A sensitive and bright single-cell resolution live imaging reporter of Wnt/ß-catenin signaling in the mouse
© Ferrer-Vaquer et al; licensee BioMed Central Ltd. 2010
Received: 21 September 2010
Accepted: 21 December 2010
Published: 21 December 2010
Understanding the dynamic cellular behaviors and underlying molecular mechanisms that drive morphogenesis is an ongoing challenge in biology. Live imaging provides the necessary methodology to unravel the synergistic and stereotypical cell and molecular events that shape the embryo. Genetically-encoded reporters represent an essential tool for live imaging. Reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of a desired reporter gene. In the case of canonical Wnt signaling, also referred to as Wnt/β-catenin signaling, since the downstream transcriptional response is well understood, reporters can be designed that reflect sites of active Wnt signaling, as opposed to sites of gene transcription, as is the case with many fluorescent reporters. However, even though several transgenic Wnt/β-catenin reporter strains have been generated, to date, none provides the single-cell resolution favored for live imaging studies.
We have placed six copies of a TCF/Lef responsive element and an hsp68 minimal promoter in front of a fluorescent protein fusion comprising human histone H2B to GFP and used it to generate a strain of mice that would report Wnt/β-catenin signaling activity. Characterization of developmental and adult stages of the resulting TCF/Lef:H2B-GFP strain revealed discrete and specific expression of the transgene at previously characterized sites of Wnt/β-catenin signaling. In support of the increased sensitivity of the TCF/Lef:H2B-GFP reporter, additional sites of Wnt/β-catenin signaling not documented with other reporters but identified through genetic and embryological analysis were observed. Furthermore, the sub-cellular localization of the reporter minimized reporter perdurance, and allowed visualization and tracking of individual cells within a cohort, so facilitating the detailed analysis of cell behaviors and signaling activity during morphogenesis.
By combining the Wnt activity read-out efficiency of multimerized TCF/Lef DNA binding sites, together with the high-resolution imaging afforded by subcellularly-localized fluorescent fusion proteins such as H2B-GFP, we have created a mouse transgenic line that faithfully recapitulates Wnt signaling activity at single-cell resolution. The TCF/Lef:H2B-GFP reporter represents a unique tool for live imaging the in vivo processes triggered by Wnt/β-catenin signaling, and thus should help the formulation of a high-resolution understanding of the serial events that define the morphogenetic process regulated by this signaling pathway.
Wnt signaling is a key, evolutionarily conserved, cellular signal transduction pathway required and reiteratively used for diverse biological functions. Precise regulation of pathway activity is required for proper embryonic development, and in adulthood, for tissue homeostasis. By contrast, impaired Wnt signaling activity can lead to embryonic defects and disease progression. Wnt proteins encompass a large family of secreted glycoproteins that trigger their outcome through different downstream cascades, among them the canonical Wnt/ß-catenin pathway, which activates transcription of target genes by the stabilization and nuclear localization of ß-catenin, a transcriptional co-activator protein.
In the absence of ligand, cytoplasmatic ß-catenin is phosphorylated and targeted for degradation by a protein complex consisting of the scaffolding proteins Axin, APC and the kinase GSK3ß. Once phosphorylated, ß-catenin is recognized by the ubiquitin ligase Trcp, which targets it for proteasomal degradation. Upon binding of the Wnt ligand to the receptor complex formed by Frizzled (Fz) and LRP5/6, Dishevelled (Dvl) is recruited by Fz leading to LRP5/6 phosphorylation and Axin recruitment. Loss of Axin from the degradation complex dismantles the complex and releases ß-catenin. Once stabilized, ß-catenin translocates to the nucleus. As a transcriptional coactivator, ß-catenin together with the T cell-specific transcription factor/lymphoid enhancer-binding factor 1 (TCF/Lef) family of transcription factors induces the transcription of downstream genes (reviewed in [1–3]).
