The author would like to thank Matthew Kelley, Tiziana Cogliati, Jessica Gumerson, Uwe Wolfrum, Rivka Levron, Viola Kretschmer and Zoe Mann and for their critical evaluation of the manuscript. This work was funded by the Sofja Kovalevskaya Award (Humbodlt Foundation) and the Johannes-Gutenberg University, Mainz, Germany.

Use and euthanize all animals in accordance with institutional and governmental guidelines and regulations, most commonly via CO2 inhalation and cervical dislocation.
1. Preparation of Reagents
NOTE: Prior to beginning, prepare all reagents using analytical grade chemicals. Make solutions using molecular grade distilled and deionized water unless otherwise specified.
<ol>
	<li>Phosphate buffered saline (1x&#160;PBS): Make 1 L of 1x PBS by dissolving 8g NaCl, 0.2g KCl, 1.44g Na2HPO<su...

<ol>
	<li>Waters, A. M., Beales, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliopathies:+an+expanding+disease+spectrum.">Ciliopathies: an expanding disease spectrum.</a> Pediatr Nephrol. 26, 1039-1056 (2011).</li><li>May-Simera, H. L., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cilia,+Wnt+signaling,+and+the+cytoskeleton.">Cilia, Wnt signaling, and the cytoskeleton.</a> Cilia. 1, 7 (2012).</li><li>Ross, A. 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+Bardet-Biedl+syndrome+ciliary+proteins+perturbs+planar+cell+polarity+in+vertebrates.">Disruption of Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates.</a> Nat Genet. 37, 1135-1140 (2005).</li><li>Ezan, J., Montcouquiol, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+planar+cell+polarity+in+the+inner+ear.">Revisiting planar cell polarity in the inner ear.</a> Seminars in cell &amp; developmental biology. 24, 499-506 (2013).</li><li>Semenov, M. V., Habas, R., Macdonald, B. T., He, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SnapShot:+Noncanonical+Wnt+Signaling+Pathways.">SnapShot: Noncanonical Wnt Signaling Pathways.</a> Cell. 131, 1378 (2007).</li><li>Wang, 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=Regulation+of+polarized+extension+and+planar+cell+polarity+in+the+cochlea+by+the+vertebrate+PCP+pathway.">Regulation of polarized extension and planar cell polarity in the cochlea by the vertebrate PCP pathway.</a> Nat Genet. 37, 980-985 (2005).</li><li>Montcouquiol, 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=Identification+of+Vangl2+and+Scrb1+as+planar+polarity+genes+in+mammals.">Identification of Vangl2 and Scrb1 as planar polarity genes in mammals.</a> Nature. 423, 173-177 (2003).</li><li>May-Simera, H. L., 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=Ciliary+proteins+Bbs8+and+Ift20+promote+planar+cell+polarity+in+the+cochlea.">Ciliary proteins Bbs8 and Ift20 promote planar cell polarity in the cochlea.</a> Development. 142, 555-566 (2015).</li><li>Jones, 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=Ciliary+proteins+link+basal+body+polarization+to+planar+cell+polarity+regulation.">Ciliary proteins link basal body polarization to planar cell polarity regulation.</a> Nat Genet. 40, 69-77 (2008).</li><li>Lim, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+structure+of+the+organ+of+Corti:+a+review.">Functional structure of the organ of Corti: a review.</a> Hearing research. 22, 117-146 (1986).</li><li>Nayak, G. D., Ratnayaka, H. S., Goodyear, R. J., Richardson, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+hair+bundle+and+mechanotransduction.">Development of the hair bundle and mechanotransduction.</a> The International journal of developmental biology. 51, 597-608 (2007).</li><li>Denman-Johnson, K., Forge, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+hair+bundle+polarity+and+orientation+in+the+deveoping+vestibular+system+of+the+mouse.">Establishment of hair bundle polarity and orientation in the deveoping vestibular system of the mouse.</a> J. Neurocytol. , 821-835 (1999).</li><li>Sobkowicz, H. M., Slapnick, S. M., August, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinocilium+of+auditory+hair+cells+and+evidence+for+its+morphogenetic+role+during+the+regeneration+of+stereocilia+and+cuticular+plates.">The kinocilium of auditory hair cells and evidence for its morphogenetic role during the regeneration of stereocilia and cuticular plates.