- Methodology article
- Open Access
A bright single-cell resolution live imaging reporter of Notch signaling in the mouse
© Nowotschin et al.; licensee BioMed Central Ltd. 2013
- Received: 9 August 2012
- Accepted: 11 April 2013
- Published: 25 April 2013
Live imaging provides an essential methodology for understanding complex and dynamic cell behaviors and their underlying molecular mechanisms. Genetically-encoded reporter expressing mouse strains are an important tool for use in live imaging experiments. Such reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of desired reporter genes. If these cis-regulatory elements are downstream targets, and thus activated as a consequence of signaling pathway activation, such reporters can provide read-outs of the signaling status of a cell. The Notch signaling pathway is an evolutionary conserved pathway operating in multiple developmental processes as well as being the basis for several congenital diseases. The transcription factor CBF1 is a central evolutionarily conserved component of the Notch signaling pathway. It binds the active form of the Notch receptor (NICD) and subsequently binds to cis-regulatory regions (CBF1 binding sites) in the promoters of Notch responsive genes. In this way, CBF1 binding sites represent a good target for the design of a Notch signaling reporter.
To generate a single-cell resolution Notch signaling reporter, we used a CBF responsive element to direct the expression of a nuclear-localized fluorescent protein. To do this, we linked 4 copies of a consensus CBF1 binding site to the basal simian virus 40 (SV40) promoter, placed this cassette in front of a fluorescent protein fusion comprising human histone H2B linked to the yellow fluorescent protein (YFP) Venus, one of the brightest available YFPs. We used the CBF:H2B-Venus construct to generate both transgenic embryonic mouse stem (ES) cell lines and a strain of transgenic mice that would report Notch signaling activity.
By using multiple CBF1 binding sites together with a subcellular-localized, genetically-encoded fluorescent protein, H2B-Venus, we have generated a transgenic strain of mice that faithfully recapitulates Notch signaling at single-cell resolution. This is the first mouse reporter strain in which individual cells transducing a Notch signal can be visualized. The improved resolution of this reporter makes it ideal for live imaging developmental processes regulated by the Notch signaling pathway as well as a short-term lineage tracer of Notch expressing cells due to the perdurance of the fluorescent reporter. Taken together, the CBF:H2B-Venus mouse strain is a unique tool to study and understand the morphogenetic events regulated by the Notch signaling pathway.
- Mouse embryo
- Live imaging
- Cell tracking
- Reporter strain
- Notch signaling
The Notch signaling pathway is one of the key signaling pathways employed during embryonic development, homeostasis and disease progression (reviewed in [1, 2]). Though first discovered in Drosophila, the Notch pathway is evolutionary conserved across many metazoan species playing an important role in various processes including cell fate specification, differentiation, tissue patterning and homeostasis, as well as stem cell self-renewal (reviewed in ).
The core components of the Notch signaling pathway include the Notch transmembrane receptors, Notch1-4 in mammals, and their five canonical membrane-bound ligands of the Delta or Serrate/Jagged family, Jagged1-2, and Delta1, Delta3 and Delta4 in mammals. Notch proteins are single-pass transmembrane proteins consisting of an extracellular domain (NECD), encompassing 29–36 tandem epidermal growth factor like repeats, a shorter membrane-spanning region and an intracellular domain (NICD) containing a transactivation domain (reviewed in ).
Cell-cell contact and the interaction of the NECD with one of the ligands expressed by a neighboring cell leads to a cascade of proteolytic events involving an S2 cleavage of the Notch receptor, and a cleavage at the S3 site by γ-secretase. This series of proteolytic cleavages results in the release of the NICD from the membrane. Thus, upon signaling the NICD translocates to the nucleus where it binds the transcription factor CBF1 (also referred to as RBPJκ, CSL or Su(H)). The binding of NICD to CBF1 replaces co-factors of the Groucho family that function to repress target genes and in doing so allows the recruitment of the co-activator mastermind-like (MAML) promoting the formation of a CBF1-NICD-MAML complex that leads to transcriptional activation of target genes including members of the Hes and Hey families of bHLH transcription factors .