Over the past fifteen years several transgenic mouse strains have been established to monitor Wnt/ß-catenin pathway activity during development, homeostasis and disease progression (reviewed in ). These reporter constructs are generally derived from the TOPFLASH design . They consist of a series of multimerized DNA binding sites for TCF/Lef (TCF/Lefn), which together with a minimal promoter (promoter min) , drive expression of a reporter gene. Thus, in principle, such TCF/Lef n -promoter min :reporter constructs label cells that are actively transducing a Wnt signal.
As a validation of their utility, several variant Wnt/ß-catenin reporter mouse strains have been characterized, are readily available and have been used to determine Wnt signaling status in a broad spectrum of applications. First generation constructs usually comprised a LacZ reporter (e.g. ), whereas later generation versions provided a quantifiable readout and promoted live imaging applications by incorporating fluorescent protein reporters such as GFP [7, 8]. Even so, because of their robust expression and resistance to fixation, LacZ reporters have often been preferable for higher resolution analysis of sectioned tissues. By contrast, native fluorescent proteins, even though desirable for live imaging applications, usually cannot be visualized at single-cell resolution, and often do not withstand fixation and post-processing. Thus, none of the existing TCF/Lef n -promoter min :reporter constructs, or derivative mouse strains, facilitate single-cell resolution imaging of Wnt/ß-catenin pathway activity that can be quantified in live as well as in fixed tissues.
We therefore sought to generate an improved, third generation, Wnt/ß-catenin reporter, that would incorporate a bright fluorescent reporter which could be live imaged at single-cell resolution and also quantified, but which would withstand fixation and therefore could also be visualized in tissue sections. To do so, we designed a reporter construct that combined the Wnt/ß-catenin signaling read-out efficiency of multimerized TCF/Lef DNA binding sites with the single-cell resolution and quantifiable reporter expression afforded by fluorescent histone fusions. Fluorescent proteins fused to histones, for example human histone H2B, are localized to the nucleus because they remain bound to chromatin, and as such allow the visualization and tracking of individual cells. H2B fusions also provide details of cell divisions, including the plane of division and identification of daughter cells. They also reveal nuclear fragmentation which is often associated with cell death [9, 10]. We placed the H2B-GFP cassette under the control of six TCF/Lef response elements and the hsp68 minimal promoter in a configuration identical to previously reported TCF/Lef-LacZ reporter mice . We then used this construct to generate a derivative TCF/Lef:H2B-GFP mouse strain.
We recovered several founder transgenic lines which exhibited equivalent expression, demonstrating that TCF/Lef:H2B-GFP reporter expression was independent of integration site. Characterization of the TCF/Lef:H2B-GFP strain of mice revealed bright single-cell resolution reporter expression that spatio-temporally recapitulated TCF/Lef-LacZ reporter expression during mouse embryonic development. Moreover, given its improved sensitivity, the TCF/Lef:H2B-GFP strain revealed additional sites of reporter expression, in the visceral endoderm and epiblast of the pre-gastrula stage mouse embryo, tissues suggested through genetic and expression analyses to possess active Wnt/ß-catenin signaling, that is not reflected by existing Wnt/ß-catenin signaling reporters.
In summary, we have generated a transgenic mouse strain that serves as a quantitative, non-invasive single-cell resolution read-out of Wnt/ß-catenin signaling in the mouse. The TCF/Lef:H2B-GFP reporter currently represents an improved tool for imaging the in vivo processes triggered by canonical Wnt signaling pathway activation.
Results and Discussion
Generation of TCF/Lef:H2B-GFP reporter mice
We subsequently cloned the TCF/Lef response elements and minimal promoter of each reporter in front of H2B-GFP to create the final construct. Transgenic mouse lines were generated from both constructs. However, since the TCF/Lef-siamois:H2B-GFP transgenic animals gave no readily detectable fluorescent signal, only the TCF/Lef-hsp68:H2B-GFP founder lines were considered for further study, and from now on are referred as TCF/Lef:H2B-GFP strain (Figure 1B).
