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Jan 2018

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Mosaic Labeling and 3-Dimensional Morphological Analysis of Single Cells in the Zebrafish Left-right Organizer
斑马鱼左右组织者中单细胞的嵌合标记和3D形态分析   

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Abstract

A transient epithelial structure called the left-right organizer (LRO) establishes left-right asymmetry in vertebrate embryos. Developmental defects that alter LRO formation result in left-right patterning errors that often lead to congenital heart malformations. However, little is known about mechanisms that regulate individual cell behaviors during LRO formation. To address this, we developed a Cre-loxP based method to mosaically label precursor cells, called dorsal forerunner cells, that give rise to the zebrafish LRO known as Kupffer’s vesicle. This methodology allows lineage tracing, 3-dimensional (3D) reconstruction and morphometric analysis of single LRO cells in living embryos. The ability to visualize and quantify individual LRO cell dynamics provides an opportunity to advance our understanding of LRO development, and in a broader sense, investigate the interplay between intrinsic biochemical mechanisms and extrinsic mechanical forces that drive morphogenesis of epithelial tissues.

Keywords: Left-right organizer (左右组织者), Dorsal forerunner cells (背侧先驱细胞), Kupffer’s vesicle (库氏泡), Mosaic labeling (嵌合标记), Zebrafish embryo (斑马鱼胚胎), 3D reconstructions (3D重构), Single cell (单细胞), Morphometric analysis (形态学分析)

Background

Recent efforts in the field of developmental biology have focused on understanding mechanisms underlying tissue and organ morphogenesis at the single-cell level. Taking advantage of the transparent zebrafish embryo–which is a useful system for conducting in vivo cell biology experiments–several approaches have been developed to analyze the dynamics of single cells in living embryos. Transient expression of injected mRNAs or transgene constructs has been widely used to mosaically label cells with fluorescent proteins for analysis of individual cells in complex environments, such as endothelial cells in the developing vasculature (Yu et al., 2015). Optogenetic approaches have used light to change the fluorescent properties of photo-convertible proteins (Schuster and Ghysen, 2013) or uncage fluorescent dextran (Clanton et al., 2011) in single cells. Taking a genetic approach, a transgene that contains three fluorescent proteins–RFP (red), YFP (yellow), and CFP (cerulean)–separated by recombination (lox) sites that was first used to engineer ‘brainbow’ mice (Livet et al., 2007) was used to generate stable ‘Zebrabow’ transgenic zebrafish (Pan et al., 2013). Expression of Cre recombinase in Zebrabow embryos generates differential fluorescent labeling of individual cells based on the stochastic recombination of the Zebrabow transgene. Each of the approaches mentioned here have been used successfully to analyze single cells during development and/or regeneration processes in zebrafish.

Kupffer’s vesicle (KV) (Figure 1A) functions as a left-right organizer (LRO) in the zebrafish embryo (Essner et al., 2005; Kramer-Zucker et al., 2005) that is analogous to the ventral node in mouse and the gastrocoel roof plate in frog (Blum et al., 2014). Motile cilia in the LRO (Figure 1B) generate asymmetric signals to establish the left-right body axis in developing vertebrate embryos, which is critical for normal development of the cardiovascular and gastrointestinal systems (Dasgupta and Amack, 2016; Grimes and Burdine, 2017). Fate-mapping studies have identified the precursor cells–called dorsal forerunner cells (DFCs)–that give rise to the zebrafish LRO (KV) (Cooper and D'Amico, 1996; Melby et al., 1996). Multiple transgenic tools, including Tg(sox17:GFP-CAAX) (Dasgupta et al., 2018) and Tg(dusp6:memGFP) (Wang et al., 2011), have been developed to visualize and quantify the behaviors of DFCs and KV cells. DFCs migrate as mesenchymal cells during gastrulation stages (Figure 2A), undergo a mesenchymal-to-epithelial transition (MET) at the end of gastrulation, and then form a rosette-like structure (Amack et al., 2007; Oteiza et al., 2008) (Figure 2B). Next, the KV lumen expands, and each KV cell extends a motile cilium into the fluid-filled lumen. During KV lumen expansion, epithelial KV cells at the middle plane of the organ that start out with similar morphologies (Figure 2C) undergo a morphogenetic process that we refer to as ‘KV remodeling’. During this process, cells in the anterior region of KV increase in size and develop columnar shapes that allow tight packing of these cells. In the posterior region of KV, cells decrease in size and become wide and thin (Figure 2D). KV remodeling creates an asymmetric distribution of motile cilia along the anteroposterior axis–with more cilia packed into the anterior region (Figure 1B)–that is necessary to generate right-to-left asymmetric fluid flows in KV and left-right patterning in the embryo (Wang et al., 2011 and 2012). Probing KV development at the single-cell level will be essential to understanding the relationship between intrinsic and extrinsic mechanisms that mediate asymmetric epithelial morphogenesis in KV.


Figure 1. Kupffer’s vesicle in the zebrafish embryo. A. A dorsal view of Kupffer’s vesicle (KV) in a live zebrafish embryo at 8-somite stage (8 ss) of development. This is a brightfield image taken using a Zeiss Discovery V12 stereomicroscope. B. A schematic diagram of KV shows cell shapes at middle focal plane, and cilia (red and blue) projecting into the lumen to drive fluid flow within the KV after remodeling at 8 ss. A = Anterior; P = Posterior, L = Left; R = Right; D = Dorsal; V = Ventral. Anterior KV cells are represented in blue and posterior KV cells are red. Arrow = strong leftward flow.


Figure 2. Behavior and 2D morphology of DFC and KV cells during development. Two transgenic zebrafish strains–Tg(dusp6:memGFP) and Tg(sox17:GFP-CAAX)–provide bright labeling of dorsal forerunner cells (DFCs) that give rise to Kupffer’s vesicle (KV) cells. A and B. Embryo diagrams (green represents DFC/KV cells) and inversed fluorescence images of membrane-localized GFP expression in DFCs in Tg(dusp6:memGFP) transgenic zebrafish at 80% epiboly stage (80% E) (A) and the tailbud stage (B) when migratory DFCs form a rosette structure. C and D. GFP expression in Tg(sox17:GFP-CAAX) transgenic zebrafish marks KV cell membranes during KV lumen formation at the 2 somite stage (2 ss) (C) and in the mature organ at 8 ss (D) after KV remodeling. A = Anterior; P = Posterior, L = Left; R = Right; D = Dorsal; V = Ventral. Anterior cells = blue, Posterior cells = red.