</a> Journal of neurocytology. 24, 633-653 (1995).</li><li>Denman-Johnson, K., Forge, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+hair+bundle+polarity+and+orientation+in+the+developing+vestibular+system+of+the+mouse.">Establishment of hair bundle polarity and orientation in the developing vestibular system of the mouse.</a> Journal of neurocytology. 28, 821-835 (1999).</li><li>van Dam, T. 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=The+SYSCILIA+gold+standard+(SCGSv1)+of+known+ciliary+components+and+its+applications+within+a+systems+biology+consortium.">The SYSCILIA gold standard (SCGSv1) of known ciliary components and its applications within a systems biology consortium.</a> Cilia. 2, 7 (2013).</li><li>Blacque, O. E., Sanders, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+within+a+compartment:+what+C.+elegans+can+tell+us+about+ciliary+subdomain+composition,+biogenesis,+function,+and+disease.">Compartments within a compartment: what C. elegans can tell us about ciliary subdomain composition, biogenesis, function, and disease.</a> Organogenesis. 10, 126-137 (2014).</li><li>Wallingford, J. B., Mitchell, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strange+as+it+may+seem:+the+many+links+between+Wnt+signaling,+planar+cell+polarity,+and+cilia.">Strange as it may seem: the many links between Wnt signaling, planar cell polarity, and cilia.</a> Genes &amp; development. 25, 201-213 (2011).</li><li>Borovina, A., Ciruna, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFT88+plays+a+cilia-+and+PCP-independent+role+in+controlling+oriented+cell+divisions+during+vertebrate+embryonic+development.">IFT88 plays a cilia- and PCP-independent role in controlling oriented cell divisions during vertebrate embryonic development.</a> Cell reports. 5, 37-43 (2013).</li><li>Huang, P., Schier, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dampened+Hedgehog+signaling+but+normal+Wnt+signaling+in+zebrafish+without+cilia.">Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia.</a> Development. 136, 3089-3098 (2009).</li><li>Ocbina, P. J., Tuson, M., Anderson, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cilia+are+not+required+for+normal+canonical+Wnt+signaling+in+the+mouse+embryo.">Primary cilia are not required for normal canonical Wnt signaling in the mouse embryo.</a> PloS one. 4, e6839 (2009).</li><li>Jones, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+electron+microscopy:+preparation+and+imaging+for+SEM.">Scanning electron microscopy: preparation and imaging for SEM.</a> Methods Mol Biol. 915, 1-20 (2012).</li><li>Curtin, J. A., 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=Mutation+of+Celsr1+disrupts+planar+polarity+of+inner+ear+hair+cells+and+causes+severe+neural+tube+defects+in+the+mouse.">Mutation of Celsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse.</a> Current biology : CB. 13, 1129-1133 (2003).</li><li>Wang, Y., Guo, N., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Frizzled3+and+Frizzled6+in+neural+tube+closure+and+in+the+planar+polarity+of+inner-ear+sensory+hair+cells.">The role of Frizzled3 and Frizzled6 in neural tube closure and in the planar polarity of inner-ear sensory hair cells.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 26, 2147-2156 (2006).</li><li>Montcouquiol, M., Jones, J. M., Sans, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+planar+polarity+proteins+in+mammalian+cochlea.">Detection of planar polarity proteins in mammalian cochlea.</a> Methods Mol Biol. 468, 207-219 (2008).</li><li>May-Simera, H., Kelley, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examining+planar+cell+polarity+in+the+mammalian+cochlea.">Examining planar cell polarity in the mammalian cochlea.</a> Methods Mol Biol. 839, 157-171 (2012).</li><li>Yin, H., Copley, C. O., Goodrich, L. V., Deans, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+phenotypes+between+different+vangl2+mutants+demonstrates+dominant+effects+of+the+Looptail+mutation+during+hair+cell+development.">Comparison of phenotypes between different vangl2 mutants demonstrates dominant effects of the Looptail mutation during hair cell development.</a> PloS one. 7, e31988 (2012).