ml streptomycin.</li>
	<li>Using forceps (#5), remove embryos from the amniotic sacs and place into a second sterile Petri dish containing cold PBS. Be careful to not touch the eyes or head area.</li>
	<li>Place embryo onto a dissecting microscope with a transillumination base. If the embryo is &#60;e17, perform steps 1.5 - 1.7. If embryos are &#62;e17, these steps may be helpful but are not essential, therefore proceed to step 1.8.</li>
	<li>Sever the head from the torso just below the lower mandible (step 1, Figure 2) using a sterile scalpel (#10 blade). Rotate head so that the mandible is now on the dissecting plate. Use forceps to stabilize the head and a scalpel to remove about &#188; of the dorsal side of brain (step 2, 1st cut, Figure 2) &#8211; this is especially important once cartilage stiffens around e15.</li>
	<li>Orient the head so that the scalpel can pierce the nose between the eyes. Make a cut through the center of the head, so that the eyes are now on separate halves (step 2, 2nd cut, Figure 2).</li>
	<li>Using two #5 forceps, take one half of the head and remove excess tissue (see step 3, Figure 2). Be sure to orient the head so the LG (located in the lower posterior corner of the eye) is not lost in this removal process. Remove excess cerebral, cartilage, and nasal tissue surrounding and below the eye. Since the gland is barely visible at this point, ensure the eye is properly oriented after excess tissue removal to avoid losing the location of the LG.</li>
	<li>Carefully grab the skin from the posterior, bottom corner of the eye with both forceps. Remove the skin by carefully pulling open with the forceps to expose the LG. Use additional illumination above and below the tissue to help visualize the LG. 
	Note: The LG, distinguishable by its appearance as a bud on a duct within a darker, condensed mesenchyme, should be visible at this point.</li>
	<li>Carefully begin to dissect the LG and associated mesenchyme (refer to diagram and images) away from the surrounding tissue using forceps, being careful not to grab the LG or its associated duct. Once the gland is free from the surrounding tissue, gently remove the entire gland by gripping the epithelia surrounding the eye at the base of the LG duct. Take care to preserve the mesenchyme surrounding the epithelium, which can be observed as a condensed mesenchyme into which the epithelium invaginates. 
	NOTE: Some additional removal of tissue (any small bone fragments, muscle, and connective tissue) may be necessary to avoid signaling/growth factors from neighboring tissue.</li>
	<li>For culture, harvest 4 - 5 glands and associated mesenchyme per plate (LGs are plated immediately upon dissection); collect a minimum of 14 glands per 100&#160;&#956;l of RNA lysis buffer for RT-PCR6.</li>
</ol>
2. Prepare Plates for Ex vivo Culture
This procedure is based on that employed for the developing salivary gland7,8.
<ol>
	<li>Add 200&#160;&#956;l of DMEM F12 (supplemented with 100&#160;U/ml penicillin and 100&#160;&#956;g/ml streptomycin) culture media with 50&#160;&#956;g/ml ascorbic acid and 50&#160;&#956;g/ml holo-transferrin to a glass-bottomed 50&#160;mm diameter microwell dish7. 
	Note: DMEM F12 was chosen as we previously found it to maximize morphogenesis in the embryonic salivary gland.</li>
	<li>Float a 13&#160;mm polycarbonate membrane filter with 0.1&#160;&#956;m pores on top of media.</li>
	<li>Optional Step: Dilute 3-D laminin 1:1 with DMEM/F12 to make a final concentration of 3&#160;mg/ml (use 200&#160;&#956;l pipette to gently mix; centrifuge for 1 min if bubbles appear). Add 15&#160;&#956;l of this diluted 3-D laminin to filter. 
	Note: This dilution was found to be optimal for maintaining the LG in its 3D state without compromising epithelial branching. However, it is not essential for culture of LG.</li>
	<li>Place 4 - 5 LG onto filter or into laminin. Culture LGs ex vivo for 24 - 48&#160;hr (Figure 3) or fix with 4% PFA for 20&#160;min for further analysis by qPCR or immunostaining (Figure 4). For qPCR analysis of embryonic glandular tissue, please refer to Rebustini et al., 2011. For immunofluorescent analysis of embryonic glandular tissues please refer to the following citations: Hoffman et al.,&#160;(2002) and Steinberg et al., (2005).</li>
</ol>
3. Mouse Postnatal and Adult Lacrimal Glands (LG): Dissection
<ol>
	<li>Euthanize the postnatal mouse according to appropriate animal ethics committee&#8217;s standards. For postnatal pups day 8 or younger, euthanize by severing the head with sterile scissors. For postnatal day 8 or older, spray fur with 70% ethanol.</li>
	<li>To locate the LG, lay mouse lateral side up under a dissecting microscope with illumination from above. Note: The post-natal and adult LG are bordered by the carotid artery, the masseter muscle and the dermis (see Figure 5).</li>
	<li>Using small dissection scissors, make small incision in the epidermis laterally from where the LG should be located.</li>
	<li>Using forceps, pull open the epidermis toward the ear to expose the LG.</li>
	<li>Use forceps to gently loosen LG from surrounding tissue.</li>
	<li>Once the LG is released from surrounding tissue, carefully remove the LG by the duct, by gripping where the duct and the epithelia surrounding the eye connect (Figure 1).</li>
	<li>Place LGs in RNA lysis buffer for RT-PCR, or fix with 4% PFA for 20&#160;min for immunostaining.</li>
</ol>