Transgenic reporters providing in vivo read-outs of Notch signaling activity have been useful for investigating the complex functions of Notch signaling across species. To date, several transgenic strains of mice have been established to monitor Notch signaling activity, each employing slightly different construct designs, but all based on the use of Notch-responsive elements driving the expression of a readily detectable genetically-encoded reporter. Of these, a transgenic mouse line, named NAS (for Notch Activity Sensor), used the TP1 minimal promoter (comprising 12 multimerized CBF binding sites), which is transactivated in a CBF/RBPJκ-dependent manner by the NICD and in doing so functions as a read-out of Notch activity. The NAS reporter comprised a modified version of the lacZ gene coding for nuclear ß-galactosidase under the control of TP1. Faithful expression regulated by Notch signaling was demonstrated by loss of reporter activity in CBF mutants . Another transgenic mouse line, CBF:EGFP, used EGFP as a reporter gene driven by the basal simian virus (SV40) promoter and a CBF1-responsive element containing four CBF1 binding sites. The CBF:EGFP strain has been used to investigate Notch signaling in a variety of contexts, including neural stem cells and intermediate neural progenitors during mouse neural development. Faithful responsiveness of the CBF:EGFP to Notch signaling activity was demonstrated using an shRNA mediated knock-down of CBF1 and the γ–secretase antagonist F18 .
A recent report developed a construct based on the promoter of the Notch target gene Hes5-1, coupled with a destabilized nuclear-localized Venus fluorescent protein and the 3’ untranslated region of Hes5-1. Using transient transgenesis by electroporation, this Notch signaling reporter was used in chick embryos to investigate endogenous Notch activity during neurogenesis. Highlighting the importance of single-cell resolution reporters to understand cell fate and lineage decisions, this reporter revealed distinct Notch signaling dynamics in different cell division modes in neural progenitors within the neuroepithelium .
Another report presented an alternative strategy to the use of transcriptional reporters as read-outs of Notch signaling activity that could also be used for monitoring Notch activation in real-time in living cells. In this newly described reporter Notch activity was recorded in a cell culture system using a luciferase complementation-based reporter that could directly monitor interactions between a specific NICD and CBF/RBPJκ, thereby allowing the non-invasive detection of protein-protein interactions in any subcellular compartment .
Since the available Notch reporter mouse strains either lack sensitivity or do not provide single-cell resolution read-outs, we sought to generate an improved reporter that would provide single-cell resolution reporter activity, as a key in understanding cell dynamics and behaviors, and greater sensitivity by incorporating a nuclear-localized bright fluorescent reporter, which could be readily detected, live imaged and quantified. To do so, we designed a construct that combined the signaling read-out efficiency of the multimerized CBF1 responsive elements  with an improved nuclear-localized fusion to a bright fluorescent protein. Fluorescent proteins fused to histones are bound to chromatin even during cell division, therefore allowing the tracking of single cells and their progeny, as well as providing information on cell division and cell death [7, 8]. We generated an H2B-Venus fusion protein, comprising human histone H2B fused to the bright yellow fluorescent protein Venus, and placed it under the control of four CBF1 responsive elements and the simian virus 40 (SV40) minimal promoter in a configuration resembling the previously reported CBF:EGFP Notch signaling reporter . We used this construct to generate CBF:H2B-Venus transgenic mouse ES cells and an equivalent strain of mice. In this way the CBF:H2B-Venus transgene was designed to function as a sensitive single-cell resolution reporter of Notch signaling, and would be comparable to the TCF/Lef:H2B-GFP single-cell resolution reporter we previously generated as a read-out of WNT/β-catenin signaling .
We recovered several founder transgenic lines, which recapitulated the spatio-temporal localization of Notch signaling activity during mouse embryonic development, as well as in adults showing among others expression in the headfolds, neural tissue of the early embryo, the vasculature, the kidney, and brain. One was analyzed in detail and is presented here. Overall, expression of CBF:H2B-Venus recapitulated the previously reported CBF:EGFP localization including the expression in the ventricular zone (VZ) and sub-ventricular zone (SVZ) regions of the cortex of the brain . We confirmed Notch signaling specificity of the reporter, both in cells and in embryos, through Notch signaling stimulation achieved by misexpression of NICD leading to reporter activation.
The CBF:H2B-Venus reporter strain produced bright single-cell resolution reporter activity. Notch responsiveness of the CBF:H2B-Venus reporter was demonstrated through reporter activation by transfection of an NICD expression construct in mouse embryo fibroblast (MEF) cells derived from transgenic embryos, and electroporation into the visceral endoderm of early post-implantation embryos. Due to its improved sensitivity, the CBF:H2B-Venus reporter revealed additional sites of Notch signaling activity, that have previously not been described, including expression in the epiblast (EPI) of the peri-implantation mouse embryo. Collectively, these data demonstrate that we have generated a transgenic mouse strain that faithfully reports Notch signaling activity and serves as a quantitative, non-invasive single-cell resolution reporter of Notch signaling. The CBF:H2B-Venus reporter currently represents an improved tool for imaging the in vivo processes triggered by canonical Notch signaling.
