Name: stimulation of notch signaling in mouse osteoclast precursors
Uploaded: 2017-02-28T15:00:00
Description: Watch this Scientific Journal Video about Stimulation of Notch Signaling in Mouse Osteoclast Precursors at JoVE.com

This work was supported by the University of Pennsylvania Center for Musculoskeletal Disorders (5 P30 AR050950-09), a grant from the AO Foundation (S-16-12A), the Philadelphia VA Medical Center Translational Musculoskeletal Research Center, and an intramural orthopaedic surgery departmental research development fund. JWA is supported by the University of Pennsylvania Postdoctoral Opportunities in Research and Teaching (PENN-PORT) fellowship funded by the National Institute of General Medical Sciences Institutional Research and Career Development Award (IRACDA; 5 K12 GM081259-08).

All research involving vertebrate animals was performed in accordance with protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC).
1. Culture Media Preparation
<ol>
	<li>Prepare &#945;-MEM by dissolving minimum essential medium (MEM) powder and 1.9 g sodium bicarbonate in 900 ml H2O and supplement with 2 mM L-glutamine, 100 units/ml penicillin, 100 &#956;g/ml streptomycin, and 100 ml heat-inactivated fetal bovine serum. Sterilize u...

<ol>
	<li>Harper, J. A., Yuan, J. S., Tan, J. B., Visan, I., Guidos, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+in+development+and+disease.">Notch signaling in development and disease.</a> Clin Genet. 64 (6), 461-472 (2003).</li><li>Andersson, E. R., Sandberg, R., Lendahl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling:+simplicity+in+design,+versatility+in+function.">Notch signaling: simplicity in design, versatility in function.</a> Development. 138 (17), 3593-3612 (2011).</li><li>Meloty-Kapella, L., Shergill, B., Kuon, J., Botvinick, E., Weinmaster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+ligand+endocytosis+generates+mechanical+pulling+force+dependent+on+dynamin,+epsins,+and+actin.">Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin.</a> Dev Cell. 22 (6), 1299-1312 (2012).</li><li>Chowdhury, F., 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=Defining+Single+Molecular+Forces+Required+for+Notch+Activation+Using+Nano+Yoyo.">Defining Single Molecular Forces Required for Notch Activation Using Nano Yoyo.</a> Nano Lett. 16 (6), 3892-3897 (2016).</li><li>Chillakuri, C. R., Sheppard, D., Lea, S. M., Handford, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+receptor-ligand+binding+and+activation:+insights+from+molecular+studies.">Notch receptor-ligand binding and activation: insights from molecular studies.</a> Semin Cell Dev Biol. 23 (4), 421-428 (2012).</li><li>Vas, V., Szilagyi, L., Paloczi, K., Uher, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+Jagged-1+is+able+to+inhibit+the+function+of+its+multivalent+form+to+induce+hematopoietic+stem+cell+self-renewal+in+a+surrogate+in+vitro+assay.">Soluble Jagged-1 is able to inhibit the function of its multivalent form to induce hematopoietic stem cell self-renewal in a surrogate in vitro assay.</a> J Leukoc Biol. 75 (4), 714-720 (2004).</li><li>Small, D., 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=Soluble+Jagged+1+represses+the+function+of+its+transmembrane+form+to+induce+the+formation+of+the+Src-dependent+chord-like+phenotype.">Soluble Jagged 1 represses the function of its transmembrane form to induce the formation of the Src-dependent chord-like phenotype.</a> J Biol Chem. 276 (34), 32022-32030 (2001).</li><li>Goncalves, R. M., Martins, M. C., Almeida-Porada, G., Barbosa, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+notch+signaling+by+immobilization+of+jagged-1+on+self-assembled+monolayers.">Induction of notch signaling by immobilization of jagged-1 on self-assembled monolayers.</a> Biomaterials. 30 (36), 6879-6887 (2009).</li><li>Varnum-Finney, B., 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=The+Notch+ligand,+Jagged-1,+influences+the+development+of+primitive+hematopoietic+precursor+cells.">The Notch ligand, Jagged-1, influences the development of primitive hematopoietic precursor cells.</a> Blood. 91 (11), 4084-4091 (1998).</li><li>Zhu, F., Sweetwyne, M. T., Hankenson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKCdelta+is+required+for+Jagged-1+induction+of+human+mesenchymal+stem+cell+osteogenic+differentiation.">PKCdelta is required for Jagged-1 induction of human mesenchymal stem cell osteogenic differentiation.