<ol>
	<li>Liu, K. C., Huynh, K., Grubbs, J., Davis, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmunity+in+the+pathogenesis+and+treatment+of+keratoconjunctivitis+sicca.">Autoimmunity in the pathogenesis and treatment of keratoconjunctivitis sicca.</a> Current allergy and asthma reports. 14, 403 (2014).</li><li>Makarenkova, H. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF10+is+an+inducer+and+Pax6+a+competence+factor+for+lacrimal+gland+development.">FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development.</a> Development. 127, 2563-2572 (2000).</li><li>Dartt, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+regulation+of+lacrimal+gland+secretory+processes:+relevance+in+dry+eye+diseases.">Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases.</a> Progress in retinal and eye research. 28, 155-177 (2009).</li><li>Nigam, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+can+the+intrinsic+power+of+branching+morphogenesis+be+used+for+engineering+epithelial+tissues+and+organs.">Concise review: can the intrinsic power of branching morphogenesis be used for engineering epithelial tissues and organs.</a> Stem cells translational medicine. 2, 993-1000 (2013).</li><li>Qu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosaminoglycan-dependent+restriction+of+FGF+diffusion+is+necessary+for+lacrimal+gland+development.">Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development.</a> Development. 139, 2730-2739 (2012).</li><li>Rebustini, I. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MT2-MMP-dependent+release+of+collagen+IV+NC1+domains+regulates+submandibular+gland+branching+morphogenesis.">MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis.</a> Developmental cell. 17, 482-493 (2009).</li><li>Hoffman, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Expression+Profiles+of+Mouse+Submandibular+Gland+Development:+FGFR1+Regulates+Branching+Morphogenesis+in+Vitro+Through+BMP-+and+FGF-Dependent+Mechanisms.">Gene Expression Profiles of Mouse Submandibular Gland Development: FGFR1 Regulates Branching Morphogenesis in Vitro Through BMP- and FGF-Dependent Mechanisms.</a> Development. 129, 24-5778 (2002).</li><li>Steinberg, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGFR2b+signaling+regulates+ex+vivo+submandibular+gland+epithelial+cell+proliferation+and+branching+morphogenesis.">FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.</a> Development. 132, 1223-1234 (2005).</li><li>Knox, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis. 329, 1645-1647 (2010).</li><li>Magenheim, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+restrain+pancreas+branching,+differentiation+and+growth.">Blood vessels restrain pancreas branching, differentiation and growth.</a> Development. 138, 4743-4752 (2011).</li><li>Jaskoll, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF10/FGFR2b+signaling+plays+essential+roles+during+in+vivo+embryonic+submandibular+salivary+gland+morphogenesis.">FGF10/FGFR2b signaling plays essential roles during in vivo embryonic submandibular salivary gland morphogenesis.</a> BMC developmental biology. 5, 11 (2005).</li><li>Takasato, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directing+human+embryonic+stem+cell+differentiation+towards+a+renal+lineage+generates+a+self-organizing+kidney.">Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney.</a> Nature cell biology. 16, 118-126 (2014).</li><li>Knox, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasympathetic+stimulation+improves+epithelial+organ+regeneration.">Parasympathetic stimulation improves epithelial organ regeneration.</a> Nature communications. 4, 1494 (2013).</li></ol>