Generation of CBF:H2B-Venusreporter construct
Reporter activation upon Notch signaling stimulation
To test for Notch responsiveness of the CBF:H2B-Venus construct, and in doing so validate the reporter as a bona fide Notch signaling reporter, we derived MEF cells from E13.5 hemizygous transgenic embryos and transfected them with an NICD expression construct (pCX:NICD)  to activate reporter expression. Subsequent FACS analysis revealed an increase in fluorescence upon transfection of pCX:NICD into CBF:H2B-Venus Tg/+ MEF cells demonstrating the Notch responsiveness of the reporter construct (Figure 1).
Generation of CBF:H2B-Venustransgenic ES cells
Since it had previously been reported that Notch signaling is involved in regulating the timing of the emergence of the different germ layers in ES cell-derived embryoid bodies (EBs), we used EB formation to test the differentiation potential of the CBF:H2B-Venus transgenic ES cells and determine whether reporter localization might correlate with the timing of germ layer formation (Figure 3B-E) . Our results revealed that Notch signaling could initially be detected in EBs cultured until Day 4 (Figure 3B-D). By day 6 however, fluorescence was downregulated in concordance with progressive differentiation. At this stage tube-like or cystic structures could be detected within EBs where Notch signaling had been downregulated (Figure 3E) .
Collectively, these data indicate that the CBF:H2B-Venus transgene is a sensitive single-cell resolution Notch signaling reporter that reveals Notch signaling activity in mouse ES cell cultures.
The CBF:H2B-Venusreporter reveals novel sites of Notch signaling within the early post-implantation epiblast
The robust expression of the CBF:H2B-Venus transgenic ES cells prompted us to investigate whether the reporter is also active in the pluripotent epiblast lineage of the pre-implantation embryo in CBF:H2B-Venus transgenic embryos. We failed to detect any fluorescent signal within the inner cell mass of E3.5-E4.5 transgenic blastocysts (data not shown), suggesting that either the CBF:H2B-Venus reporter was not sufficiently sensitive for direct visualization at these stages, or that Notch signaling is not active in the epiblast lineage at pre-implantation stages.
To further characterize the localization of the CBF:H2B-Venus reporter and thus the activation of Notch signaling, we analyzed its distribution in early post-implantation stages (Figure 3F-I). After implantation into the maternal uterus around embryonic day (E) 4.5, the three blastocyst cell lineages (epiblast, primitive endoderm and trophoblast) are expanded and the axes of the embryo (proximal-distal/P-D and anterior-posterior/A-P) are specified at early post-implantation (~E5.5-E6.0). The visceral endoderm (VE) encapsulates the pluripotent epiblast distally, and extra-embryonic ectoderm proximally, and close apposition of these adjacent tissues facilitates cross talk between them, resulting in the formation of the distal visceral endoderm (DVE). Subsequently, translocation of the DVE population to generate the anterior visceral endoderm (AVE) leads to the establishment of the A-P axis (reviewed in ).
At the earliest post-implantation stages that we could recover (~E5.0-5.25), reporter expression was detected in the epiblast of the CBF:H2B-Venus embryos (Figure 3F, F1, F2, G, G2, H, H2, I and I2). Expression in the epiblast persisted until the onset of gastrulation (Figure 3F and data not shown). In addition, for a very brief window of time, at around ~ E6.0, reporter expression was detected in the extra-embryonic ectoderm (Figure 3F). This extra-embryonic expression was downregulated by ~ E6.5 (data not shown). Of note, reporter expression in the EPI at early post-implantation stages looks like a salt-and-pepper distribution (Figure 3F1), begging the question whether this distribution represents different cell populations or whether these could be fluctuations in Notch signaling. Future studies will address this question. Epiblast-specific location was confirmed by co-localization with the epiblast marker, Oct4 (Figure 3H). Localization of the pan-VE marker Gata6 (Figure 3G), and pan-emVE marker Eomes in CBF:H2B-Venus embryos revealed that the reporter expression was excluded from the VE (Figure 3G, I). The epiblast-specific site of Notch reporter expression has not been previously reported and may presage the future requirement for Notch signaling and subsequently reporter expression in neural tissue.