Herein we describe a genetic mosaic labeling strategy to fluorescently label individual KV cells and provide a guide to analyze 3D data obtained from imaging live mosaic-labeled embryos using Imaris software. We have generated stable transgenic Tg(sox17:CreERT2)sny120 zebrafish, in which a sox17 promoter drives the expression of a tamoxifen-inducible Cre recombinase (CreERT2) (Feil et al., 1997) in the DFC/KV cell lineage and endodermal cells. To take advantage of Cre-loxP based cell labeling in Zebrabow embryos, we created double transgenic fish to express the Tg(sox17:CreERT2) transgene in a Tg(ubi:Zebrabow-M)a131 background (Pan et al., 2013) in which the zebrafish ubiquitin (ubi) promoter drives the expression of the Zebrabow transgene in all cells (Figure 3A). We next determined a dose of 4-hydroxytamoxifen (4-OHT) that induces low levels of Cre activity in DFCs, and reliably results in mosaic labeling of DFC/KV cells in Tg(sox17:CreERT2); Tg(ubi:Zebrabow) embryos (Figure 3B). The low Cre activity switches default RFP expression to CFP or YFP expression in a subset of cells (Figures 3C and 3D). Confocal images of single mosaic-labeled cells in live embryos can be used to reconstruct and quantify 3D cellular morphology (Figure 3E). This approach provides a simple and efficient method to stochastically label individual DFC/KV cells for analysis of cell behaviors in real-time during morphogenesis of the zebrafish LRO.


Figure 3. Mosaic labeling and 3D rendering of single KV cells. A. Double transgenic Tg(sox17:CreERT2); Tg(ubi:Zebrabow) zebrafish are incrossed to obtain embryos. B. Time course of mosaic labeling of KV cells. Brief treatment of double transgenic Tg(sox17:CreERT2); Tg(ubi:Zebrabow) embryos with 4-OHT from the dome stage (4 h post-fertilization) to the shield stage (6 hpf) generates low levels of Cre activity that changes expression of default RFP to expression of CFP or YFP in a subset of KV cells. C. Structure of the ubi:zebrabow and sox17:CreERT2 transgenes and the possible recombination outcomes of the Zebrabow transgene by Cre recombinase activity in KV cell lineages. Cre can mediate the deletion of sequences flanked by loxP sites (orange triangles) or variant lox2272 sites (blue triangles), leaving behind single loxP or lox2272 sites that are not cross-compatible with each other. D. Mosaic labeled YFP+ KV cells (pseudo-colored green) at the middle plane of KV at tailbud stage and 8 somite stage (8 ss). Scale bars = 20 μm. E. 3D reconstructions of single KV cells (green) using Imaris software at tailbud and 8 ss. Dashed line indicates KV lumen surface. Scale bars = 10 μm.

Materials and Reagents

  1. Petri dish (VWR, catalog number: 25384-088)
  2. 12-well clear flat bottom not treated multiwell cell culture plate (Falcon, catalog number: 351143)
  3. Glass bottom microwell dishes, 35 mm Petri dish, 14 mm microwell, No. 1.5 coverglass (0.16-0.19 mm) (MatTek, catalog number: P35G-1.5-14-C)
  4. Glass transfer pipet (Fisher, catalog number: 63A183-624)
  5. Double transgenic Tg(sox17:CreERT2); Tg(ubi:Zebrabow) zebrafish (available upon request from the Amack lab: amackj@upstate.edu)
    Note: The Tg(sox17:CreERT2); Tg(ubi:Zebrabow) strain is maintained by selecting embryos to raise that have green fluorescent hearts (cmlc2:GFP expression is a marker for the sox17: CreERT2 transgene) and bright ubiquitous RFP expression from the ubi:Zebrabow transgene.
  6. (Z)-4-Hydroxytamoxifen (4-OHT) (Sigma, catalog number: H7904)
    Note: Reconstituted to a stock solution of 10 mM in 1% DMSO and stored at -20 °C in single-use aliquots.
  7. 1% low-melting point (LMP) agarose (Invitrogen, catalog number: 15517-014) prepared in embryo medium and maintained at 50 °C
  8. 1% agarose (VWR, catalog number: 0710-500G) prepared in embryo medium that will be used to coat the bottom of Petri dishes and 12-well plates
  9. Dimethyl sulfoxide (DMSO) (VWR, catalog number: 0231-500mL)
  10. Sodium chloride (NaCl) (Fisher, catalog number: BP358-212)
  11. Potassium chloride (KCl) (J. T. Baker, catalog number: 3040-01)
  12. Calcium chloride dihydrate (CaCl2•2H2O) (J. T. Baker, catalog number: 1332-01)
  13. Magnesium sulfate heptahydrate (MgSO4•7H2O) (Fisher, catalog number: BP214-500)
  14. Methylene blue (Fisher, catalog number: BP117-100)
  15. Embryo medium (see Recipes)

Equipment

  1. Tweezers (Electron Microscopy Sciences, catalog number: 0103-5-PS)
    Note: We use Dumont Tweezer style 5.
  2. Incubator set at 28.5 °C for culturing zebrafish embryos (VWR, catalog number: 35960-056)
  3. Stereomicroscope with epifluorescent light source (Zeiss, model: Stereo Discovery V12)
    Note: We use a Zeiss Stereo Discovery V12.
  4. Confocal microscope (Nikon, model: Eclipse Ti)
    Note: We use a Perkin-Elmer UltraVIEW Vox spinning disc confocal system equipped with 488 nm and 561 nm solid-state lasers (for YFP and RFP excitation) mounted on a Nikon Eclipse Ti inverted microscope with a Hamamatsu C9100-50 EM-CCD camera. We use a 20x oil-immersion objective. The microscope is equipped with a temperature control chamber for live imaging. It is likely that this protocol can be adapted for use with a laser scanning confocal microscope, but we have not tested different microscope platforms. 
  5. Freezer