</a> Stem Cells. 31 (6), 1181-1192 (2013).</li><li>Yang, B., 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=Effect+of+radiation+on+the+Notch+signaling+pathway+in+osteoblasts.">Effect of radiation on the Notch signaling pathway in osteoblasts.</a> Int J Mol Med. 31 (3), 698-706 (2013).</li><li>Urs, S., 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=Effect+of+soluble+Jagged1-mediated+inhibition+of+Notch+signaling+on+proliferation+and+differentiation+of+an+adipocyte+progenitor+cell+model.">Effect of soluble Jagged1-mediated inhibition of Notch signaling on proliferation and differentiation of an adipocyte progenitor cell model.</a> Adipocyte. 1 (1), 46-57 (2012).</li><li>LeComte, M. D., Shimada, I. S., Sherwin, C., Spees, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1-STAT3-ETBR+signaling+axis+controls+reactive+astrocyte+proliferation+after+brain+injury.">Notch1-STAT3-ETBR signaling axis controls reactive astrocyte proliferation after brain injury.</a> Proc Natl Acad Sci U S A. 112 (28), 8726-8731 (2015).</li><li>Kostianovsky, A. M., Maier, L. M., Baecher-Allan, C., Anderson, A. C., Anderson, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+gene+related+to+anergy+in+lymphocytes+is+associated+with+Notch-mediated+human+T+cell+suppression.">Up-regulation of gene related to anergy in lymphocytes is associated with Notch-mediated human T cell suppression.</a> J Immunol. 178 (10), 6158-6163 (2007).</li><li>Teitelbaum, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclasts:+what+do+they+do+and+how+do+they+do+it?.">Osteoclasts: what do they do and how do they do it?.</a> Am J Pathol. 170 (2), 427-435 (2007).</li><li>Ma, 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=Disruption+of+the+transcription+factor+RBP-J+results+in+osteopenia+attributable+to+attenuated+osteoclast+differentiation.">Disruption of the transcription factor RBP-J results in osteopenia attributable to attenuated osteoclast differentiation.</a> Mol Biol Rep. 40 (3), 2097-2105 (2013).</li><li>Bai, S., 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=NOTCH1+regulates+osteoclastogenesis+directly+in+osteoclast+precursors+and+indirectly+via+osteoblast+lineage+cells.">NOTCH1 regulates osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblast lineage cells.</a> J Biol Chem. 283 (10), 6509-6518 (2008).</li><li>Yamada, 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=Regulation+of+osteoclast+development+by+Notch+signaling+directed+to+osteoclast+precursors+and+through+stromal+cells.">Regulation of osteoclast development by Notch signaling directed to osteoclast precursors and through stromal cells.</a> Blood. 101 (6), 2227-2234 (2003).</li><li>Sethi, N., Dai, X., Winter, C. G., Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-derived+JAGGED1+promotes+osteolytic+bone+metastasis+of+breast+cancer+by+engaging+notch+signaling+in+bone+cells.">Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.</a> Cancer Cell. 19 (2), 192-205 (2011).</li><li>Sekine, C., 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=Differential+regulation+of+osteoclastogenesis+by+Notch2/Delta-like+1+and+Notch1/Jagged1+axes.">Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes.</a> Arthritis Res Ther. 14 (2), R45 (2012).</li><li>Ashley, J. W., Ahn, J., Hankenson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+Signaling+Promotes+Osteoclast+Maturation+and+Resorptive+Activity.">Notch Signaling Promotes Osteoclast Maturation and Resorptive Activity.</a> J Cell Biochem. 116 (11), 2598-2609 (2015).</li><li>Fukushima, H., 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=The+association+of+Notch2+and+NF-kappaB+accelerates+RANKL-induced+osteoclastogenesis.">The association of Notch2 and NF-kappaB accelerates RANKL-induced osteoclastogenesis.</a> Mol Cell Biol. 28 (20), 6402-6412 (2008).</li><li>Canalis, E., Schilling, L., Yee, S. P., Lee, S. K., Zanotti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hajdu+Cheney+Mouse+Mutants+Exhibit+Osteopenia,+Increased+Osteoclastogenesis,+and+Bone+Resorption.">Hajdu Cheney Mouse Mutants Exhibit Osteopenia, Increased Osteoclastogenesis, and Bone Resorption.</a> J Biol Chem. 291 (4), 1538-1551 (2016).</li></ol>