</li><li>Rachel, R. A., 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=Combining+Cep290+and+Mkks+ciliopathy+alleles+in+mice+rescues+sensory+defects+and+restores+ciliogenesis.">Combining Cep290 and Mkks ciliopathy alleles in mice rescues sensory defects and restores ciliogenesis.</a> J Clin Invest. 122, 1233-1245 (2012).</li><li>Jagger, 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=Alstrom+Syndrome+protein+ALMS1+localizes+to+basal+bodies+of+cochlear+hair+cells+and+regulates+cilium-dependent+planar+cell+polarity.">Alstrom Syndrome protein ALMS1 localizes to basal bodies of cochlear hair cells and regulates cilium-dependent planar cell polarity.</a> Human molecular genetics. 20, 466-481 (2011).</li><li>Collin, G. 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+Alstrom+Syndrome+Protein,+ALMS1,+Interacts+with+alpha-Actinin+and+Components+of+the+Endosome+Recycling+Pathway.">The Alstrom Syndrome Protein, ALMS1, Interacts with alpha-Actinin and Components of the Endosome Recycling Pathway.</a> PloS one. 7, e37925 (2012).</li><li>Copley, C. O., Duncan, J. S., Liu, C., Cheng, H., Deans, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+refinement+of+auditory+hair+cell+planar+polarity+deficits+occurs+in+the+absence+of+Vangl2.">Postnatal refinement of auditory hair cell planar polarity deficits occurs in the absence of Vangl2.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 33, 14001-14016 (2013).</li><li>Sorusch, N., Wunderlich, K., Bauss, K., Nagel-Wolfrum, K., Wolfrum, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usher+syndrome+protein+network+functions+in+the+retina+and+their+relation+to+other+retinal+ciliopathies.">Usher syndrome protein network functions in the retina and their relation to other retinal ciliopathies.</a> Advances in experimental medicine and biology. 801, 527-533 (2014).</li><li>Hebert, J. M., McConnell, 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=Targeting+of+cre+to+the+Foxg1+(BF-1)+locus+mediates+loxP+recombination+in+the+telencephalon+and+other+developing+head+structures.">Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures.</a> Dev Biol. 222, 296-306 (2000).</li><li>Willott, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+8,+Measurement+of+the+auditory+brainstem+response+(ABR)+to+study+auditory+sensitivity+in+mice.">Chapter 8, Measurement of the auditory brainstem response (ABR) to study auditory sensitivity in mice.</a> Current protocols in neuroscience. , Unit8 21B (2006).</li><li>Martin, G. K., Stagner, B. B., Lonsbury-Martin, B. L., 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=Chapter+8,+Assessment+of+cochlear+function+in+mice:+distortion-product+otoacoustic+emissions.">Chapter 8, Assessment of cochlear function in mice: distortion-product otoacoustic emissions.</a> Current protocols in neuroscience. , Unit8 21C (2006).</li><li>Finetti, 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=Intraflagellar+transport+is+required+for+polarized+recycling+of+the+TCR/CD3+complex+to+the+immune+synapse.">Intraflagellar transport is required for polarized recycling of the TCR/CD3 complex to the immune synapse.</a> Nature cell biology. 11, 1332-1339 (2009).</li><li>Sedmak, T., Wolfrum, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraflagellar+transport+molecules+in+ciliary+and+nonciliary+cells+of+the+retina.">Intraflagellar transport molecules in ciliary and nonciliary cells of the retina.</a> J Cell Biol. 189, 171-186 (2010).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+horizons:+ciliary+proteins+reach+beyond+cilia.">Expanding horizons: ciliary proteins reach beyond cilia.</a> Annual review of genetics. 47, 353-376 (2013).</li><li>Tarchini, B., Jolicoeur, C., Cayouette, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+blueprint+at+the+apical+surface+establishes+planar+asymmetry+in+cochlear+hair+cells.">A molecular blueprint at the apical surface establishes planar asymmetry in cochlear hair cells.</a> Developmental cell. 27, 88-102 (2013).</li><li>Ezan, 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=Primary+cilium+migration+depends+on+G-protein+signalling+control+of+subapical+cytoskeleton.">Primary cilium migration depends on G-protein signalling control of subapical cytoskeleton.</a> Nature cell biology. 15, 1107-1115 (2013).</li></ol>