Software

  1. Volocity (PerkinElmer) for image acquisition
  2. Imaris (BitPlane) for 3D rendering and morphometric analysis

Procedure

  1. Fluorescent mosaic labeling of DFC/KV cells
    1. Set up crosses of homozygous double transgenic Tg(sox17:CreERT2); Tg(ubi:Zebrabow) zebrafish in breeding tanks with dividers that separate males from females. Remove dividers at the desired time to allow fish to breed and synchronize embryo development. 
    2. Collect Tg(sox17:CreERT2); Tg(ubi:Zebrabow) embryos and culture them in embryo medium in a Petri dish at 28.5 °C until they reach the dome stage of development ~4 h post-fertilization (hpf).
    3. Carefully remove embryos from their chorion using fine tweezers (we use Dumont Tweezer style 5 from Electron Microscopy Sciences) in a Petri dish coated with 1% agarose. The agarose prevents the yolk of embryo from sticking to the plastic surface of Petri dish. To coat the dish, pipet enough hot liquid 1% agarose to cover the bottom of the dish, and then allow it to cool and solidify.
    4. Transfer dechorionated embryos using a glass transfer pipet (fire polish the tip) to a 12-well flat bottom cell culture plate coated with 1% agarose. Replace the embryo medium with fresh embryo medium containing 5 μM 4-hydroxytamoxifen (abbreviated here as 4-OHT) and 0.1% DMSO (dimethyl sulfoxide). The DMSO aids in cell permeability and drug delivery. Treat control embryos with 0.1% DMSO alone. We recommend that each well contain 5-6 dechorionated embryos. 
    5. Incubate embryos in 5 μM 4-OHT medium from the dome stage (4 hpf) to the shield stage (6 hpf) at 28.5 °C. 
    6. At the shield stage, transfer treated embryos to fresh embryo medium without 4-OHT and gently swirl. Repeat this step 3 times with fresh embryo medium to wash out 4-OHT. 
    7. Return rinsed embryos to 28.5 °C to allow development to the desired stage for imaging labeled DFC/KV cells.
      Note: Results from our work (Dasgupta et al., 2018) indicate Cre activity is not spatially biased, but randomly labels cells throughout the KV. In addition, we found on average that imaging 12 embryos will result in ~20 anterior KV cells and ~20 posterior KV cells to analyze. 

  2. Immobilization of mosaic labeled embryos for imaging using an inverted microscope
    1. At the desired stage of development, carefully transfer an embryo to a glass-bottom (MatTek) dish using a glass transfer pipet. To analyze DFC behaviors, embryos can be prepared at any stage during epiboly. To visualize KV morphogenesis, we prepare embryos between the 1-2 somite stages.
      Note: Accumulation of YFP expression is time-dependent following Cre activation (4-OHT treatments). Thus, YFP fluorescence is weak at early (epiboly) stages and brighter at later (somite) stages.
    2. After transferring the embryo to the MatTek dish, remove most of the embryo medium and then cover the embryo with liquid 1% low-melting point (LMP) agarose that was maintained at 50 °C. 
    3. While the agarose solidifies, use a stereomicroscope to orient the embryo so that the DFC/KV cells face the glass-bottom (Figure 4; Video 1). 


      Figure 4. Immobilization of mosaic labeled embryos for live imaging. Schematic representing embryo immobilization technique used for live imaging with an inverted confocal microscope. A live embryo is covered with liquid low-melting point (LMP) agarose in a MatTek dish and then positioned such that DFC/KV cells are close to the glass bottom. Once solidified, the agarose is covered by embryo medium.

    4. Once the agarose has solidified, and the embryo is immobilized, add embryo medium to the dish to cover the sample and prevent the sample from drying out.
      Note: It is recommended to repeat this process to mount 5+ embryos for screening to identify the embryo(s) with the degree of mosaic labeling that is appropriate for the designed experiment.

      Video 1. Immobilization of a live embryo for confocal microscopy. This video demonstrates Steps 1-4 of Procedure B.

  3. Imaging mosaic labeled DFC/KV cells in live embryos
    1. Position the MatTek dish containing the immobilized live embryo on an inverted confocal microscope. We use a 20x objective on a Perkin-Elmer UltraVIEW Vox spinning confocal disc confocal system with an environmental chamber maintained at 32 °C to image live embryos.
      Note: The Tg(ubi:Zebrabow) transgene drives RFP expression in all cells by default, which we excite using a 561 nm laser. If Cre-mediated recombination has occurred in a cell, we observe YFP expression (excited with a 488 nm laser).
    2. Select an embryo with bright YFP+ mosaic labeling that allows individual cells to be distinguished from their neighbors (Figure 3D). Laser power and exposure time will depend on signal intensity. Typically, we use 488 nm laser power between 30% and 50%, and exposure times between 500 ms and 800 ms using the 20x objective. Power for the 561 nm laser is typically 30% with an exposure time of 100-300 ms.
      Note: Laser power and exposure time should be minimized to prevent photo-toxicity. To achieve this, we suggest selecting mosaic labeled embryos with the brightest YFP expression.
    3. To analyze a single time point, we capture a Z-series through the entire KV using 2 µm Z steps. The typical distance is ~35 Z steps (70 μm) at the 2 somite stage and ~45 Z steps (90 μm) at the 8 somite stage. 
    4. For time-lapse imaging, we capture Z-stacks through the entire KV every 5 min during KV morphogenesis.
      Note: We have imaged a single embryo (using 5 min intervals) for up to 3 h (between 2 ss and 8 ss) without detecting photo-damage or deleterious effects on embryo development. 