Critical steps within the protocol
Culture and in vitro differentiation of osteoclast precursors provides a useful platform for investigation of molecular mechanisms of osteoclastogenesis and identification of therapeutic targets for bone regeneration and preservation of bone mass. When culturing mouse osteoclast precursors, the most critical element is maintenance of precursors in a na&#239;ve state. As macrophage-like cells, osteoclast precursors are primed to respond to bacterial comp...

The mammalian Notch signaling pathway is homologous to the same pathway in Drosophila melanogaster and consists of four transmembrane Notch receptors (Notch1-4) and five membrane-bound ligands of the Jagged (JAG1 &#38; JAG2) and Delta-like (DLL1, 3,&#38; 4) families1. Notch signaling is initiated when Notch receptor on a receiving cell is bound by ligand on a transmitting cell2. During this trans-activation, the membrane-bound Notch ligand produces a stretching force on the membrane-bound Notch receptor3,4. The stretching force of ligand binding induces conformational changes in the Notch receptor that facilitate extracellular cleavage of the receptor by TNFalpha converting enzyme (TACE) followed by an intracellular cleavage event mediated by a presenilin-containing gamma-secretase complex (&#947;-secretase). &#947;-secretase releases the Notch intracellular domain (NICD) which translocates into the nucleus where it forms a transcriptional activation complex with CBF-1-Su(H)-Lag-1 (CSL), mastermind-like (MAML), and cell type-specific factors to drive expression of target genes5.
The mechanical elements of Notch signaling activation result in the need for unique methods of Notch pathway activation in vitro. Soluble Notch ligands can bind to Notch receptors, but fail to produce stretching forces necessary for NICD release while at the same time competitively inhibiting binding of cell-associated Notch ligands. Thus, addition of soluble Notch ligands to culture medium can attenuate normal Notch signaling6,7. Fortunately, Notch ligands can induce NICD release if they are fixed to a suitably rigid substrate5,8,9,10. Seeding cells on ligand-coated culture substrates or applying ligand-coated beads to cells can both activate Notch signaling, and the choice between them depends primarily on the desired timing of Notch stimulation. For immediate, temporary Notch signaling activation, as would be desired during the midpoint of a functional or differentiation assay, Notch ligand can be bound to agarose beads, applied to cultured cells, and washed out at any time. For more sustained Notch signaling from the beginning of a culture period, tissue culture plates can be coated with ligand prior to cell seeding.
For the purposes of this protocol, methods are carried out using mouse osteoclast precursors, but the methods and variations on the methods described here are applicable to a wide variety of cell types6,11,12,13,14. Osteoclasts are terminally differentiated hematopoietic linage cells that are responsible for the resorption of bone tissue, and they are implicated in multiple disorders of bone loss15. Thus, in vitro study of the differentiation of osteoclasts from their monocyte/macrophage-lineage precursors and the molecular mechanisms controlling their function is essential to better understanding of osteoclasts and development of new bone-regenerating therapies. While it is now generally accepted that Notch signaling plays a positive role in the differentiation and function of osteoclasts, variations in both Notch signaling stimulation and osteoclast precursor culture and differentiation led to initially contradictory findings16,17,18,19. Closer examination of the differences in methods and use of genetic models have greatly clarified the role of Notch signaling in osteoclastogenesis, but application of standardized Notch stimulation and culture methods could prevent such controversies in future studies of Notch signaling in other cell types20,21,22,23.
There are multiple methods for culturing and differentiating mouse osteoclast precursors, and, as with varying methods for stimulating Notch signaling, the best method will depend upon the experimental question. Herein, our preferred method of culturing adherent and non-adherent fractions of marrow cells flushed from mouse long bones will be presented. This method has the advantage of requiring essentially no specialized equipment and producing cells that are applicable to a variety of differentiation methods.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Recombinant mouse M-CSF</td>
			<td>Biolegend</td>
			<td>576402</td>
			<td>Available from multiple suppliers, test activity before experiments</td>
		</tr>
		<tr>
			<td>Recombinant mouse RANKL</td>
			<td>Shenandoah Biotechnology</td>
			<td>200-04</td>
			<td>Available from multiple suppliers, test activity before experiments</td>
		</tr>
		<tr>
			<td>Recombinant human Jagged1-Fc</td>
			<td>R&#38;D Systems</td>
			<td>1277-JG-050</td>
			<td>Available from multiple suppliers, test activity before experiments</td>
		</tr>
		<tr>
			<td>Protein G agarose beads</td>
			<td>InvivoGen</td>
			<td>gel-agg-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgG Fc</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-001-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium powder</td>
			<td>Sigma-Aldrich</td>
			<td>M0894</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase cell dissociation reagent</td>
			<td>ThermoFisher</td>
			<td>A1110501</td>
			<td>Used to lift osteoclast precursors</td>
		</tr>
		<tr>
			<td>Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A-1KT</td>
			<td>Used to stain differentiated osteoclasts</td>
		</tr>
	</tbody>
</table>