When preparing cochlear tissue for analysis, there are a few key points to bear in mind. Firstly, differences in genetic background can modify the cochlear phenotype, making it necessary to analyze and compare only littermate controls. Secondly, complete removal of the tectorial membrane is required to obtain the best images with immunohistochemistry and is essential for SEM. The tectorial membrane is an opaque structure and can obscure the cells of the sensory epithelium directly beneath it, making imaging more challeng...

Primary cilia are long microtubule-based appendages that extend from the surface of most mammalian cells. Primary cilia are often confused with motile cilia, of which there are always multiple per cell, and whose purpose is to move fluid across membrane surfaces. Primary cilia, in contrast, adopt sensory roles and are consequently also referred to as sensory cilia. Once long forgotten, this organelle has recently been &#39;rediscovered&#39; as a result of its association with a multitude of human genetic diseases1. Ideally positioned as a signaling organelle, the primary cilium has been shown to regulate numerous signaling pathways, many of which are important not only in tissue homeostasis and disease, but also during development2.
One of the first signaling pathways shown to be associated with cilia dysfunction was the non-canonical Wnt signaling pathway, known also as the planar cell polarity (PCP) pathway3. This signaling cascade initially identified in Drosophila, is critical for embryogenesis; in particular for convergence and extension processes and for the correct orientation of cells in the plane of epithelia4. The sequential signaling of a core set of regulatory proteins translates directional cues which ultimately lead to cytoskeletal rearrangements and result in the coordinated polarization of epithelial cells in a plane5. The process of convergence and extension is absolutely required for the cochlear duct to elongate and for correct cellular patterning6. As this is regulated via activation of the PCP pathway, one of the most striking phenotypes of cochlea PCP mutants is a shortened cochlear duct with disorganized sensory epithelia7. Similarly, mouse mutants, which lack cilia, also exhibit such a convergence and extension phenotype8,9, though precisely how this is regulated remains to be elucidated.
Because convergence and extension processes are critical for the outgrowth of the cochlear duct, and cellular patterning of the sensory epithelia within the cochlear duct, the developing cochlea is an ideal organ in which to examine PCP signaling during vertebrate development. The organ of Corti, the term given to the specialized sensory epithelium that lines the cochlear duct, is comprised of non-sensory supporting cells and mechanosensory hair cells which must be uniformly oriented for the cochlea to function10. The mechanosensory hair cells are so called because of the stereociliary bundles that extend from the cuticular plate (apical surface) of each sensory hair cell11. These act as primary transducers of mechanosensation and despite their nomenclature as stereocilia, are actually comprised of modified actin filament-based microvilli. Within each chevron-shaped hair bundle, three rows of stereocilia are organized in a highly ordered and regular pattern in a stair case-like manner. Real microtubule-based cilia, termed kinocilia, are required for the development and orientation of the stereociliary bundles12. Upon each hair cell, one single kinocilium is physically attached to the stereocilia bundle, located centrally adjacent to the tallest row of stereocilia. The precise function of the kinocilium is unclear, and one hypothesis is that the kinocilium &#39;pulls&#39; the stereocilia into shape as they mature from microvilli12. In vertebrates, kinocilia in the cochlea are only present transiently and retract from the hair cells in mice prior to the onset of hearing11,13,14.
Complete loss of cilia in the developing cochlea results in severely shortened cochlear ducts, mis-formed and mis-oriented stereociliary bundles, as well as mis-positioned basal bodies8,9. A functional cilium is not just comprised of the ciliary axoneme. Many proteins associated with cilia function occur in complexes localized to cilia-related subdomains such as the basal body, transition zone, or ciliary axoneme15. The basal body, derived from the mother centriole of the centrosome, is also a microtubule-organizing center for microtubules extending away from the cilium into the cell body and can regulate intracellular trafficking as well as ciliary trafficking. The ciliary transition zone is another region where ciliary function is regulated in terms of organizing import and export of ciliary compounds16.
Multiple studies have identified a connection between cilia and non-canonical Wnt (PCP signaling), though the precise mechanism is unclear17. Redundancy of ciliary and PCP genes and the sensitivity of cell polarity to generalized cellular abnormalities, make it difficult to directly link a mutation to PCP-specific deficits. One of the read outs of PCP signaling is the positioning of the basal body and primary cilium, therefore segregating the primary from the secondary defects is challenging. Some studies in zebrafish and mouse mutants have suggested no connection between cilia and Wnt signaling18-20. Discrepancies in the data may reflect species, tissue, or temporal-dependent differences in ciliary contributions towards Wnt signaling. Furthermore, normal Wnt responsiveness might be retained if basal bodies remain functional. A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