Failure to detect TCF/Lef:H2B-GFP reporter expressing cells at preimplantation stages of embryonic development
Considerable debate has surrounded the issue of Wnt/ß-catenin signaling at preimplantation stages of mouse development, and specifically at the blastocyst stage. In support of a transcriptional read-out of canonical Wnt signaling, previous reports have suggested possible transient nuclear-localization of ß-catenin in a minor population of cells at the blastocyst stage. In our hands and with our TCF/Lef:H2B-GFP reporter, which we believe to exhibit increased sensitivity over existing reporter strains, we were unable to detect convincing reproducible nuclear-localized GFP fluorescence at any stage of preimplantation development in either successively staged or time-lapse imaged embryos. We take this to suggest that either the TCF/Lef:H2B-GFP reporter is not sufficiently sensitive, or that any Wnt/ß-catenin pathway signaling response is non-transcriptional.
TCF/Lef:H2B-GFP reporter reveals sites of Wnt/ß-catenin signaling in the early postimplantation embryo not previously detected with reporter strains
At the late blastocyst stage (E4.5) the embryo implants into the maternal uterus. This event is followed by lineage expansion, which results in formation of a cup-shaped egg cylinder. Close apposition of epiblast, extraembryonic endoderm and visceral endoderm (VE) facilitates signaling cross-talk between these layers, leading to the formation of the distal visceral endoderm (DVE), migration of the anterior visceral endoderm (AVE) and establishment of the anterior-posterior axis of the embryo . Canonical Wnt signaling has been implicated in both formation of anterior-posterior axis and AVE migration. Specifically, a population of cells which are proposed to actively migrate within the VE epithelium, a morphogenetic movement which results in the formation of the AVE, have been proposed to move away from a region of high WNT activity . Although β-catenin localization can be detected throughout VE, Wnt/β-catenin reporter strains generated to date have not exhibited activity at these early postimplantation stages making it difficult to determine the spatiotemporal dynamics of in vivo signaling within the VE.
Tracking TCF/Lef:H2B-GFP reporter expressing cells in the visceral endoderm
One of the advantages of H2B fusions as live imaging reporters is that they facilitate the identification and tracking of single cells while at the same time permitting visualization of an entire population. Indeed, how a group or population of cells can move collectively and in doing so radically change the structure and function of a tissue, is a central question in developmental biology, and underscores many integral morphogenetic cell behaviors driving embryonic development. Since the TCF/Lef:H2B-GFP transgenic is the first Wnt reporter line to reveal the dynamics of Wnt signaling activity in cells of the visceral endoderm of E5.5 embryos, we focused on this stage to study the behavior of cells expressing the reporter. We assumed that GFP-positive cells were either actively signaling, or had recently been transducing a Wnt signal and remained GFP-positive due to perdurance of the GFP protein (Additional file 1).
3D time-lapse movies of embryos pre-streak (PS) stage embryos (Figure 5, Additional file 1) confirmed that the number of GFP-positive cells increased within the VE as development proceeded, and confirmed the heterogeneity of GFP reporter levels among GFP-positive cells, as had been observed in sequentially staged embryos (Figure 4). Tracking of reporter-expressing VE cells in E5.5 embryos for over 9 hours, a period of time during which the AVE would have migrated, revealed extensive cell division (over 50% of cells tracked divided), and the conservation of nearest-neighbor relationships between GFP-positive cells. No change was observed in the relative position GFP-positive cells that were tracked, suggesting that the TCF/Lef:H2B-GFP reporter might not be labeling cells of the DVE/AVE.
In the data depicted in Figure 5, a total of 22 GFP-positive VE cells were identified and tracked. Color-coded open circles identify individual cells in Figure 5, and color-coded closed spheres identify individual tracked cells in Additional file 2. About half the tracked population (12 cells) divided during the 9 hours of time-lapse (Figure 5A - color-coded open circles with white outline indentify cells having divided since previous time-point shown). In nearly all cases, nearest-neighbor relationships were preserved both in regard to individual cells, as well as their relative positions within the group.