  4. Viewing confocal images using Imaris software
    1. To open a confocal image (Z-stack) in Imaris software, the raw data will need to be converted to an OME TIFF (.ome) file. In Volocity software, select the image to convert, and then right-click to export the file. Save the file as an OME TIFF.
    2. In Imaris (we have used version 8.4.0), click the Assay icon to create a new project folder. Next, click the Group icon to create a new group within the assay. Finally, click the Image icon and use the open file window to add an image (Z-stack in .ome file format) to the group.
    3. Double click on the .ome file icon to open the image in Imaris. This will open in the Surpass view.
    4. Next, open the Image Properties window (CTRL-I). Set the desired color for each channel (e.g., green, red, blue, etc.).
    5. Open the Geometry tab within the Image Properties window. Set the voxel size using pixel dimensions and Z-step size used to capture the image. Pixel dimensions are measured manually for each objective using a micrometer. For example:
      Spinning disc confocal 20x objective: X = 0.33 (1 pixel = 0.33 μm)

      Y = 0.33

      Z = step size (2 μm) used to acquire Z-stack
    6. Open the Adjustment window (CTRL-D) to adjust signal levels. 
    7. Click Store to save the processed image.

  5. 3D rendering of mosaic labeled DFC/KV cells using Imaris software
    1. To 3D render an object (e.g., single cell), click Surface under the 3D view menu.
    2. To define the region of interest (ROI) for rendering, go to the create tab and check Segment only a region of interest box.
    3. Click the next (blue) button at the bottom of the menu sidebar. The ROI bounding box appears (Figure 5A).
    4. To change the size of the bounding box, the cursor must be in the Select mode. Use the ESC key to toggle between Navigate and Select modes.
    5. In Select mode, click and drag arrowheads to re-size the bounding box in X, Y and Z around a single cell that you would like to reconstruct (Figure 5B).
      Note: you cannot zoom or rotate in Select mode; you must toggle to Navigate mode.
    6. Click next.
    7. Select source signal (e.g., green channel that includes YFP+ cells).
    8. Set surface detail. A higher number is more smooth and less detail (e.g., 1 = smooth).
    9. Set thresholding. Fifteen micrometer works well for KV cells. Smaller background signals are ignored.
      Note: The thresholding number should be based on the size of the object (cell) that you are rendering. The length and width of the cell can be measured manually in the 2D slice view.
    10. Click next.
    11. The slider can be used to manually adjust level of 3D rendering of the cell. Use navigate mode to check the rendering in X, Y, and Z.
    12. Optional: If a cell of interest is in contact with another labeled cell, use Split Touching Objects function. Check Enable and set the Seed Point Diameter. A value of 8 is often good for KV cells.
    13. Click next.
    14. Dots show how many labeled cells the software detects. 
    15. If necessary, go back and change Seed Point Diameter until the number of cells is accurate.
    16. Click the next button to finish. This completes the 3D rendering a single KV cell completes (Figure 5C). 
    17. To edit a 3D rendering, select edit (pencil icon in the lower menu bar). 
    18. Select the cell you wish to analyze and click duplicate in the edit tab. This creates a new surface file with only the selected 3D rendered cell.
    19. To obtain measurements of the 3D rendering, click statistics (graph icon in the lower menu bar), select detailed tab, select specific values and use the drop-down menu to select a measurement (e.g., area, volume, intensity, etc.). 
    20. Use clipping plane (scissors icon) to slice through the 3D rendered image. This tool can be used to slice 3D surface rendered KV cells to measure cell cross-sectional areas. 
    21. Use snapshot to capture an image (3D rendering, cross-section, etc.). Under the file tab, make a copy and save.
    22. Representative data are shown in Figure 6.


      Figure 5. 3D rendering of a single cell using a region of interest in Imaris software. A. A region of interest (ROI) bounding box (yellow box) around an entire 3D image of mosaic-labeled KV cells. Scale bar = 30 μm. B. The ROI bounding box can be re-sized to include only a single cell. Scale bar = 20 μm. C. The software 3D renders only the cell included in the ROI. Scale bar = 30 μm.


      Figure 6. Quantification of 3D rendered KV cells using Imaris. A. 3D renderings of cells in the anterior or posterior region of KV at the 2 somite stage (2 ss) or 8 somite stage (8 ss). Cell height (h), length (l), and width (w) measurements are shown at 2 ss. Dashed line indicates KV lumen surface. Scale bars = 10 μm. B-C. Box and whisker plots showing quantification of length to width ratio (LWR) that describes the shape of KV anterior or posterior cells (B), and quantification of cell volumes (C). n = 27 anterior cells at 2 ss; n = 25 posterior cells at 2 ss; n = 21 anterior cells at 8 ss; n = 22 posterior cells at 8 ss. Anterior and posterior KV cells have similar shapes and volumes at 2 ss, and then undergo asymmetric morphological changes that result in different cell shapes and volumes at 8 ss. n = number of cells analyzed. NS = not significant; ****P < 0.001. These images and results are modified from Dasgupta et al. (2018). 

Data analysis

Taking measurements of 3D rendered DFC/KV cells using Imaris software.

  1. Click new measurement points icon in the top menu bar with small icons.
  2. In the settings tab, select sphere (check the box).
  3. For line mode check pairs.
  4. Select edit tab.
  5. Under intersect with, select surface of an object.
  6. In Select mode, a box appears at the cursor.
  7. Hold down the shift key and click to mark the first point of the line.
  8. Repeat to mark the second point of the line.
  9. To adjust the line, select one point (turns yellow), hold down shift and drag to new location.
  10. Use settings tab to change font or color of the line.
  11. Click statistics icon to obtain measurements in the detailed tab. Results can be saved as an excel file by clicking the export statistics (floppy disc icon) at the bottom of the menu sidebar.
  12. Use snapshot to capture an image. Under the file tab, make a copy and save.

Notes

We realized Imaris is expensive software that is not available at all institutions. Alternative software packages such as Volocity (Perkin Elmer) or FIJI/ImageJ (free download at https://imagej.net/Fiji/Downloads) can be used to visualize confocal data sets in 3D, and generate 3D reconstructions for analysis. We focus this protocol on using Imaris because of its capability to segment a region of interest and 3D reconstruct a single cell (Figure 5).

Recipes

  1. Embryo medium

Acknowledgments

We thank Sharleen Buel for technical assistance and zebrafish care, Madeline Clark for video demonstration, and Peu Santra for assistance with data presentation. The Tg(ubi:Zebrabow-M)a131 strain was a kind gift from the Alex Schier Lab (Harvard University). This protocol corresponds to experiments presented in (Dasgupta et al., 2018). This work was supported by NIH grants R01HL095690 and R01GM117598.