<table>
	<tbody>
		<tr>
			<td>Tools/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone elastomere - Sylgard 184</td>
			<td>Sigma-Aldrich</td>
			<td>761028-5EA</td>
			<td>See Note 2</td>
		</tr>
		<tr>
			<td>Micro dissecting scissors-straight blade</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps (no. 5 and 55) and blunt forceps</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope.</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Uncoated glass microscope slides</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover slips (22 mm &#215; 40 mm &#215; 0.15 mm)</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Minutien pins</td>
			<td>&#160;Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM sample holder</td>
			<td>tousimis</td>
			<td>8762</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscopy studs</td>
			<td>TED PELLA</td>
			<td>16111</td>
			<td></td>
		</tr>
		<tr>
			<td>PELCO Tabs: Carbon adhesive</td>
			<td>TED PELLA</td>
			<td>16084-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Critical Point Dryer</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slides</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe Tissue&#160;</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Paint Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1&#215; Phosphate buffered saline (PBS)</td>
			<td>Gibco/Life Technologies</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde&#160; (PFA) (EM Grade Required for EM)</td>
			<td>Various</td>
			<td></td>
			<td>Prepare a 4% solution in 1&#215; PBS made fresh each time. EM Grade Required for EM.</td>
		</tr>
		<tr>
			<td>2.5% Glutaraldehyde Grade1</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl (pH 7.5)</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl 2</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AffiniPure Fab Fragment Donkey Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-007-003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G Mounting media</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>10&#215; Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td>Gibco/Life Technologies</td>
			<td>14065</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Gibco/Life Technologies</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide (OsO4 )</td>
			<td>Sigma-Aldrich/Fluka Analytical</td>
			<td>75632</td>
			<td></td>
		</tr>
		<tr>
			<td>Tannic acid</td>
			<td>Sigma-Aldrich</td>
			<td>403040</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti Arl13b</td>
			<td>Protein Tech</td>
			<td>17711-1-AP</td>
			<td>Suggested concentration 1:1000</td>
		</tr>
		<tr>
			<td>anti acetylated tubulin (611-B1)</td>
			<td>Sigma-Aldrich</td>
			<td>T6793</td>
			<td>Suggested concentration 1:800</td>
		</tr>
		<tr>
			<td>anti gamma tubulin (GTU-88)</td>
			<td>Sigma-Aldrich</td>
			<td>T6557</td>
			<td>Suggested concentration 1:200</td>
		</tr>
		<tr>
			<td>anti Zo_1&#160;</td>
			<td>Invitrogen</td>
			<td>40-2300</td>
			<td>Suggested concentration 1:500</td>
		</tr>
		<tr>
			<td>Myosin VI</td>
			<td>Proteus Biosciences</td>
			<td>25-6791</td>
			<td>Suggested concentration 1:1000</td>
		</tr>
		<tr>
			<td>Myosin VIIa</td>
			<td>Proteus Biosciences</td>
			<td>25-6790</td>
			<td>Suggested concentration 1:1000</td>
		</tr>
		<tr>
			<td>anti Vangl2</td>
			<td>Merk Millipore</td>
			<td>ABN373</td>
			<td>Suggested concentration 1:250</td>
		</tr>
		<tr>
			<td>anti G&#945;i3</td>
			<td>Sigma-Aldrich</td>
			<td>G4040</td>
			<td>Suggested concentration 1:250</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#174; 488 Phalloidin</td>
			<td>Invitrogen/Life Technologies</td>
			<td>A12379</td>
			<td>Suggested concentration 1:300-1000</td>
		</tr>
		<tr>
			<td>Alexa Fluor&#174; 568 Phalloidin</td>
			<td>Invitrogen/Life Technologies</td>
			<td>A12380</td>
			<td>Suggested concentration 1:300-1000</td>
		</tr>
	</tbody>
</table>

evaluation of planar-cell-polarity phenotypes in ciliopathy mouse mutant cochlea

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest signaling pathways shown to be associated with ciliary function was the non-canonical Wnt signaling pathway, also referred to as planar cell polarity (PCP) signaling. One of the best places in which to study the effects of planar cell polarity (PCP) signaling during vertebrate development is the mammalian cochlea. PCP signaling disruption in the mouse cochlea disrupts cochlear outgrowth, cellular patterning and hair cell orientation, all of which are affected by cilia dysfunction. The goal of this protocol is to describe the analysis of PCP signaling in the developing mammalian cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy. Defects in convergence and extension are manifested as a shortening of the cochlear duct and/or changes in cellular patterning, which can be quantified following dissection from developing mouse mutants. Changes in stereociliary bundle orientation and kinocilia length or positioning can be observed and quantitated using either immunofluorescence or scanning electron microscopy (SEM). A deeper insight into the role of ciliary proteins in cellular signaling pathways and other biological phenomena is crucial for our understanding of cellular and developmental biology, as well as for the development of targeted treatment strategies.