stimulation of notch signaling in mouse osteoclast precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

The aim of this method is to culture and stimulate Notch signaling in osteoclast precursors. When properly cultured, osteoclast precursors exhibit a primarily elongated spindle-shaped morphology with smooth cytoplasm (Figure 1A). Care should be taken to avoid immunological activation of the osteoclast precursors. Upon activation, precursors spread and become flattened with foamy cytoplasm (Figure 1B). These &#34;fried egg&#34; cells are resistant to RANK ...

Watch this Scientific Journal Video about Stimulation of Notch Signaling in Mouse Osteoclast Precursors at JoVE.com

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

The overall goal of this procedure is to observe the affects of notch signaling stimulation at varying points during the osteoclast differentiation process. This method can help answer key questions in the field of cell biology, such as:How does the role of notch signaling change during a cellular process? The main advantage of this technique is its marginality, which allows it to be adapted to other cells or notch ligands.While this method can provide insight into osteoclast differentiation, it can also be applied to other processes such as cell survival, proliferation, or to the differentiation of other cell types. Begin the procedure by securing a mouse in the supine position and disinfecting the skin with 70 percent ethanol. Next, make an incision along the anterior surface of one hind limb to expose the tibia and femur.Followed by an incision through the knee joint to free the distal end of the femur. Remove the femur by cutting through the end of the bone close to the pelvis and clear away any of the attached soft tissue. Cut through the ankle to remove the tibia and remove the soft tissue.Then, use forceps to hold the leg bone over a 15 milliliter conical tube and insert a 25 and five-eighths gauged needle attached to a 10 milliliter syringe into the uncut end of the bone to flush two point five milliliters of room temperature alpha MEM through the cut end of the bone into the tube. When all of the bone marrow has been removed, the bone will turn a clear, beige color. After collecting the marrow from each bone, allow any large debris to settle to the bottom of the tube and transfer the debris-free, bone marrow cell containing supernatant into a new 15 milliliter tube.Pellet the cells by centrifugation. Then lyse the red blood cells in zero point five milliliters of ACK Lysis buffer at 37 degrees Celsius for three minutes. At the end of the incubation, restore the isotonicity with 10 milliliters of PBS and collect the cells by centrifugation.Resuspend the pellet in 10 milliliters of fresh Alpha MEM for plating. In the 100 milliliter tissue culture treated dish, promouse. And incubate the cells overnight at 37 degrees Celsius in a humidified tissue-culture incubator.After enriching for the osteoclast precursors, wash the cells in five milliliters of PBS and treat them for three to five minutes with one milliliter of cell-dissociation reagent at room temperature until the osteoclast precursors can be dislodged with gentle tapping. Next, add four milliliters of Alpha MEM to the dish and transfer the cells to a 15 milliliter conical tube. After counting, dilute the precursors to a five times 10 to the fourth cells per milliliter concentration in Alpha MEM.And treat them with MCSF and RANKL, then immediately seed one milliliter of cells into each well of a 24 well tissue-treated plate. Further incubation and 37 degrees Celsius in the humidified tissue culture incubator. After two days, replace the medium with one milliliter of fresh osteoclast medium to continue the differentiation.For continuous notch signaling simulation, coat single well of a 24 well plate with 250 microliters of goat anti-human, IGG FC antibody in PBS. After one hour at room temperature, remove any unbound antibody with three washes and one milliliter of PBS. Next, add jagged1-FC fusion protein in PBS to the well for a two hour incubation at room temperature.At the end of