We selected 4 cells that did not divide during the time-lapse as a reference set (closed yellow circles in Figure 5, yellow spheres in Additional file 2), and documented their relative distances during the period of the time-lapse (white lines connecting yellow circles in Figure 5A, red lines connecting yellow spheres in Additional file 2). Notably, the distance between these non-dividing reference cells doubled as their individual positions changed relative to each other. Despite this fact, their nearest-neighbor relationships remained predominantly unchanged, as did their position relative to their neighbors, suggesting that the topology of cells within the VE was constant.
We tracked dividing reporter-expressing VE cells. We plotted the orientation of the division planes, as well as the final position of daughter cells at the last time-point for which we generated image data (Figure 5). Our data suggest that any migration of reporter-expressing cells may result from oriented divisions, as well as a general increase in the size of the embryo, rather than by an active movement of cells. However, additional data and statistical analyses will be required to determine if cell divisions within this population exhibit a prevalent orientation, as our data might suggest.
As reporter-expressing VE cells proliferated, their progeny did not reorganize, but retained their relative positions. A minor reorganization or 'jostling' of cells could serve to alter cell geometry, but facilitate conservation of cell topology, within the VE epithelium and could be driven by differential regional proliferation within the VE. Our data support a model whereby active cell migration driving AVE formation , and cell proliferation may together result in a reorganization of cells within the VE epithelium. These morphogenetic cell rearrangements are also likely influenced by, and are expected to accommodate, the rapid growth of the embryo, notably the adjacent epiblast, at this stage. Further detailed analyses will be important in extending these observations and determining the respective roles of proliferation and cell signaling within the VE and its neighboring tissues, and importantly how these coordinated cell behaviors in association with cell signaling might direct the morphogenesis of the early mouse embryo.
The TCF/Lef:H2B-GFP reporter marks the primitive streak and nascent mesoderm at gastrulation
Gastrulation is the event that results in the generation of the three primary germ layers (ectoderm, definitive endoderm and mesoderm) from the pluripotent epiblast, and the elaboration of the axes (anterior-posterior, dorsal-ventral and left-right). In the mouse, the onset of gastrulation is marked by the appearance of the primitive streak (the source of mesoderm and definitive endoderm) which represents a morphologically-distinct structure which breaks the bilateral symmetry of the epiblast, and in doing so, defines the posterior of the embryo at E6.5. The site of primitive streak formation has been proposed to be regulated by Nodal and Wnt3 signaling activities likely emanating from the overlying visceral endoderm [17–20]. This data combined with the expression pattern of Wnt3 suggest an essential role for Wnt signaling in primitive streak specification, and the maintenance of gastrulation. By E7.5 Wnt3 is downregulated while the related gene, Wnt3a, is activated. Embryos lacking Wnt3a exhibit a complete absence of paraxial mesoderm, the cell type emerging from the primitive streak starting at E7.5. These genetic data support an essential role for Wnt/ß-catenin signaling associated with the site of the primitive streak, in the initiation and progression of gastrulation in the mouse.
By late bud/early headfold (~E7.5) stages, a second population of H2B-GFP-positive cells located in the proximal part of the conceptus within the extra-embryonic region emerged. These small patches of H2B-GFP-positive cells resembled the pools of primitive erythroid cells, or "blood islands", a site at which Wnt signaling has been proposed to play a role in specifying hematopoietic cell populations (Figure 6H, 6I; yellow arrowhead) [21–23].
TCF/Lef:H2B-GFP reporter during midgestation
TCF/Lef:H2B-GFP reporter expression at midgestation to later fetal stages
Widefield fluorescence imaging at E12.5 revealed high levels of reporter expression within the spinal cord and limbs (Figure 8I). In the brain, expression of the transgene was localized to discrete regions such as the infundibulum, whereas it was broader in the spinal cord (Figure 8M, 8L, 8R). Close-up of the otic region showed increased levels of fluorescence in the developing semicircular canals (Figure 8J). In sections through the inner ear, transgene expression was detected in the dorsomedial otic epithelium that will give rise to the vestibular structures (Figure 8O). Previous studies have also described Wnt activity in the olfactory epithelium (Figure 8N) and oral epithelium (Figure 8P) . High magnification wholemount views of the limb revealed continued expression of the transgene at the AER (Figure 8K), while sections showed regions positive for the transgene outside cartilage primordia (Figure 8T). Moreover, high levels of transgene were also detected in the lung epithelia (Figure 8U), where Wnt signaling is proposed to play a key role in branching morphogenesis .