The CBF:H2B-Venusreporter marks the neural plate and cardiogenic region at gastrulation
During gastrulation, which in the mouse takes place between E6.5 and E8.0, three germ layers, ectoderm, mesoderm and definitive endoderm, are formed from the pluripotent epiblast lineage. Mesoderm and definitive endoderm arise when epiblast cells undergo an epithelial-to-mesenchymal transition (EMT), and in doing so lose their epithelial character, ingress through the primitive streak (PS), which forms at the posterior end of the embryo, and migrate away as the so-called wings of mesoderm [15, 16].
In addition, robust reporter expression was detected in the blood islands (Figure 4F), a cell population of maturing erythrocytes surrounded by developing endothelial cells that will give rise to the mesoderm layer of the yolk sac , another site of persistent specific and strong Notch reporter expression (Figure 4H white arrowhead).
Surprisingly, we failed to observe expression of the CBF:H2B-Venus reporter in the presomitic mesoderm (PSM), a tissue of known and dynamic Notch activity . However, several previously characterized reporters have also failed to detect Notch activity at this site of Notch activity.
CBF:H2B-Venusreporter expression at mid-gestation and to later fetal stages
We determined that this observed site of reporter expression was in endothelial cells of the vasculature, by analyzing the localization of PECAM (CD31) a marker of endothelial cells in CBF:H2B-Venus embryos and noting the co-localization of the PECAM antibody and Venus (Figure 6K-O). Note, the nuclear expression of the reporter and membrane expression of PECAM in the same cells (Figure 6O1).
Localization of CBF:H2B-Venus expression at later fetal stages (E14.5, Figure 5) revealed reporter expression in a variety of tissues. In the lens, H2B-Venus could be detected in cells of the cuboidal epithelium that comprises the anterior lens (red arrowhead), as well as in cells in the equatorial region (red arrow), the region where anterior capsular epithelial cells become integrated into the lens-proper and form the lens fibers. In the retina of the eye, strong expression could be found in the retinal pigmented epithelium, RPE (Figure 5B and B1). Reporter expression was also detected in mesenchymal cells of the lung (Figure 5C and C1), in cells that will form the valves of the heart (Figure 5D and D1), in cells of the gut (Figure 5E and E1) and liver (Figure 5F and F1), in the vasculature of the digits (Figure 5G and G1), as well as in cells of thymus (Figure 5H and H1), and in cells surrounding the nasal process likely being vascular cells (Figure 5I and I1).
CBF:H2B-Venusreporter expression in post-natal tissues
CBF:H2B-Venus reporterexpression in the kidney
To validate the CBF:H2B-Venus reporter as an improved tool for visually dissecting morphogenetic processes, we chose to look at reporter expression in the kidney in detail. The importance of the evolutionary conserved Notch signaling pathway is not just illustrated by the fact that it is indispensable for kidney development across phyla , but it is also involved in the pathophysiology of congenital diseases, such as the Alagille syndrome [24, 25], glomerular diseases, in tissue repair after acute renal injuries , as well as in the prevention of renal epithelial cancers .
CBF:H2B-Venusreporter expression in the brain
We have generated a novel single-cell resolution transgenic strain of mice that serves as a transcriptional read-out of Notch signaling. The reporter comprises multimerized CBF1 DNA binding sites that drive the expression of a subcellular-localized, genetically-encoded fluorescent fusion protein, H2B-Venus. We have characterized the expression of the CBF:H2B-Venus reporter in detail in mouse ES cells, post-implantation embryos and adult tissues. Specificity of this putative Notch signaling reporter was demonstrated by its activation upon transfection with an NICD expression construct in transgenic MEF cells grown in culture and in vivo in embryos, as well as by documenting regions of reporter expression that correspond to those previously reported as sites of Notch signaling activity. Collectively, these data suggest that CBF:H2B-Venus reporter expression acts as a faithful read-out of Notch signaling.
In addition to previously reported sites of expression, the sensitivity of the reporter identified new sites of Notch signaling in embryos, in particular within the epiblast of the early post-implantation embryo. These novel sites of expression are supported by previous observations of Notch signaling function made in mouse embryonic stem (ES) cells.
Even though the reporter described in this report provides a read-out of the transcription resulting from pathway activation, and is not a read-out of the localization of one of the core components of the Notch signaling pathway, we cannot exclude some perdurance of the fluorescent protein. As such, the complete domain of reporter expression, that we have characterized, may encompass cells actively signaling as well as those that have recently been actively signaling but currently not signaling. Future efforts will be aimed at generating more sensitive reporters and will include the development of dual reporters that combine short-term expression, using a destabilized fluorescent protein with a more perdurant fluorescent protein as has recently been described in zebrafish .