Competing interests

The authors have no conflicts of interest to declare.

References

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简介

称为左右组织者(LRO)的瞬时上皮结构在脊椎动物胚胎中建立左右不对称性。改变LRO形成的发育缺陷导致左右图案化错误,这常常导致先天性心脏畸形。然而,关于在LRO形成期间调节个体细胞行为的机制知之甚少。为了解决这个问题,我们开发了一种基于Cre- loxP 的方法,用于对前体细胞进行马赛克标记,称为背部先行细胞,这种细胞产生斑马鱼LRO,称为Kupffer's囊泡。该方法允许谱系追踪,三维(3D)重建和活体胚胎中单个LRO细胞的形态测定分析。能够可视化和量化个体LRO细胞动力学提供了一个机会,以促进我们对LRO发展的理解,并在更广泛的意义上,研究内在生化机制和驱动上皮组织形态发生的外在机械力之间的相互作用。

【背景】最近在发育生物学领域的努力集中于理解单细胞水平的组织和器官形态发生的潜在机制。利用透明斑马鱼胚胎 - 这是一种用于进行体内细胞生物学实验的有用系统 - 已经开发了几种方法来分析活胚胎中单细胞的动态。注射mRNA或转基因构建体的瞬时表达已被广泛用于用荧光蛋白标记细胞以分析复杂环境中的个体细胞,例如发育中的脉管系统中的内皮细胞(Yu et al。,2015 )。光遗传学方法使用光来改变光可转换蛋白(Schuster和Ghysen,2013)的荧光特性或在单细胞中破坏荧光葡聚糖(Clanton et al。,2011)。采用遗传方法,含有三种荧光蛋白的转基因-RFP(红色),YFP(黄色)和CFP(天蓝色) - 通过重组( lox )位点分离,最初用于工程'脑卒中小鼠(Livet et al。,2007)用于产生稳定的'Zebrabow'转基因斑马鱼(Pan et al。,2013)。 Zebrabow胚胎中Cre重组酶的表达基于Zebrabow转基因的随机重组产生单个细胞的差异荧光标记。这里提到的每种方法都已成功用于分析斑马鱼发育和/或再生过程中的单个细胞。

Kupffer的囊泡(KV)(图1A)在斑马鱼胚胎中起到左右组织者(LRO)的作用(Essner et al。,2005; Kramer-Zucker et al。,2005)这类似于小鼠的腹侧节点和青蛙的胃体顶板(Blum et al。,2014)。 LRO中的运动纤毛(图1B)产生不对称信号以在发育中的脊椎动物胚胎中建立左右体轴,这对于心血管和胃肠系统的正常发育是至关重要的(Dasgupta和Amack,2016; Grimes和Burdine,2017) 。命运映射研究已经确定了前体细胞 - 称为背侧前驱细胞(DFCs) - 引起斑马鱼LRO(KV)(Cooper和D'Amico,1996; Melby et al。,1996 )。多种转基因工具,包括 Tg(sox17:GFP-CAAX)(Dasgupta et al。,2018)和 Tg(dusp6:memGFP)( Wang et al。,2011),已被开发用于可视化和量化DFC和KV细胞的行为。 DFCs在原肠胚形成期间作为间充质细胞迁移(图2A),在原肠胚形成结束时经历间充质 - 上皮转换(MET),然后形成玫瑰花结样结构(Amack 等。 ,2007; Oteiza et al。,2008)(图2B)。接下来,KV腔扩张,并且每个KV细胞将活动的纤毛延伸到充满液体的腔中。在KV腔扩张期间,器官中间平面的上皮KV细胞以相似的形态开始(图2C)经历形态发生过程,我们称之为'KV重塑'。在此过程中,KV前部区域的细胞尺寸增大并形成柱状,允许这些细胞紧密堆积。在KV的后部区域,细胞尺寸减小并变得宽而薄(图2D)。 KV重塑产生沿前后轴的运动纤毛的不对称分布 - 更多纤毛填充到前部区域(图1B) - 这是在KV中产生从右到左的不对称流体流动和胚胎中的左右图案化所必需的。 (Wang et al。,2011和2012)。探索单细胞水平的KV发育对于理解介导KV中不对称上皮形态发生的内在和外在机制之间的关系至关重要。


图1. Kupffer在斑马鱼胚胎中的囊泡。 A.在8个体节阶段(8 ss)发育的活斑马鱼胚胎中Kupffer囊泡(KV)的背视图。这是使用Zeiss Discovery V12立体显微镜拍摄的明场图像。 B.KV的示意图显示了在中焦平面处的细胞形状,并且纤维(红色和蓝色)突出到内腔中以在8s重塑后驱动KV内的流体流动。 A =前部; P =后部,L =左; R =对; D =背部; V =腹侧。前KV细胞以蓝色表示,后KV细胞为红色。箭头=强大的向左流动。
图2.发育过程中DFC和KV细胞的行为和2D形态。两种转基因斑马鱼菌株 - Tg(dusp6:memGFP)和 Tg(sox17:GFP- CAAX) - 提供产生Kupffer囊泡(KV)细胞的背部前驱细胞(DFCs)的明亮标记。 A和B.胚胎图(绿色代表DFC / KV细胞)和在80%外胚层阶段的 Tg(dusp6:memGFP)转基因斑马鱼中DFC中膜定位GFP表达的反向荧光图像(80%) E)(A)和尾部阶段(B)当迁移的DFC形成玫瑰花结构时。 C和D.GFP在 Tg(sox17:GFP-CAAX)转基因斑马鱼中的表达标记在2个体节阶段(2s)(C)和成熟器官中的KV腔形成期间的KV细胞膜。 KV重塑后8 ss(D)。 A =前部; P =后部,L =左; R =对; D =背部; V =腹侧。前部细胞=蓝色,后部细胞=红色。