The authors have nothing to disclose.

The versatility to culture and manipulate the LG ex vivo provides significant advantages for studying its development. This includes the speed at which the researcher is able to test hypotheses and the multitude of perturbations that can be performed to assess how epithelial, neuronal, endothelial and mesencyhmal cells interact to form the organ. However, there are a number of caveats when utilizing this model. First, by virtue of its isolation the gland is no longer connected to the peripheral vasculature or nervous system, which may affect the signaling pathways being tested if they work in conjunction with signals delivered by the nerves or blood vessels9,10. Second, the surrounding non-LG tissues may provide factors that regulate LG development4. To avoid this other non-mesenchymal tissue components must be removed prior to culture. Third, at present we are not able to provide the conditions necessary for complete cytodifferentiation into a fully functional tissue. This is the case for all organs cultured ex vivo and is likely due to multiple reasons including the absence of functional blood vessels and nerves and/or mechanical forces from surrounding tissues. Despite these caveats, mechanisms discovered in ex vivo culture have shown to be recapitulated in subsequent in vivo experiments, making this a robust system for testing new hypotheses8,11. 
It has been shown that developmental and regenerative pathways significantly overlap, indicating that the ex vivo culture system can also be employed to study organ regeneration12. Although we have not demonstrated this aspect here we, and others, have previously shown the embryonic salivary gland can be used to model the effects of therapeutic radiation on organ damage and regeneration13. Future studies are needed to explore the use of the LG ex vivo system as a model for tissue regeneration.

The lacrimal gland (LG) is responsible for aqueous tear secretion critical for visual acuity and the health, maintenance, and protection of the cells of the ocular surface. LG dysfunction results in one of the most common and debilitating ocular disorders: aqueous deficient Dry Eye Disease, which is characterized by ocular irritation, light sensitivity and decreased vision1. In the human the LG resides in the orbit above the lateral end of the eye where 3 - 5 excretory ducts deposit tears onto the ocular surface. The mouse has three pairs of major ocular glands, the most studied of which is the lacrimal gland (LG) located anterior and ventral to the ear (exorbital) with tears traveling to the eye via a single excretory duct. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts (Figure 1)2. During development the epithelium becomes vascularized as well as heavily innervated by the parasympathetic nerves of the pterygopalatine ganglion and to a lesser extent by sympathetic nerves from the superior cervical ganglion3. Interactions between each of these cells types i.e. neuronal, epithelial, endothelial and mesenchymal cells, are essential to the function and maintenance of the adult tissue. However, the underlying molecular mechanisms coordinating LG development and regeneration as well as how inter-cell type communication guides these processes remains unclear.
The advent of embryonic ex vivo culture techniques has allowed the identification of developmental and regenerative pathways in multiple branching organs4. Culturing ex vivo gives the researcher the ability to manipulate the organ (mechanical, genetic or chemical) under defined conditions as well as to characterize organ development and cell-cell interactions in real time. The exorbital LG of the mouse is highly amenable to this technique and recent studies have defined signaling systems that regulate its development2,5. However, despite the need to understand molecular cues underpinning LG development and regeneration, it currently remains understudied, likely due to the technical difficulties in isolating the organ. In this paper, we describe how to isolate and perform ex vivo culture of the embryonic murine LG to define developmental programs.

