Live imaging of individual cell divisions in mouse neuroepithelium shows asymmetry in cilium formation and Sonic hedgehog response
© Piotrowska-Nitsche and Caspary; licensee BioMed Central Ltd. 2012
Received: 21 November 2011
Accepted: 2 May 2012
Published: 2 May 2012
Primary cilia are microtubule-based sensory organelles that play important roles in developmental signaling pathways. Recent work demonstrated that, in cell culture, the daughter cell that inherits the older mother centriole generates a primary cilium and responds to external stimuli prior to its sister cell. This asynchrony in timing of cilia formation could be especially critical during development as cell divisions are required for both differentiation and maintenance of progenitor cell niches.
Here we integrate several fluorescent markers and use ex vivo live imaging of a single cell division within the mouse E8.5 neuroepithelium to reveal both the formation of a primary cilium and the transcriptional response to Sonic hedgehog in the daughter cells.
We show that, upon cell division, cilia formation and the Sonic hedgehog response are asynchronous between the daughter cells.
Our results demonstrate that we can directly observe single cell divisions within the developing neuroepithelium and concomitantly monitor cilium formation or Sonic hedgehog response. We expect this method to be especially powerful in examining whether cellular behavior can lead to both differentiation and maintenance of cells in a progenitor niche.
Keywordscell division ex vivo live imaging imaging neuroepithelium primary cilia Shh
Primary cilia are critical for a number of signaling pathways linked to cell proliferation and differentiation [1–3]. They are often thought of as cellular antennae because they send and receive signals [4–6]. In dividing cells, the cilium must be generated anew after each cell division. The cilium projects from the older centriole of the centrosome, so generation of the cilium is tightly linked to centriole duplication and to the cell cycle . Recent work demonstrated that, in cell culture, the daughter cell that inherits the older mother centriole generates a primary cilium and responds to external stimuli before its sister cell . This asynchrony implies that cell fate may be controlled, in part, by the timing of cilia formation.
In this study, we focus on mouse neural tube patterning, specifically on the role of primary cilia in Sonic hedgehog (Shh) signaling . Shh specifies the distinct ventral neural cell fates [9–11]. In order to examine the relative timing of cilia formation and Shh signaling response at a physiological level, we developed a system that integrates live imaging of fluorescent markers in cultured slices of embryonic mouse neuroepithelium. Here we show that this method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response.
The mouse kidney cell line, IMCD3, stably expressing somatostatin receptor 3 (SsTR3)-GFP in cilia (a kind gift from Greg Pazour) was seeded at low density on the 35 mm glass bottom dish (MatTek, Ashland, MA, USA, part No. P35GC-0-10-C) and grown in DMEM high glucose media without serum at 37°C in 5% CO2. After 7.5 hours of serum starvation, cells were cultured in media with 10% FBS at 37°C with 5% CO2 during the imaging. Cells were imaged for 15 hours in total. Images were obtained in 15-image z-stack series at 0.4 μm intervals so that 90 images were taken every 10 minutes.
Whole mouse embryo culture
Embryos at embryonic day E7.5 and E8.5 were dissected in pre-warmed wash medium containing DMEM/F12 (1:1) (GIBCO, Grand Island, NY USA) supplemented with 10% newborn calf serum (Lonza, Lawrenceville, GA USA) and 1% penicillin/streptomycin (Sigma, Saint Louis, MO USA) . Directly after dissection, E8.5 embryos still surrounded by yolk sac were placed on the 37°C heating stage under the fluorescent microscope and identified as GFP and/or dsRed positive. Up to two selected embryos were transferred into a 500-μL drop of pre-equilibrated culture media containing 50% Sprague-Dawley male rat serum (Harlan Bioproducts, Tampa, FL USA) and 50% DMEM/F12 (1:1) without phenol red supplemented with L-glutamine (GIBCO, Grand Island, NY USA) and 1% of 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid in 0.85% NaCl (BioWhittaker, Walkersville, MD USA) and penicillin/streptomycin (Sigma, Saint Louis, MO USA) . A thin layer of equilibrated light mineral oil (Sigma) was placed over the medium to prevent evaporation and the culture dish containing the embryos was transferred to the incubator set at 37°C and 5% CO2.