Cochlea Dissection and Tissue Preparation
After removal of the brain, post mid-line sagittal dissection of a P0 mouse head, the bony labyrinth, seen from behind, can be visualized (Figure 1A, white arrow) and removed. Figure 1B shows the isolated bony labyrinths with the cochlear hair facing upwards, ventral, (left) and from behind, dorsal (right). The white arrow points to the ...

Watch this Scientific Journal Video about Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea at JoVE.com

Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron microscopy.

In recent years, primary cilia have emerged as key regulators in development and disease by influencing numerous signaling pathways. One of the earliest ...

The overall goal of this protocol is to examine ciliary mutant mouse cochlea for planar-cell-polarity phenotypes. These techniques allow for an extensive detailing of the cochlear phenotype, which will lead to a more precise understanding of the ciliary involvement in establishing vertebrate PCP signaling. The main advantage of this protocol is that once mastered, the dissected cochlear epithelia can be used for a variety of different types of phenotypic analysis.After preparing reagents and tissue from experimental animals, at developmental stage E16.5 to p3, use a scalpel blade or a small pair of scissors to dissect the head along the sagittal midline beginning at the nose and extending cotyle. Remove the brain from each half of the skull with a pair of forceps. Then, identify the temporal bones which contain the developing bony labyrinths of the inner ear.While working with the sample under a dissection microscope, use a pair of forceps to prize the bony labyrinths away from the skull by running the forceps gently underneath the bony labyrinths. Following dissection, use the tip of the dissecting forceps to clear the oval and round windows and make a small hole in the apex of the cochlear spiral. Next, fix the bony labyrinths in 1.5 milliliters of 4%paraformaldehyde for five minutes on ice to allow for easy removal of the tectorial membrane.Now, place the bony labyrinths in a black silicone elastomer-coated dissecting dish containing PBS to facilitate dissection. Use number five forceps to remove the outer cartilage, exposing the cochlear duct. Start at the oval window, insert the bottom tip of the forceps into the oval window, and gently pry open the cartilage, slowly moving upwards towards the apex.Next, use a fine pair of forceps to pinch the Reissner's membrane at the base of the cochlear duct, and peel it off in an upward motion. Visualize the dorsal aspect of the cochlear duct, including the sensory epithelium. Then, use number 55 forceps to pinch the tectorial membrane at the base of the cochlea, and peel upwards towards the apex.Complete removal of the tectorial membrane is required to obtain the best images with immunohistochemistry, and is essential for scanning electron microscopy. After exposing the organ of Corti, further fix the tissue and perform immunohistochemistry, or prepare for scanning electron microscopy, as detailed in the written portion of the protocol. After the tissue has been stained, place the sample in a black silicone elastomer dish, containing PBS.Using fine forceps, remove the vestibular region from the cochlear spiral. Then, very carefully remove the underlying cartilage and mesenchyme from the cochlear spiral. The remaining cochlear spiral contains the inner sulcus, organ of Corti, and outer sulcus.Transfer the cochlear spiral into a drop of PBS placed on a microscope slide. Wick away the PBS with an absorbent tissue or piece of filter paper, and gently re-position the cochlear spiral if the epithelia has shifted and overlaps onto itself. Add a drop of mounting media directly onto the cochlea sample, and gently place a cover slip directly onto the sample, taking care to avoid air bubbles.Use an epifluorescent or laser scanning confocal microscope equipped with high magnification and high numerical aperture objectives, to capture images of the apical surface of the cochlear hair cell. Use lower objectives to take overlapping images of the cochlear spiral to quantify the length of the cochlear duct. The following images show immunofluorescent staining of basal turn, post-natal day one type cochlea.Phalloidin labels filamentous actin in stereocilia, and cortical actin at the cell periphery. Myosin VIIA is a marker for inner and outer hair cells. ZO_1 labels the tight junctions in between cells, making it a great marker to distinguish cell boundaries.Acetylated alpha-tubulin is used as a marker for the kinocilium at the vertex of the bundle, as indicated by the white arrow. Internal microtubules are also labeled by acetylated alpha-tubulin, which are particularly abundant in pillar cells, as indicated by the white asterisks. This image of dissected cochlear ducts at E18.5, stained with acetylated tubulin, demonstrates marked shortening of IFT20 conditional knockout cochlear ducts compared to control.The following image is a whole-mount preparation of basal cochlear turn, in a BBS8 knockout mice, at post natal day zero. Stereociliary bundles in BBS8 knockout cochleae are variably rotated, as indicated by the right arrow, or flattened and mis-localized, as indicated the middle arrow. The kinocilia are mis-localized, or axonemes are missing, as indicated by the left arrow.When preparing cochlear phenotype for analysis, it's important to remember that differences in genetic background can modify the cochlear phenotype. Therefore, it's important to use littermate controls for analysis. Further analyses include audiometric testing and culturing of cochlear explants, which allows for treatment with various signaling activators or inhibitors, thereby contributing to mechanistic understanding of developmental processes involved.After watching this video, you should have a good understanding of how to prepare cochlear tissue for analyzing the role of ciliary components and their distinct effects on PCP signaling.