<table><tbody><tr><td>DMEM/F12 without HEPES</td><td>SH3027101</td><td>Thermo Scientific</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>P0781</td><td>Sigma-Aldrich</td><td></td></tr><tr><td>Albumin solution from bovine serum</td><td>A9576</td><td>Sigma-Aldrich</td><td></td></tr><tr><td>Paraformaldehyde 16% Solution</td><td>15710</td><td>Electron Microscopy Sciences</td><td>Diluted to 4% in 1x PBS</td></tr><tr><td>Phosphate Buffered Saline 10x</td><td>BP665-1</td><td>Fisher Scientific</td><td></td></tr><tr><td>Phosphate Buffered Saline 1x</td><td></td><td></td><td>Prepared from 10x stock</td></tr><tr><td>holo-Transferrin bovine</td><td>T1283</td><td>Sigma-Aldrich</td><td>25&amp;nbsp;mg/ml stock solution in water. Freeze single-use aliquots at -20&amp;nbsp;&amp;deg;C. Add to DMEM/F12 media to a final concentration of 50&amp;nbsp;&amp;mu;g/ml.</td></tr><tr><td>L-ascorbic acid (Vitamin C)</td><td>A4544</td><td>Sigma-Aldrich</td><td>25&amp;nbsp;mg/ml stock solution in water. Freeze single-use aliquots at -20&amp;nbsp;&amp;deg;C. Add to DMEM/F12 media to a final concentration of 50&amp;nbsp;&amp;mu;g/ml.</td></tr><tr><td>BioLite 100 mm Tissue Culture Treated Dishes</td><td>130182</td><td>Thermo Scientific</td><td>Non-tissue culture-treated plates can also be used.</td></tr><tr><td>Stereo Microscope with substage iluminator</td><td>Stemi 2000</td><td>Carl Zeiss</td><td>Any stereo dissecting microscope can be used that has a transmitted light base.</td></tr><tr><td>Substage Illuminator Base for Stereo Microscope</td><td>TLB 4000</td><td>Diagnostics Instruments, Inc.</td><td></td></tr><tr><td>Falcon 35&amp;nbsp;mm Tissue Culture Treated Dishes</td><td>353001</td><td>Corning</td><td>Non-tissue culture-treated plates can also be used.</td></tr><tr><td>50 mm uncoated glass bottom dishes</td><td>P50G-1.5-14-F</td><td>MatTek Corporation</td><td></td></tr><tr><td>Widefield fluorescence microscope</td><td>Axio Observer Z1</td><td>Carl Zeiss</td><td>Any fluorescence microscope (upright, inverted or stereo dissecting microscope) can be used to monitor GFP expression at low magnification with an attached digital camera.</td></tr><tr><td>Confocal Microscope</td><td>TCS SP5</td><td>Leica Microsystems</td><td>Confocal microscopy is necessary to see detailed cell structures. Any confocal microscope can be used.</td></tr><tr><td>SWISS Micro-Fine Forceps, #5 (11.2&amp;nbsp;cm)</td><td>17-305X</td><td>Integra LifeSciences Corporation</td><td>Fine tips are required for removing mesenchyme from epithelium. Tungsten needles can also be used.</td></tr><tr><td>Dumont Standard Tip Forceps, #5 (11&amp;nbsp;cm)</td><td>91150-20</td><td>Fine Science Tools (USA), Inc.</td><td>Ideal for harvesting glands from embryos.</td></tr><tr><td>Reusable Plastic Surgical Knife Handles, style no. 3.</td><td>4-30</td><td>Integra LifeSciences Corporation</td><td></td></tr><tr><td>Stainless Steel Sterile Surgical Blades, no. 10</td><td>4-310</td><td>Integra LifeSciences Corporation</td><td></td></tr><tr><td>RNAqueous-Micro Total RNA Isolation Kit</td><td>AM1931</td><td>Life Technologies</td><td></td></tr><tr><td>Cultrex 3D Culture Matrix Laminin I</td><td>3446-005-01</td><td>Trevigen</td><td>6 mg/ml stock diluted 1:1 with DMEM/F12</td></tr><tr><td>Timed-pregnant Crl:CD1(ICR) mice</td><td></td><td>Charles River Labs</td><td>Embryos are harvested on day 14 (with day of plug discovery designated as day 0).</td></tr><tr><td>Nucleopore Track-Etched Hydrophilic Membranes, 0.1&amp;nbsp;&amp;mu;m pore size, 13&amp;nbsp;mm</td><td>110405</td><td>Whatman</td><td></td></tr><tr><td>Timed-pregnant Pax6-Cre, GFP mice</td><td>Tg(Pax-Cre, GFP)1Pgr</td><td>The Jackson Laboratory</td><td>Optional mice for learning how to locate the LG.</td></tr></tbody></table>