在本文中,我们描述了用于荧光标记单个KV细胞的遗传镶嵌标记策略,并提供了使用Imaris软件分析从活体镶嵌标记的胚胎成像获得的3D数据的指南。我们已经产生稳定的转基因 Tg(sox17:Cre ERT2) sny120 斑马鱼,其中 sox17 启动子驱动在DFC / KV细胞谱系和内胚层细胞中表达他莫昔芬诱导的Cre重组酶(Cre ERT2)(Feil 等人,,1997)。为了利用Zebrabow胚胎中基于Cre- loxP 的细胞标记,我们创建了双转基因鱼来表达 Tg(sox17:Cre ERT2) Tg(ubi:Zebrabow-M) a131 背景中的转基因(Pan et al。,2013),其中斑马鱼遍在蛋白(ubi)启动子驱动Zebrabow转基因在所有细胞中的表达(图3A)。我们接下来确定了一种4-羟基三苯氧胺(4-OHT)的剂量,其在DFC中诱导低水平的Cre活性,并且可靠地导致在 Tg中的DFC / KV细胞的镶嵌标记(sox17:Cre ERT2); Tg(ubi:Zebrabow)胚胎(图3B)。低Cre活性将默认RFP表达切换为细胞子集中的CFP或YFP表达(图3C和3D)。活胚胎中单个镶嵌标记细胞的共聚焦图像可用于重建和量化3D细胞形态(图3E)。该方法提供了一种简单有效的方法,用于随机标记单个DFC / KV细胞,用于在斑马鱼LRO的形态发生过程中实时分析细胞行为。


图3.单个KV细胞的马赛克标记和3D渲染。 A.双转基因 Tg(sox17:Cre ERT2); Tg(ubi:Zebrabow)斑马鱼正在接受以获得胚胎。 B. KV细胞的镶嵌标记的时间过程。简单处理双转基因 Tg(sox17:Cre ERT2);从穹顶阶段(受精后4小时)到屏蔽阶段(6 hpf)的具有4-OHT的Tg(ubi:Zebrabow)胚胎产生低水平的Cre活性,其将默认RFP的表达改变为CFP的表达。或KFP细胞子集中的YFP。 C. ubi:zebrabow 和 sox17:Cre ERT2 转基因的结构以及Kb重组酶活性对Zebrabow转基因可能的重组结果细胞谱系。 Cre可以介导删除侧翼为 loxP 位点(橙色三角形)或变体 lox2272 位点(蓝色三角形)的序列,留下单个 loxP 或 lox2272 彼此不互相兼容的网站。 D.马赛克标记YFP + KV细胞(伪色绿色)在KV的中间平面处于尾部阶段和8个体节阶段(8s)。比例尺=20μm。 E.使用Imaris软件在尾巴和8秒处对单个KV细胞(绿色)进行三维重建。虚线表示KV流明表面。比例尺=10μm。

关键字:左右组织者, 背侧先驱细胞, 库氏泡, 嵌合标记, 斑马鱼胚胎, 3D重构, 单细胞, 形态学分析

材料和试剂

  1. 培养皿(VWR,目录号:25384-088)
  2. 12孔透明平底未处理多孔细胞培养板(Falcon,目录号:351143)
  3. 玻璃底微孔培养皿,35 mm培养皿,14 mm微孔,1.5号盖玻片(0.16-0.19 mm)(MatTek,目录号:P35G-1.5-14-C)
  4. 玻璃移液管(Fisher,目录号:63A183-624)
  5. 双转基因 Tg(sox17:Cre ERT2); Tg(ubi:Zebrabow)斑马鱼(可向Amack实验室索取: amackj@upstate.edu )< br /> 注意: Tg(sox17:Cre ERT2 );通过选择具有绿色荧光心脏的胚胎来维持Tg(ubi:Zebrabow) 菌株(cmlc2:GFP表达是sox17的标记:Cre ERT2 转基因)和来自ubi:Zebrabow转基因的明亮无处不在的RFP表达。
  6. (Z)-4-羟基三苯氧胺(4-OHT)(Sigma,目录号:H7904)
    注意:重新配制成10 mM在1%DMSO中的储备溶液,并在-20°C下以一次性等分试样储存。
  7. 在胚胎培养基中制备并保持在50℃的1%低熔点(LMP)琼脂糖(Invitrogen,目录号:15517-014)
  8. 在胚胎培养基中制备的1%琼脂糖(VWR,目录号:0710-500G)将用于涂覆培养皿底部和12孔板
  9. 二甲基亚砜(DMSO)(VWR,目录号:0231-500mL)
  10. 氯化钠(NaCl)(Fisher,目录号:BP358-212)
  11. 氯化钾(KCl)(J.T.Baker,目录号:3040-01)
  12. 氯化钙二水合物(CaCl 2•2H 2 O)(J。T. Baker,目录号:1332-01)
  13. 硫酸镁七水合物(MgSO 4•7H 2 O)(Fisher,目录号:BP214-500)
  14. 亚甲蓝(Fisher,目录号:BP117-100)
  15. 胚胎培养基(见食谱)

设备

  1. 镊子(Electron Microscopy Sciences,目录号:0103-5-PS)
    注意:我们使用Dumont Tweezer样式5。
  2. 孵化器设置在28.5°C,用于培养斑马鱼胚胎(VWR,目录号:35960-056)
  3. 具有落射荧光光源的立体显微镜(Zeiss,型号:Stereo Discovery V12)
    注意:我们使用Zeiss Stereo Discovery V12。
  4. 共聚焦显微镜(尼康,型号:Eclipse Ti)
    注意:我们使用配有488 nm和561 nm固态激光器(用于YFP和RFP激发)的Perkin-Elmer UltraVIEW Vox旋转盘共聚焦系统,安装在带有Hamamatsu C9100-50 EM的Nikon Eclipse Ti倒置显微镜上-CCD相机。我们使用20倍油浸物镜。显微镜配有温度控制室,用于实时成像。该协议可能适用于激光扫描共聚焦显微镜,但我们尚未测试不同的显微镜平台。&nbsp;
  5. 冰柜