Live imaging and time-lapse confocal microscopy
Live cell imaging was performed using the Nikon A1R Laser Scanning Confocal Inverted Microscope (Nikon, Tokyo, Japan) equipped with a hybrid scanner that allows for traditional confocal imaging as well as high speed imaging. The excitation wavelengths were 488 and 561 nm. The imaging system was equipped with an environmental chamber that regulates temperature, set to 37°C, and 5% CO2. Nikon Perfect Focus was used to ensure absence of focus drift when imaging living cells. The 60× oil-immersion objective was used to record GFP-labeled cilia and dsRed-positive cells, while the 40× oil-immersion objective was used to monitor oligodendrocyte transcription factor 2 (Olig2)-GFP- and dsRed-positive cells. Every 10 minutes we acquired z-stacks of up to 25 μm with spacing of 1.5 μm (40× objective) and up to 8 μm with a spacing of 0.4 μm (60× objective). A separate workstation was equipped with Imaris x64 7.2.3 (Bitplane Inc, South Windsor, CT USA) three-dimensional reconstruction software to analyze recorded data.
Mice were cared for according to animal protocols approved by Emory University. Mice used were: Z/RED line (STOCK Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J, Jackson Laboratory, Bar Harbor, ME USA), CAGGCreER™ and the modified bacterial artificial chromosome (BAC) transgenic Olig2-enhanced GFP (eGFP) line (STOCK Tg(Olig2-EGFP)EK23Gsat/Mmcd, MMRRC) which were re-derived [13, 14].
The tamoxifen was dissolved in 100% ethanol. To initiate recombination, we performed intraperitoneal injections of 2.5 mg tamoxifen (Sigma) per 40 g of body weight into pregnant females at 6.5 days postcoitum. Mouse embryos were collected and analyzed 48 hours post-injection.
Neural tube slice preparation for live imaging
In order to record dividing neuroepithelial cells at the E8.5 embryonic stage, the neural tube was dissected in pre-warmed wash medium using a micro-knife (Electron Microscopy Sciences, Hatfield, PA USA), size 0.025 mm, on a 1% agar-coated dish. Next, the sample was placed ventral side down in a 150-μL drop of equilibrated culture medium without phenol red on the 35-mm poly-L-lysine coated glass bottom dish (MatTek) and covered with a thin layer of equilibrated light mineral oil (Sigma). To avoid sample movement during imaging, the isolated neural tube was mounted between small amounts of a 1:1 mixture made from 100% pure petroleum jelly and wax, and gently pressed by a narrow piece of glass coverslip in order to immobilize the neural tube.
Sstr3-GFP lentivirus was packaged and titered by the Emory Viral Core. Approximately two million virions (5 to 10 μL) of virus were added to a 500-μL equilibrated drop of culture medium containing an E8.5 embryo. After culturing at 37°C for 18 hours, the embryo was washed three times in culture media without virus prior to initiating the imaging.
Embryos were dissected, fixed and frozen, with sections prepared and stained as previously described . Antibodies and their concentrations were: rat monoclonal anti-red fluorescent protein (5F8) 1:200 (Chromotek, San Diego, CA USA); rabbit anti-Arl13b serum 1:1500 and mouse monoclonal anti-Arl13b 1:5 (295B/54, both NeuroMab, University of California, Davis, USA); rabbit anti-Olig2 1:300 (Chemicon, Temecula, CA USA); mouse monoclonals paired box gene 6 (Pax6), Shh and Nkx2.2- all 1:10; (Developmental Hybridoma Bank, Iowa City, IA USA); and rabbit polyclonal Ki67 1:500 (Abcam, Cambridge, MA USA) . Secondary antibodies Alexa Fluor 488, 568 and 350 (Molecular Probes, Eugene, OR USA) to the appropriate species were used at 1:200 concentration. Hoechst 1:3000 (Molecular Probes, Eugene, OR USA) or TO-PRO-3 1:500 (T3605, Molecular Probes, Eugene, OR USA) were used to stain nuclei. Slides were mounted in 80% glycerol and viewed within 24 hours. Images of neural tube sections were collected with a Leica DM6000B upright fluorescence microscope (Leica Microsystems, Inc., Buffalo Grove, Il USA) and processed using the SimplePCI program (Hamamatsu Corporation, Sewickley, PA USA).
We used a paired Student's t test to compare the differences between groups. A P value < 0.05 was considered statistically significant.
Results and discussion
Live imaging of cilia formation in cultured cells
Additional file 1: Cilia formation in cultured cells: IMCD3 stably expressing SsTR3-GFP in cilia. White arrows point to two dividing cells and follow daughter cell expressing SsTR3-GFP prior to the other daughter cell. (MOV 13 MB)
Post-implantation whole mouse embryo culture - ex vivo system
Live imaging of single cell divisions within the neuroepithelium
Under the low-density conditions of cell culture, it was relatively easy to follow single cell divisions; however, the close packed cells of the neuroepithelium required us to label the cells. We used genetic lineage tracing since it is well established to indelibly label an individual cell and all subsequent progeny. We took advantage of two existing mouse lines: an ubiquitous tamoxifen-inducible Cre line, CAGGCreER™and a dsRed Cre reporter line, with a loxP-βgeo-STOP-pA-loxP cassette upstream of dsRed [13, 14]. When these two lines are crossed, Cre-mediated recombination of the loxP sites excises the βgeo-STOP-pA, resulting in dsRed expression in all cell progeny. By injecting pregnant female mice with tamoxifen when the embryos were at E6.5, we induced Cre expression and dsRed labeling in a small subset of cells. This density of labeling enabled us to observe single cell divisions 48 hours later during ex vivo imaging.