manipulating the murine lacrimal gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The LG develops through the process of epithelial branching morphogenesis. Brightfield images of embryonic LGs dissected at e14, e15, e16, e17, and P2 illustrate this event.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51970/51970fig1highres.jpg" width="600" /> 
Figure 1. The LG develops through the process of epithelial branching morphogenesis. Brightfield images of embryonic LGs freshly dissected at e14, e15, e16, e17 and P2. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51970/51970fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Note that as the epithelium increases in size, the amount of mesenchyme decreases. The experimental steps for microdissection of the embryonic LGs are depicted in Figure 2. 
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51970/51970fig2highres.jpg" width="600" /> 
Figure 2. Schematic of steps involved in LG microdissection from mouse embryos. Schematic diagram of the dissection and removal of the LG from the embryonic mouse. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51970/51970fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For LG culture we routinely utilize e16 embryos due to the consistency in ex vivo growth. As shown in Figure 3, LG in ex vivo culture conditions develop in a similar manner to the in vivo gland (compare to Figure 1).
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51970/51970fig3highres.jpg" width="600" /> 
Figure 3. Ex vivo culture of e16 LGs recapitulates in vivo morphogenesis. e16 LGs were derived from Pax6-Cre, GFP embryos, where GFP is expressed by the epithelium. Consecutive fluorescent images were taken of this single gland at 0, 3, 6, 12, 24, and 48 hr. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51970/51970fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here we employed Pax6-Cre, GFP (also called Le-Cre5) embryos to highlight epithelial cells, allowing us to take consecutive fluorescent images of the branching epithelium over the 48 hr culture period. Mice harboring the Pax6-Cre, GFP transgene can be obtained from JAX: Tg(Pax6-Cre, GFP)1Pgr but are not necessary for gland dissection and culture.
Cultures or freshly dissected LG can then be fixed for immunofluorescent analysis. Figure 4 shows the visualization of 4 cellular compartments, mesenchyme, epithelium (EpCAM), nerves (Tubb3) and blood vessels (PECAM), in an e16 LG from a wild type (CD1) embryo.
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51970/51970fig4highres.jpg" width="600" /> 
Figure 4.The LG is composed of multiple cell types including epithelial, neuronal and endothelial cells. e16 LG was immunostained for epithelial cells (Epcam, green), neurons (Tubb3, red) and endothelial cells (Pecam, cyan). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51970/51970fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For freshly dissected tissue it is easier to first embed the LG in laminin, as done for culture, so as to immobilize the gland for fixation and subsequent handling. Figure 5 shows a schematic for dissection of the postnatal/adult LG.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51970/51970fig5highres.jpg" width="500" /> 
Figure 5. Schematic of steps involved in LG microdissection from post-natal and adult mouse. Schematic diagram of the dissection and removal of the LG from either post-natal or adult mouse. A Pax6-Cre,GFP post-natal day 30 mouse was used to visualize the position of the LG and associated duct. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51970/51970fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The position of the post-natal/adult LG has been visualized by using the Pax6-Cre, GFP mouse.