软件

  1. Volocity(PerkinElmer)用于图像采集
  2. Imaris(BitPlane)用于3D渲染和形态测量分析

程序

  1. DFC / KV细胞的荧光镶嵌标记
    1. 建立纯合双转基因 Tg的杂交(sox17:Cre ERT2); Tg(ubi:Zebrabow)斑马鱼在繁殖池中具有将雄性与雌性分开的分隔物。在所需的时间去除分隔物,让鱼类繁殖并同步胚胎发育。&nbsp;
    2. 收集 Tg(sox17:Cre ERT2); Tg(ubi:Zebrabow)胚胎并在培养皿中于28.5℃在胚胎培养基中培养它们直至它们在受精后~4小时达到发育的圆顶阶段(hpf)。
    3. 使用细镊子(我们使用电子显微镜科学公司的Dumont Tweezer样式5)在涂有1%琼脂糖的培养皿中小心地从绒毛膜中取出胚胎。琼脂糖可以防止胚胎的蛋黄粘在培养皿的塑料表面上。为了涂上培养皿,吸取足够的热液1%琼脂糖以覆盖培养皿的底部,然后使其冷却并固化。
    4. 使用玻璃转移移液管(对尖端进行抛光)将去绒毛胚转移至涂有1%琼脂糖的12孔平底细胞培养板。用含有5μM4-羟基三苯氧胺(此处缩写为4-OHT)和0.1%DMSO(二甲基亚砜)的新鲜胚胎培养基替换胚胎培养基。 DMSO有助于细胞渗透性和药物递送。用0.1%DMSO单独处理对照胚胎。我们建议每个孔含有5-6个去绒毛胚胎。&nbsp;
    5. 将胚胎在5μM4-OHT培养基中从圆顶阶段(4hpf)孵育至28.5℃的屏蔽阶段(6hpf)。&nbsp;
    6. 在屏蔽阶段,将处理过的胚胎转移到没有4-OHT的新鲜胚胎培养基中并轻轻旋转。用新鲜胚胎培养基重复该步骤3次以洗去4-OHT。&nbsp;
    7. 将冲洗过的胚胎恢复到28.5°C,以便开发到标记的DFC / KV细胞成像所需的阶段。
      注意:我们工作的结果(Dasgupta et al。 ,2018)表明Cre活动不是空间偏差,而是随机标记整个KV的细胞。此外,我们平均发现成像12个胚胎将导致~20个前KV细胞和~20个后KV细胞进行分析。&nbsp;

  2. 使用倒置显微镜固定马赛克标记的胚胎成像
    1. 在期望的发育阶段,使用玻璃移液管将胚胎小心地转移到玻璃底(MatTek)培养皿中。为了分析DFC行为,可以在外包期间的任何阶段制备胚胎。为了可视化KV形态发生,我们在1-2个体节阶段之间制备胚胎。
      注意:在Cre活化(4-OHT处理)后,YFP表达的累积是时间依赖性的。因此,YFP荧光在早期(外包)阶段较弱,在后期(体节)阶段较亮。
    2. 将胚胎转移到MatTek培养皿后,除去大部分胚胎培养基,然后用保持在50℃的液体1%低熔点(LMP)琼脂糖覆盖胚胎。&nbsp;
    3. 当琼脂糖固化时,使用立体显微镜定向胚胎,使DFC / KV细胞面向玻璃底(图4;视频1)。&nbsp;


      图4.马赛克标记胚胎的固定化,用于实时成像。 表示用于倒置共聚焦显微镜实时成像的胚胎固定技术的示意图。将活胚胎在MatTek培养皿中用液体低熔点(LMP)琼脂糖覆盖,然后定位使得DFC / KV细胞靠近玻璃底部。一旦固化,琼脂糖被胚胎培养基覆盖。

    4. 一旦琼脂糖凝固,胚胎被固定,将胚胎培养基加入培养皿中以覆盖样品并防止样品变干。
      注意:建议重复此过程以安装5+胚胎进行筛选,以确定具有适合设计实验的镶嵌标记程度的胚胎。


      视频1.用于共聚焦显微镜的活胚胎的固定化。该视频演示了程序B的步骤1-4。&nbsp;

  3. 在活胚胎中成像马赛克标记的DFC / KV细胞
    1. 将含有固定化活胚胎的MatTek培养皿置于倒置共聚焦显微镜上。我们在Perkin-Elmer UltraVIEW Vox旋转共焦圆盘共聚焦系统上使用20x物镜,环境室保持在32°C,以对活胚胎进行成像。
      注意:Tg(ubi:Zebrabow)转基因默认驱动所有细胞中的RFP表达,我们使用561 nm激光激发。如果在细胞中发生Cre介导的重组,我们观察到YFP表达(用488nm激光激发)。
    2. 选择具有明亮YFP +镶嵌标记的胚胎,允许将单个细胞与其邻居区分开(图3D)。激光功率和曝光时间取决于信号强度。通常,我们使用30x物镜时488 nm激光功率在30%和50%之间,曝光时间在500 ms和800 ms之间。 561 nm激光的功率通常为30%,曝光时间为100-300 ms。
      注意:应尽量减少激光功率和曝光时间,以防止光毒性。为实现这一目标,我们建议选择具有最亮YFP表达的镶嵌标记胚胎。
    3. 为了分析单个时间点,我们使用2μmZ步骤通过整个KV捕获Z系列。典型距离在2个体节阶段为~35 Z阶(70μm),在8体节阶段为〜45 Z阶(90μm)。&nbsp;
    4. 对于延时成像,我们在KV形态发生期间每5分钟通过整个KV捕获Z-堆叠。
      注意:我们已经对单个胚胎(间隔5分钟)进行了长达3小时(2秒到8秒)的成像,但未发现光损伤或对胚胎发育的有害影响。