Even so, the CBF:H2B-Venus reporter is, at present, the most sensitive reporter of Notch signaling, and due to the use of the human histone H2B fusion, it allows the live visualization of individual cells in cohorts. It is presently the only reporter facilitating the analysis of cellular dynamics, including tracking of single cells in Notch responsive populations. Live imaging has become an essential part of understanding developmental processes; therefore this reporter should be a valuable tool to understand cellular events in development and disease regulated by canonical Notch signaling.
Generation of Notch reporter constructs and transgenic animals
To generate the CBF:H2B-Venus construct, a CBF responsive element (CBFRE) including 4 binding sites for CBF1 together with basal simian virus 40 (SV40) promoter was amplified from the CBFRE:EGFP plasmid (purchased from Addgene, plasmid #17705), which has been described previously . The amplified CBF responsive element was then subcloned into the AseI/NheI sites of pCMV:H2B-Venus. Recombinant clones were confirmed with restriction digest analysis as well as sequencing. Transgenic mice were generated by pronuclear injection following standard protocols. Animals were genotyped by PCR using either of two alternative strategies. Primers used for the PCR amplification of the CBF responsive element (CBFRE, 350bp) were CBF AseI Fw: GCTGATTAATCGAGATCTGGTGTAAACAC and SV40 NheI Rev: GCGAGCTAGCCAGCTTTTTGCAAAAGCCTAG, or alternatively, a 475bp fragment from the Venus cassette was amplified using primers IMR872 Fw: AAGTTCATCTGCACCACCG and IMR873 Rev: TGCTCAGGTAGTGGTTGTCG. Several transgenic founder lines exhibiting comparable expression were identified. One transgenic founder line (CBF:H2B-Venus #5) was analyzed in detail and presented in this report. Subsequent generations exhibited Mendelian transgene inheritance, stable transgene activity and comparable levels of reporter expression. The transgene could be homozygozed without incurring any phenotype. Animals were maintained in accordance with National Institute of Health (NIH) guidelines for the care and use of laboratory animals and under the approval of the Memorial Sloan Kettering Cancer Center (MSKCC) Institutional Animal Care and Use Committee (IACUC).
Generation of transgenic ES cells
The CBF:H2B-Venus construct was tested in R1 ES cells . R1 ES cells were cultured in standard conditions . Transgenic R1 ES cells were generated by nucleofecting a FspI linearized CBF:H2B-Venus construct followed by selection of clones under Neomycin selection according to standard protocols.
Transfection of MEFs
MEFs were derived from E13.5 transgenic embryos and passaged 3 times in Dulbecco's Modified Eagle Medium (DMEM; Gibco) containing 15% serum (Gibco) and 50 mg/mL each of penicillin/streptomycin (Gibco). They were subsequently seeded onto a 12-well plate (BD Falcon) and transfected with 2 μg pCX:NICD (purchased from Addgene, plasmid #26891) using 10 μL Lipofectamine LTX (Invitrogen) and 2 μL Plus Reagent according to the manufacturer's protocol. Cells were then cultured for 36 h and analyzed using flow cytometry.
Generation of embryoid bodies (EBs)
ES cells were passaged off MEFs, as previously described , then plated as hanging drops at a concentration of 15,000 cells/mL (300 cells per hanging drop) in Iscove's Modified Dulbecco Medium (IMDM; Gibco) containing 20% serum (Gibco), 50 mg/mL each of penicillin/streptomycin (Gibco), 200 mg/mL apo-transferrin (Sigma), 5% protein-free hybridoma medium (PFHMII; Gibco), 0.5 mM 1-thioglycerol (Sigma), and 0.5 mM ascorbic acid (Sigma). Ebs were harvested after 1, 2, 4 and 6 days of culture and imaged.
MEF cells were harvested 36hrs after transfection by trypsinization with 0.025% trypsin/EDTA (Invitrogen) and analyzed by flow cytometry using a FACS Fortessa flow cytometer (BD Biosciences). The live single cell population was selected for further analysis on the basis of Forward Scatter (FSC) and Side Scatter (SSC) characteristics as well as with Propidium Iodide (PI) staining to evaluate cell viability. Histogram plots of the data were generated using FlowJo (Tree Star, Inc).