evaluation-planar-cell-polarity-phenotypes-ciliopathy-mouse-mutant

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JoVE Journal

Medicine

10.3K Views.  Johannes Gutenberg-Universität Mainz. The overall goal of this protocol is to examine ciliary mutant mouse cochlea for planar-cell-polarity phenotypes. These techniques allow for an extensive detailing of the cochlear phenotype, which will lead to a more precise understanding of the ciliary involvement in establishing vertebrate PCP signaling. The main advantage of this protocol is that once mastered, the dissected cochlear epithelia can be used for a variety of different types of phenotypic analysis.After preparing reagen...

Video: Evaluation of Planar-Cell-Polarity Phenotypes in Ciliopathy Mouse Mutant Cochlea

Primary cilia influence various signaling pathways. The mammalian cochlea is ideal for examining planar cell polarity (PCP) signaling. Cilia dysfunction affects cochlear outgrowth, cellular patterning and hair cell orientation, readouts of PCP. Our goal is to analyze PCP signaling in mouse cochlea via phenotypic analysis, immunohistochemistry and scanning electron...

dextran labeling and uptake in live and functional murine cochlear hair cells

Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells

Here, we present a method for visualizing the uptake of 3 kDa Texas Red-labeled dextran in auditory hair cells with functional mechanotransduction channels. In addition, dextrans of 3&#8211;10 kDa can be used to study endocytosis in hair and supporting cells of the organ of...

morphological and functional evaluation of ribbon synapses at specific frequency regions of the mouse cochlea

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also...

whole mount dissection and immunofluorescence of the adult mouse cochlea

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the...

use of in vivo imaging to screen for morphogenesis phenotypes in candida albicans mutant strains during active infection in a mammalian host

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy.

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

measurement of strial blood flow in mouse cochlea utilizing an open vessel-window and intravital fluorescence microscopy

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological...

single-cell rna sequencing of mutant whole mouse embryos: from the epiblast to the end of gastrulation

Author Spotlight: A Pipeline to Analyze Lineage-Specific Mutant Embryos at Single-Cell Resolution

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

en face endocardial cushion preparation for planar morphogenesis analysis in mouse embryos

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve...

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

cell-type specific protein purification and identification from complex tissues using a mutant methionine trna synthetase mouse line

Cell-Type Specific Protein Purification and Identification from Complex Tissues Using a Mutant Methionine tRNA Synthetase Mouse Line

This protocol describes how to perform cell-type-specific protein labeling with azidonorleucine (ANL) using a mouse line expressing a mutant L274G-Methionine tRNA synthetase (MetRS*) and the necessary steps for labeled cell-type-specific proteins isolation. We outline two possible ANL administration routes in live mice by (1) drinking water and (2) intraperitoneal injections.

quantification of orofacial phenotypes in xenopus

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and...

Quantification of Orofacial Phenotypes in Xenopus

imaging the aging cochlea with light-sheet fluorescence microscopy

Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy

A light-sheet microscope was developed to image and digitize whole...

preparation of a micropatterned substrate to study schwann cell phenotypes

The video describes the preparation of a micropatterned substrate to study cell phenotype specification. A protein-coated micropatterned PDMS stamp is used to transfer the micropattern on a PDMS-coated coverslip. The coverslip is then treated with a surfactant to hinder cell adhesion at the unwanted surfaces. Cells are then added to settle in the pattern and incubated for further...