Additional file 2: Individual cell expressing dsRed undergoes division within the neuroepithelium. White arrow indicates dividing dsRed cell. (MOV 1 MB)
Live imaging of cilia formation and Shh response
Additional file 3: Neural tube infected with fluorescent cilia marker, SsTR3-GFP. GFP expressed in cilia is pointed by the white arrow. (MOV 1 MB)
Additional file 4: Neural tube of the Olig2-GFP embryo expresses Shh in individual cells. Two dividing cells that express Olig2-GFP are followed by the white arrow. (MOV 3 MB)
Asynchrony in cilia formation in dividing cells of the developing neural tube
Additional file 5: Asynchrony in cilia formation in dividing dsRed cell infected with the SsTR-GFP lentivirus within the neuroepithelium. GFP is expressed in cilia in one daughter cell following division and is pointed by the white arrow. (MOV 2 MB)
In order to be sure that the Sstr3-GFP expression we were using to visualize cilia was not interfering with any underlying biological process, we confirmed these results in fixed samples that we stained with antibodies against dsRed and the ciliary protein, Arl13b . After imaging 178 pairs of daughter cells in which at least one cell showed a cilium, we observed a single ciliated daughter cell in 88% of the pairs and dual ciliated daughter cells in 12% of the pairs (Figures 6F, G, H and 7A). Taken together, these data indicate that cilia formation between pairs of daughter cells is asynchronous in the developing mouse neural tube.
Asynchrony in Shh signaling in daughter cells of the developing neural tube
Additional file 6: Asynchrony in Shh signaling in daughter cells of the dsRed and Olig2BAC-GFP positive embryo within the neuroepithelium. Olig2-eGFP is expressed in one daughter cell following division and is pointed by the white arrow. (MOV 4 MB)
Our results demonstrate that we can directly observe single cell divisions within the developing neuroepithelium and concomitantly monitor cilium formation or Shh response. We showed that when a cell divides in the developing mouse neural tube, both cilia formation and the Shh response are asynchronous between the resulting daughter cells. Thus, data generated using our ex vivo system are consistent with previous results from in vitro cultured cells . This suggests that the experiments using cultured cells provide physiologically relevant data.
While we were unable to image cilia formation and Shh response in the exact same cell using our ex vivo method, three lines of evidence suggest that the daughter cell in which the cilium first forms is also the cell that first responds to Shh. First, the live imaging data show that, in the majority of cases, after a cell division only one cell has a cilium (92%) and only one cell displays a Shh-response (83%), making it likely that they are the same cell in most pairs (two-tailed P = 0.1695; degrees of freedom = 1). Second, cells without cilia cannot transduce Shh signaling [1, 22, 23]. Finally, our numbers are quite consistent with previous experiments in immortalized cell lines where the authors observed cilia formation and Shh signal transduction in the same cell . Their study also demonstrated that the daughter cell that inherits the older mother centriole will be first to form a cilium and transduce a Shh response. Although our studies did not directly address this question, the consistency between their in vitro and our in vivo data would be consistent with the older centriole being inherited by the daughter cell that first forms a cilium and responds to Shh within the neuroepithelium as well. While formal proof will require further work, our method provides tools with which the field can more immediately monitor cell behavior. Coupled with the rich resources of mouse mutants, we expect this method to be especially powerful in examining whether cellular behavior can lead to both differentiation and maintenance of cells in a progenitor niche.
bacterial artificial chromosome
(Dulbecco's) modified Eagle's medium
fetal bovine serum
green fluorescent protein
oligodendrocyte transcription factor 2
paired box gene 6
somatostatin 3 receptor fused to GFP.
This research project was supported by an ARRA Supplement, 5 R01 NS056380. Additional support was provided through the Viral Vector Core and the Microscopy Core of the Emory Neuroscience NINDS Core Facilities grant, P30NS055077. We thank the Emory Transgenic Mouse and Gene Targeting Core for deriving the mouse line from GENSAT; Greg Pazour for the stable SsTR3-GFP IMCD3 cell line; and Bradley Yoder for the SsTR3-GFP lentiviral construct. Monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD, and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. We thank all the members of the Caspary laboratory for helpful suggestions on the manuscript. All animal procedures were approved by the IACUC and the Biosafety Committee at Emory University.
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