TCF/Lef:H2B-GFP reporter expression at postnatal stages
TCF/Lef:H2B-GFP reporter expression in the kidney
TCF/Lef:H2B-GFP reporter expression in the brain
We have generated a single-cell resolution fluorescent transgenic Wnt reporter strain of mice and have performed an in depth characterization of its expression in embryos and several postnatal stages. The reporter design comprises multimerized TCF/Lef DNA binding sites driving expression of H2B-GFP, a subcellularly-localized fusion of a genetically-encoded fluorescent protein to human histone H2B, which by labeling active chromatin enables the visualization of single cells within a cohort. H2B fusion reporters not only facilitate the visualization and tracking of labeled cells over time, but also provide spatiotemporal information on cell proliferation and apoptosis dynamics. Moreover, to test canonical Wnt read-out efficiency of the TCF/Lef:H2B-GFP reporter line, we compared our line with the well characterized LacZ-based TCF/Lef-LacZ mouse strain, confirming specific GFP reporter expression in all expected sites of canonical Wnt signaling activity. Even thought Wnt reporter lines based on the multimerization of Lef/TCF binding sites which act as transcriptional reporters, might not be ideal readouts of active Wnt signaling, they are at present some of the best tools available to accurately identify the major sites of Wnt activity.
In conclusion, we demonstrate the specificity of the TCF/Lef:H2B-GFP transgenic line as a faithful readout of canonical Wnt signaling activity and its resolution at the single cell level. The increased brightness and sensitivity of the reporter has already revealed additional sites of reporter expression. While in readily detectable or robust sites of reporter expression reduced laser power is likely to be required for image acquisition, which is advantageous for live imaging applications. Since live imaging approaches critical for the study of cell dynamics, this reporter line represents a valuable tool to further our understanding of the events triggered by canonical Wnt signaling pathway activation during mouse development, tissue homeostasis or disease progression.
Generation of Reporter Constructs and Transgenic Animals
To generate the pTCF/Lef:H2B-GFP construct, six copies of the TCF/Lef response elements together with the hsp68 minimal promoter from the TCF/Lef-LacZ reporter construct  were inserted into the AseI/Nhe1 sites of pCMV::H2B-GFP. Transgenic mice were generated by pronuclear injection following standard protocols. Animals were genotyped by PCR. Primers used for the PCR reaction were GFPGenotFW: ACAACAAGCGCTCGACCATCAC; GFPGenotRW: AGTCGATGCCCTTCAGCTCGAT. Two transgenic founder lines (TCF/Lef:H2B-GFP #16, TCF/Lef:H2B-GFP #61) were established both exhibiting similar patterns and levels of reporter expression. However, only one line (TCF/Lef:H2B-GFP #61) was further characterized in detail. Subsequent generations exhibited Mendelian transgene inheritance, stable transgene activity and comparable levels of reporter expression. Animals were maintained in accordance with National Institute of Health guidelines for the care and use of laboratory animals and under the approval of the Memorial Sloan-Kettering Cancer Center Institutional Animal Care and Use Committee.
Embryo collection and strains used
Embryos were dissected in DMEM/F12 (1:1) containing 5% calf serum and fixed in 4% paraformaldehyde. Presomitic embryos were staged by morphological landmarks according to Downs and Davies ; somite stage embryos were staged according to their somite number. Additional mouse strains used were TCF/Lef-LacZ  and TOPGAL  reporters.
For ex utero culture embryos were dissected in DMEM/F12 (1:1) containing 5% calf serum and cultured in medium comprising 50% DMEM/F12 (1:1), 50% rat serum and 1% Penicillin/Streptomycin .