Preparation of a Micropatterned Substrate to Study Schwann Cell Phenotypes

transgenic rodent assay for quantifying male germ cell mutant frequency

Transgenic Rodent Assay for Quantifying Male Germ Cell Mutant Frequency

De novo mutations in the male germline may contribute to adverse health outcomes in subsequent generations. Here we describe a protocol for the use of a transgenic rodent model for quantifying mutations in male germ cells induced by environmental...

extracting the cochlea from a human temporal bone: a cadaveric protocol

Extracting the Cochlea from a Human Temporal Bone: A Cadaveric Protocol

This article presents a reliable method for extracting the human cochlea from the cadaver temporal bone by drill-out while following distinct anatomical landmarks.

an endothelial planar cell model for imaging immunological synapse dynamics

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC...

evaluation of mammary gland development and function in mouse models

Evaluation of Mammary Gland Development and Function in Mouse Models

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction...

shifting zebrafish lethal skeletal mutant penetrance by progeny testing

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

To confirm the efficacy of the micropattern, dry the surface of the PDMS stamps using a filtered airstream, and pipette 50 micrograms per milliliter BSA solution to cover the entire patterned side of the stamp. Incubate the stamps with the BSA solution for 1 hour at room temperature to allow for protein adsorption, then air-dry the stamps to remove the BSA solution. Use the filtered airstream to dry the surface of the tunable...

evaluation of the spindle assembly checkpoint integrity in mouse oocytes

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse...

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (na&iuml;ve) mice (13% decrease, p&lt;0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p&lt;0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Longitudinal Evaluation of Mouse Hind Limb Bone Loss After Spinal Cord Injury using Novel, in vivo, Methodology

A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. Dysregulated glucose metabolism is an important manifestation of the disease and is the key to its pathogenesis. Therefore, studies involving evaluation of glucose metabolism in PCOS are of utmost importance. Very few studies have quantified hepatic glucose production directly in PCOS models using non-radioactive glucose tracers. In this study, we discuss step-by-step instructions for the quantification of the rate of hepatic glucose production in a PCOS mouse model by measuring M+2 enrichment of [6,6-2H2]glucose, a stable isotopic glucose tracer, via gas chromatography - mass spectrometry (GCMS). This procedure involves creation of stable isotopic glucose tracer solution, use of tail vein catheter placement and infusion of the glucose tracer in both fasting and glucose-rich states in the same mouse in tandem. The enrichment of [6,6-2H2]glucose is measured using pentaacetate derivative in GCMS. This technique can be applied to a wide variety of studies involving direct measurement of the rate of hepatic glucose production.

This study describes the direct measurement of hepatic glucose production in a polycystic ovary syndrome mouse model by using a stable isotopic glucose tracer via tail vein in both fasting and glucose-rich states in tandem.

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. ...

Repetitive Blood Sampling from the Subclavian Vein of Conscious Rat

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to detect drug exposure. The rat blood sampling method determines the quality of the plasma and further affects the precision of the test results. The subclavian vein blood collection method described in this protocol collects blood samples repeatedly in the conscious state of animals to meet the needs of PK and TK tests. The skills of restraint handling and appropriate procedure of needle incision ensure the success rate of blood collection. It is easy to operate while ensuring the quality of plasma and at the same time catering to animal welfare. However, this method requires skilled operation, and an improper one may cause animal weakness, pain, lameness, and even mortality. The current method has been used in the testing facility for a 4-week oral toxicity study in Sprague Dawley (SD) rats with TK. The maximum amount of blood collected within 24 h did not exceed 20% of the animal's total blood. The animals' body weight was more than 200 g for males and females. The data showed that the animals' bodyweight increased steadily every week, and the clinical observation was normal after repetitive sample collection.

The present protocol describes a simple and efficient method for collecting blood from the subclavian vein in rats. It enables rapid, timely, and easily identifiable sampling without anesthesia and obtains high-quality blood through repetitive sample collection.

Rats are widely used in pharmacokinetics (PK) and toxicokinetic (TK) studies that need to collect a certain amount of blood at specific time points to ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...