Watch this Scientific Journal Video about Manipulating the Murine Lacrimal Gland at JoVE.com

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Nanyang Technological University

Research

JoVE Journal

Biology

20.8K Views.  University of California San Francisco.  De traanklier (LG) is een vertakking orgaan dat de waterige componenten van tranen nodig produceert voor het behoud van de visie en de gezondheid van het oog. Hier beschrijven we muizen LG dissectie en ex vivo cultuur technieken te ontcijferen signaalwegen betrokken bij LG ontwikkeling. 

Video: Manipulating the Murine Lacrimal Gland

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...

Medicine

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Scopolamine-Induced Lacrimal Gland Dysfunction in Rats

A Rat Dry Eye Model with Lacrimal Gland Dysfunction Induced by Scopolamine

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal tear production can lead to a disturbance in the ocular surface environment, including corneal damage and inflammation. In severe cases, ADDE can cause vision loss or even blindness. Currently, dry eye treatment is limited to eye drops or physical therapy, which can only alleviate eye discomfort symptoms and cannot fundamentally cure dry eye syndrome. To restore the function of the lacrimal gland in dry eye, we have created an animal model of lacrimal gland dysfunction in rats induced by scopolamine. Through the comprehensive evaluation of the lacrimal gland, corneas, conjunctivas, and other factors, we aim to provide a full understanding of the pathological changes of ADDE. Compared with the current dry eye mouse model, this ADDE animal model includes a functional evaluation of the lacrimal gland, providing a better platform for studying lacrimal gland dysfunction in ADDE.

Author Spotlight: Challenges in Developing Dry Eye Animal Models and Future Research Directions

Here, we establish a rat model of lacrimal gland dysfunction to provide a basis for the study of aqueous-deficient dry eye.

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal ...

Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture method to obtain a sufficient number of LG stem cells (LGSCs) is one obstacle for further research and application. The three-dimensional (3D), serum-free culture method for adult mouse LGSCs is well established and shown here. The LGSCs could be continuously passaged and induced to differentiate to acinar or ductal-like cells.
For the LGSC primary culture, the LGs from 6-8-week-old mice were digested with dispase, collagenase I, and trypsin-EDTA. A total of 1 &#215; 104 single cells were seeded into 80 &#181;L of matrix gel-lacrimal gland stem cell medium (LGSCM) matrix in each well of a 24-well plate, precoated with 20 &#181;L of matrix gel-LGSCM matrix. The mix was solidified after incubation for 20 min at 37 &#176;C, and 600 &#181;L of LGSCM added.
For LGSC maintenance, LGSCs cultured for 7 days were disaggregated into single cells by dispase and trypsin-EDTA. The single cells were implanted and cultured according to the method used in the LGSC primary culture. LGSCs could be passaged over 40 times and continuously express stem/progenitor cell markers Krt14, Krt5, P63, and nestin. LGSCs cultured in LGSCM have self-renewal capacity and can differentiate into acinar or ductal-like cells in vitro and in vivo.

The three-dimensional, serum-free culture method for adult lacrimal gland (LG) stem cells is well established for the induction of LG organoid formation and differentiation into acinar or ductal-like cells.

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture ...

Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its underlying mechanisms. Therefore, we developed a mouse model of dry eye disease by surgically removing both the extraorbital lacrimal glands (ELG) and intraorbital lacrimal glands (ILG) to investigate the ocular surface pathology in the context of aqueous deficiency dry eye. Two weeks post operation, the mice exhibited severe dry eye manifestations, including reduced tear secretion, corneal epithelial irregularities, positive fluorescein sodium staining, and neovascularization. Histological examination via hematoxylin and eosin staining revealed inflammatory cell infiltration and corneal epithelium dysplasia. Immunofluorescence staining and quantitative reverse-transcription polymerase chain reaction revealed decreased expression of the normal corneal epithelial biomarkers K12 and Pax6 and increased expression of Sprr1b in the corneal epithelium. These ocular manifestations indicated abnormal corneal epithelial differentiation. Furthermore, immunofluorescence staining of Ki67 revealed the increasing cell proliferation. In conclusion, the ELG plus ILG excision model proved suitable for studying changes in the ocular surface and elucidating the mechanisms underlying aqueous deficiency dry eye.

Here, we present a method to establish a mouse model of aqueous-deficient dry eye by excising the extraorbital and infraorbital lacrimal glands and evaluate the changes in the ocular surface in aqueous deficiency dry eye.

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its ...

Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from desiccation stress and external insults. Little is known about lacrimal gland (patho)physiology because of the lack of adequate in vitro models. Organoid technology has proven itself as a useful experimental platform for multiple organs. Here, we share a protocol to establish and maintain mouse and human lacrimal gland organoids starting from lacrimal gland biopsies. By modifying the culture conditions, we enhance lacrimal gland organoid functionality. Organoid functionality can be probed through a "crying" assay, which involves exposing the lacrimal gland organoids to selected neurotransmitters to trigger tear release in their lumen. We explain how to image and quantify this phenomenon. To investigate the role of genes of interest in lacrimal gland homeostasis, these can be genetically modified. We thoroughly describe how to genetically modify lacrimal gland organoids using base editors-from guide RNA design to organoid clone genotyping. Lastly, we show how to probe the regenerative potential of human lacrimal gland organoids by orthotopic implantation in the mouse. Together, this comprehensive toolset provides resources to use mouse and human lacrimal gland organoids to study lacrimal gland (patho)physiology.

This protocol describes how to establish, maintain, genetically modify, differentiate, functionally characterize, and transplant lacrimal gland organoids derived from primary mouse and human tissue.

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from ...

Superior Lacrimal Gland Removal: A Surgical Technique to Remove the Palpebral and Orbital Parts of Superior Lacrimal Gland from Rabbit Model

Source: Honkanen, R. A. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/60126">Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits.</a> J. Vis. Exp. (2020).
This video describes the surgical procedure to remove the two parts: palpebral and orbital of the superior lacrimal gland from a rabbit to create a dry eye disease model. This rabbit model is suitable for studies of ocular surface homeostasis, pathophysiology and ocular therapeutics.

Source: Honkanen, R. A. et al., Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits. J. Vis. Exp. (2020).
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JoVE Encyclopedia of Experiments

Large Animal Models

Leporine

Inferior Lacrimal Gland Removal From Rabbit Model: A Surgical Procedure to Remove the Larger Inferior Tear Gland From Rabbit Eye Orbit

Source: Honkanen, R. A., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/60126">Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits.</a> J. Vis. Exp. (2020).
In this video, we describe a surgical procedure to remove the large inferior lacrimal gland from a rabbit model to study the effect on tear production in the animal.&#160;

Source: Honkanen, R. A., et al. Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits. J. Vis. Exp. (2020).
In this video, we ...

Performing Injection into Palpebral Portion of the Superior Lacrimal Gland: A Technique for Inducing Dry Eye Disease in Rabbit Model

Source: Honkanen, R. A., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59631">A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies.</a> J. Vis. Exp. (2020).
In this video, we show a procedure to induce aqueous-deficient dry eye disease in a rabbit model by injecting the palpebral portion of the superior lacrimal gland with Concanavalin A. This model is useful for pharmacokinetic and biodistribution assays of drugs for treating dry eye disease.

Manipulating the Murine Lacrimal Gland

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