Embryo collection and culture
Early post-implantation mouse embryos were dissected in DMEM/F12 (1:1) containing 5% newborn calf serum and fixed in 4% paraformaldehyde. E13.5, E 16.5, P1 were dissected in PBS and fixed in 4% paraformaldehyde.
Electroporation and culture of postimplantation embryos
E6.5 embryos were dissected and kept in culture medium (50% DMEM/F12, 50% rat serum). Then embryos were transferred into a drop of PBS. Highly concentrated plasmid DNA of pCX:NICD (purchased from Addgene, plasmid #26891) and/or pCX:mCherry was added and incubated for 5min. Subsequently, embryos were transferred into Tyrode’s ringer saline for electroporation. Embryos were pulsed using a ECM830 electroporator (BTX, Harvard Instruments). Settings were as follows: Voltage: 0017V, Length: 050ms; Pulse: 5, Interval 1s). Embryos were then cultured for 8hrs or over night in roller culture and then imaged. pCX:mCherry was generated by PCR of mCherry from pRSET:mCherry using the 5’ oligo CCGGAATTCCGGATGGTGAGCAAGGGCGAGGAGGATAAC and the 3’oligo CCGGAATTCCGGTTACTTGTACAGCTCGTCCATGCCGCCGGT, both containing EcoRI restriction sites. The resulting PCR product cloned into pCAGGS  using the EcoRI restriction enzyme.
For cryosections, samples were equilibrated in 10% sucrose in PBS, followed by 30% sucrose in PBS overnight and OCT (Tissue-Tek) overnight, and then snap-frozen in OCT. Sections were cut with a Leica cryostat at 12 μm and counterstained with Hoechst 33342 (Invitrogen) to label nuclei. Sections were mounted in Fluoromount-G (Southern Biotech) and imaged through glass coverslips.
ES cells cultured on coverslips were immunostained as previously described . Primary antibodies used were: NANOG (1:200, Cosmo Bio) and OCT4 (1:200, Santa Cruz). Embryos were permeabilized in 0.5% Triton X-100 in PBS for 20 minutes, washed in 0.1% Triton X-100 in PBS (PBT) and blocked in horse serum in PBT for 1 hr at 4C. Primary antibodies used were: anti-Eomes (1:500, Abcam), anti-Oct4 (1:200, Santa Cruz) anti-Gata6 (1:100, RD Systems) and anti-PECAM (CD31) (1:250, BD Pharmingen). Secondary Alexa Fluor conjugated antibodies were used at a dilution of 1/1000. Nuclei were visualized using Hoechst (Invitrogen). Phalloidin was used to label F-Actin.
Widefield and epifluorescence images were acquired using a Zeiss Axiocam MRc CCD camera coupled to a Leica M165FC dissecting scope. Laser scanning confocal data were acquired using a Zeiss LSM510META or a Zeiss LSM700 using a Fluar 5×/NA0.25, a PlanApo 10×/NA 0.45 and PlanApo 20×/NA0.75 objective. Fluorophores were excited using a 405 nm diode laser (Hoechst), 488 nm Argon laser (GFP) or 543 nm HeNe laser (Alexa 546) on the LSM 510 META, and 405 nm (Hoechst), 488 nm (GFP), 555 nm (Alexa 546) solid-state lasers on the LSM 700.
Embryos and ES cells were imaged in whole mount in MatTek dishes (Ashland). Confocal images were acquired as z-stacks of xy images taken at 2 μm z-intervals (20×) 10 μm z-intervals (10×) and 40 μm z–intervals (5×). Raw data were processed using the Zen 2010 software (Zeiss) and assembled in Adobe Photoshop CS4 (Adobe).
The CBF:H2B-Venus transgenic strain described in this report has been accepted for inclusion in, and will be made available from, The Jackson Laboratory Mouse Repository as JAX Stock No. 020942 STOCK Tg(Cp-HIST1H2BB/Venus)#Hadj/J.
This study was made possible through use of the MSKCC Mouse Genetics and Flow Cytometry Core facilities. We thank Evan Weiner for technical assistance; Anna Ferrer-Vaquer and Aitana Perea-Gomez for discussions and comments on the manuscript; Celia Andreu-Agulló for expert advice on neuroanatomy and neuronal expression of the reporter, and Cristina Cebrian for advice on renal expression of the reporter. Work in our lab is supported by the HFSP and NIH (RO1-HD052115 and RO1-DK084391). SN is the recipient of a Muscular Dystrophy Association (MDA) development grant (186552).
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