For vibratome sections, embryos were embedded 0.5% gelatin/15% BSA/20% sucrose (gelatin/albumin mix) and 4.5% glutaraldehyde in PBS and cut on a Leica vibrating microtome (VT1000) at 35-175 μm. For cryosections, samples were equilibrated in PBS/10% sucrose followed by PBS/30% sucrose overnight and snap-frozen in OCT (Tissue-Tek). Sections were cut in a Leica cryostat at 12 μm and counterstained with Hoechst for nuclei (5 μg/ml, Molecular Probes) and Phalloidin Alexa546 for F-actin (Invitrogen).
Mouse embryos were dissected in PBS, fixed for 20 min in 0.35% glutaraldehyde in PBS and processed as whole mounts or embedded in the gelatin/albumin mix and sectioned. Samples were washed 3 times with X-Gal rinse (0.02% NP-40, 0.01% Sodium deoxycholate, 2 mM MgCl2, 100 mM sodium phosphate pH 7.3) for 20 min and incubated in 1 mg/ml X-gal, 5 mM K3Fe(CN)6, 5 mM K4Fe(Cn)6, 0.02% NP-40, 0.01% Sodium deoxycholate, 2 mM MgCl2, 100 mM sodium phosphate pH7.3 overnight. They were finally rinsed with PBS and fixed for 30 min in 4% paraformaldehyde.
Embryos were permeabilised in 0.55% Triton X-100 in PBS for 20 minutes and blocked in 10% fetal bovine serum in PBS for 1 hour. Primary antibodies used were: anti-Cer1 (R&D Systems), anti-Eomes (abcam), anti-GFP (Invitrogen), anti-HNF4a (Santa Cruz), and anti-Oct4 (Santa Cruz). Secondary Alexa Fluor (Invitrogen)-conjugated antibodies were used at a dilution of 1/500. DNA was visualized using Hoechst 33342 (5 μg/ml, Molecular Probes).
Widefield and epifluorescence images were acquired using a Zeiss Axiocam MRc camera coupled to a Leica M165FC dissecting scope or a Zeiss A1 Axioscope. Laser scanning confocal data were acquired using a Zeiss LSM510 META using a PlanApo 20×/NA0.75 objective. Fluorophores were excited using a 405 nm diode laser (Hoechst), 488 nm Argon laser (GFP, GFP) or 543 nm HeNe laser (Alexa 546). Embryos were imaged whole mount in MatTek dishes (Ashland). Spinning disc confocal 3D timelapse data was acquired using Volocity acquisition software http://www.improvision.com/ and a Perkin-Elmer RS3 Nipkow-type scan head mounted on a Zeiss Axiovert 200 M with Hamamatsu C4742-80-12AG camera. GFP was excited using a 488 nm Argon laser. Images were acquired using a Zeiss plan-Neofluar 25×/0.8 DIC korr objective. 10-20 xy planes were acquired, separated by 3-4 μm. Time intervals between z-stacks were 15 minutes. For live imaging experiments, embryos were maintained in a humidified, temperature-controlled chamber with 5% CO2 atmosphere. Sections were mounted in Fluoromount-G (Southern Biotech) and imaged through glass coverslips. Confocal images were acquired as z stacks of xy images taken at 1 μm z -intervals.
The Jackson Laboratory Induced Mutant Resource http://www.jax.org/imr/index.html will be distributing the TCF/Lef:H2B-GFP mouse line. The provisional reference name for the strain is: STOCK Tg(TCF/Lef1-HIST1H2BB/EGFP)61Hadj/J.
We thank the MSKCC Mouse Genetics Core Facility for pronuclear injections to generate transgenic lines; Celia Andreu-Agulló for expert advice on neuroanatomy and neuronal expression of the reporter; Celia Andreu-Agulló, Ann Foley, Alfonso Martinez-Arias, Sonja Nowotschin and Silvia Muñoz-Descalzo for discussions and/or comments on the manuscript. Work in AKH's lab is supported by the NIH (RO1-HD052115 and RO1-DK084391) and NYSTEM.
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