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 ne...

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 border="0" cellpadding="0" cellspacing="0" width="1767">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Name</td>
			<td style="width:129px;">Catalog #.</td>
			<td style="width:213px;">Company</td>
			<td style="width:997px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12 without HEPES</td>
			<td>SH3027101</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>P0781</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin solution from bovine serum</td>
			<td>A9576</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 16% Solution</td>
			<td>15710</td>
			<td>Electron Microscopy Sciences</td>
			<td>Diluted to 4% in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline 10X</td>
			<td>BP665-1</td>
			<td>Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline 1X</td>
			<td></td>
			<td></td>
			<td>Prepared from 10X stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">holo-Transferrin bovine</td>
			<td>T1283</td>
			<td>Sigma-Aldrich</td>
			<td>25 mg/ml stock solution in water. Freeze single-use aliquots at -20C. Add to DMEM/F12 media to a final concentration of 50 ug/mL.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">L- Ascorbic acid (Vitamin C)</td>
			<td>A4544</td>
			<td>Sigma-Aldrich</td>
			<td>25 mg/ml stock solution in water. Freeze single-use aliquots at -20C. Add to DMEM/F12 media to a final concentration of 50 ug/mL.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioLite 100mm 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 height="21">
			<td height="21" style="height:21px;">Stereo Microscope with substage iluminator</td>
			<td>Stemi 2000</td>
			<td>Carl Zeiss</td>
			<td rowspan="2">Any stereo dissecting microscope can be used that has a transmitted light base.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Substage Illuminator Base for Stereo Microscope</td>
			<td>TLB 4000</td>
			<td>Diagnostics Instruments, Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 35mm Tissue Culture Treated Dishes</td>
			<td>353001</td>
			<td>Corning</td>
			<td>Non-tissue culture-treated plates can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mm uncoated glass bottom dishes</td>
			<td>P50G-1.5-14-F</td>
			<td>MatTek Corporation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Widefield fluorescence microscope</td>
			<td>Axio Observer Z1</td>
			<td style="width:213px;">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 height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>TCS SP5</td>
			<td style="width:213px;">Leica Microsystems</td>
			<td>Confocal microscopy is necessary to see detailed cell structures. Any confocal microscope can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td>Ideal for harvesting glands from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SWISS Micro-Fine Forceps, #5 (11.2cm)</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 height="21">
			<td height="21" style="height:21px;">Dumont Standard Tip Forceps, #5 (11cm)</td>
			<td>91150-20</td>
			<td>Fine Science Tools (USA), Inc.</td>
			<td>Ideal for harvesting glands from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reusable Plastic Surgical Knife Handles, style no. 3.</td>
			<td>4-30</td>
			<td colspan="2">Integra LifeSciences Corporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless Steel Sterile Surgical Blades, no. 10</td>
			<td>4-310</td>
			<td colspan="2">Integra LifeSciences Corporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAqueous-Micro Total RNA Isolation Kit</td>
			<td>AM1931</td>
			<td>Life Technologies</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;width:428px;">Timed-pregnant Crl:CD1(ICR) mice</td>
			<td></td>
			<td style="width:213px;">Charles River Labs</td>
			<td>Embryos are harvested on day 14 (with day of plug discovery designated as day 0).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nucleopore Track-Etched Hydrophilic Membranes, 0.1 um pre size, 13mm</td>
			<td>110405</td>
			<td>Whatman</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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, e1...

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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 ...

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20.7K Views.  University of California San Francisco. 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.