the incubation, wash the well three times with fresh PBS as just demonstrated. After the third wash, remove the PBS and immediately plate two point six times 10 to the fourth osteoclast precursors per centimeter squared onto the coated surface. Then transfer the plate to the tissue culture incubator for continuous notch signaling for up to three days.For temporary notch signaling stimulation, first add one microgram of jagged1-FC with 40 microliters of protein G Agarose beads in one point five milliliters of PBS for two hour incubation with rotation at four degrees Celsius. At the end of the incubation, collect the beads by centrifugation followed by two washes and one point five milliliters of PBS under the same centrifuge conditions. After the second wash, re-suspend the jagged1 conjugated beads in 130 microliters per centimeter squared of culture medium and add the entire volume of beads directly onto two point six times 10 to the fourth osteoclast precursors in one well of a 24 well plate for their cold culture at 37 degrees Celsius and five percent carbon dioxide.After the appropriate experimental stimulation period, remove the beads with several washes of PBS. Notch signaling activation can then be assayed by measuring the target gene transcription. When properly cultured, osteoclast precursors exhibit a primarily elongated, spindle-shaped morphology with a smooth cytoplasm.Upon activation, the precursors spread and become flattened with foamy cytoplasm obtaining a resistance to RANK signaling and do not efficiently differentiate into mature osteoclasts. Under the correct culture and differentiation conditions, enriched osteoclast precursors will differentiate and infuse into large, TRAP-positive, multi-nuclear osteoclasts within three days of treatment. Seeding osteoclast precursors onto jagged1 FC-coated surfaces for their continuous stimulation induces the upper regulation of the expression of specific notch-target genes within 24 hours, while the expression of other notch target genes is not significantly altered by the stimulation.Temporary notch signaling stimulation, via jagged1 FC coated beads with as little as 600 nanograms of jagged1 FC protein also induces a significant increase in the expression of the specific notch target genes. While attempting this procedure, it's important to remember that a common cause of osteoclast precursor differentiation failure is by accidental activation during handling. This can be assessed by measuring specific activation markers such as TNF to Inos.After watching this video, you should have a good idea of how to differentiate osteoclast precursors and also to stimulate notch signaling, both transiently and continuously.

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Research

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Developmental Biology

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within the bone marrow cavity alongside hematopoietic stem cells (HSCs), which can give rise to red blood cells, immune progenitors, and osteoclasts. Thus, extractions of cell populations from the bone marrow results in a very heterogeneous mix of various cell populations, which can present challenges in experimental design and confound data interpretation. Several isolation and culture techniques have been developed in laboratories in order to obtain more or less homogeneous populations of BMSCs and HSCs invitro. Here, we present two methods for isolation of BMSCs and HSCs from mouse long bones: one method that yields a mixed population of BMSCs and HSCs and one method that attempts to separate the two cell populations based on adherence. Both methods provide cells suitable for osteogenic and adipogenic differentiation experiments as well as functional assays.

In this article, we present methods to isolate and differentiate bone marrow stromal cells and hematopoietic stem cells from mouse long bones. Two different protocols are presented yielding different cell populations suitable for expansion and differentiation into osteoblasts, adipocytes, and osteoclasts.

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...