  4. 使用Imaris软件查看共聚焦图像
    1. 要在Imaris软件中打开共焦图像(Z-stack),需要将原始数据转换为OME TIFF( .ome )文件。在Volocity软件中,选择要转换的图像,然后右键单击以导出文件。将文件另存为OME TIFF。
    2. 在Imaris(我们使用的是版本8.4.0)中,单击 Assay 图标以创建新的项目文件夹。接下来,单击组图标以在分析中创建新组。最后,单击图像图标,然后使用打开的文件窗口将图像(.ome文件格式的Z-stack)添加到组中。
    3. 双击.ome文件图标以在Imaris中打开图像。这将在 Surpass 视图中打开。
    4. 接下来,打开图像属性窗口(CTRL-I)。为每个通道设置所需的颜色(例如,绿色,红色,蓝色,等。)。
    5. 在图像属性窗口中打开几何标签。使用像素尺寸和用于捕获图像的Z步长来设置体素大小。使用千分尺手动测量每个物镜的像素尺寸。例如:
      class =“ke-zeroborder”bordercolor =“#000000”style =“width:700px;” border =“0”cellspacing =“0”cellpadding =“2”>旋转盘共焦20x物镜: X = 0.33(1像素=0.33μm)

      Y = 0.33

      Z =用于获得Z-堆叠的步长(2μm)
    6. 打开调整窗口(CTRL-D)以调整信号电平。&nbsp;
    7. 点击存储以保存已处理的图像。

  5. 使用Imaris软件标记DFC / KV细胞的马赛克的3D渲染
    1. 要3D渲染对象(例如,单个单元格),请单击3D视图菜单下的曲面。
    2. 要定义要渲染的感兴趣区域(ROI),请转到创建标签,然后选中细分 仅感兴趣的区域框。
    3. 点击菜单侧栏底部的下一步(蓝色)按钮。出现ROI边界框(图5A)。
    4. 要更改边界框的大小,光标必须处于选择模式。使用 ESC 键在导航和选择模式之间切换。
    5. 在选择模式下,单击并拖动箭头以围绕要重建的单个单元格重新调整X,Y和Z中的边界框的大小(图5B)。
      注意:您无法在选择模式下缩放或旋转;您必须切换到导航模式。
    6. 点击下一步。
    7. 选择源信号(例如,包含YFP +单元格的绿色通道)。
    8. 设置表面细节。数字越大越平滑,细节越少(例如,1 =平滑)。
    9. 设置阈值。十五微米适用于KV细胞。较小的背景信号被忽略。
      注意:阈值编号应基于要渲染的对象(单元格)的大小。可以在2D切片视图中手动测量单元格的长度和宽度。
    10. 点击下一步。
    11. 滑块可用于手动调整单元格的3D渲染级别。使用导航模式检查X,Y和Z中的渲染。
    12. 可选:如果感兴趣的细胞与另一个标记的细胞接触,请使用拆分触摸对象功能。选中启用并设置种子点直径。对于KV细胞,值8通常是有益的。
    13. 点击下一步。
    14. 点显示软件检测到的标记单元格数。&nbsp;
    15. 如有必要,返回并更改种子点直径,直到单元格数准确。
    16. 点击下一步按钮完成操作。这样就完成了单个KV单元的3D渲染(图5C)。&nbsp;
    17. 要编辑3D渲染,请选择编辑(下方菜单栏中的铅笔图标)。&nbsp;
    18. 选择您要分析的单元格,然后在修改标签中点击重复。这将创建一个仅包含所选3D渲染单元格的新曲面文件。
    19. 要获得3D渲染的测量值,请单击统计信息(下方菜单栏中的图形图标),选择详细选项卡,然后选择特定 值并使用下拉菜单选择度量(例如,区域,音量,强度,等。)。&nbsp;
    20. 使用剪裁平面(剪刀图标)切割3D渲染图像。此工具可用于切割3D表面渲染的KV细胞以测量细胞横截面积。&nbsp;
    21. 使用快照捕获图像(3D渲染,横截面,等。)。在文件选项卡下,进行复制并保存。
    22. 代表性数据如图6所示。


      图5.使用Imaris软件中感兴趣的区域对单个细胞进行3D渲染。 :一种。在镶嵌标记的KV细胞的整个3D图像周围的感兴趣区域(ROI)边界框(黄色框)。比例尺=30μm。 B.可以将ROI边界框重新调整大小以仅包括单个单元。比例尺=20μm。 C.软件3D仅呈现ROI中包括的单元。比例尺=30μm。


      图6.使用Imaris对3D渲染的KV细胞进行定量。 :一种。在2体节阶段(2 ss)或8 somite阶段(8 ss),KV前部或后部区域的细胞三维渲染。单元高度(h),长度(l)和宽度(w)测量结果以2秒显示。虚线表示KV流明表面。比例尺=10μm。公元前。框和晶须图显示了长宽比(LWR)的量化,其描述了KV前或后细胞的形状(B),以及细胞体积的定量(C)。 n = 27个2s的前细胞; n = 25个后细胞,2s; n = 21个前细胞,8s; n = 22个后部细胞,8s。前后KV细胞在2s时具有相似的形状和体积,然后经历不对称的形态变化,导致8s时不同的细胞形状和体积。 n =分析的细胞数。 NS =不显着; **** P &lt; 0.001。这些图像和结果是根据Dasgupta 等(2018)修改的。&nbsp;

数据分析

使用Imaris软件测量3D渲染的DFC / KV细胞。

  1. 点击顶部菜单栏中带有小图标的新测量点图标。
  2. 在设置标签中,选择球体(选中此框)。
  3. 对于线路模式,请检查对。
  4. 选择修改标签。
  5. 在与交叉下,选择对象的表面。
  6. 在选择模式下,光标处会出现一个框。
  7. 按住 shift 键并单击以标记该行的第一个点。
  8. 重复以标记线的第二个点。
  9. 要调整线条,请选择一个点(变为黄色),按住shift并拖动到新位置。
  10. 使用设置标签更改线条的字体或颜色。
  11. 点击统计信息图标,在详细标签中获取测量结果。通过单击菜单侧栏底部的导出统计信息(软盘图标),可以将结果另存为Excel文件。
  12. 使用快照捕获图像。在文件选项卡下,制作副本并保存。

笔记

我们意识到Imaris是一种昂贵的软件,并非所有机构都可以使用。其他软件包如Volocity(Perkin Elmer)或FIJI / ImageJ(可在 https://imagej.net/Fiji免费下载) / Downloads )可用于在3D中可视化共聚焦数据集,并生成用于分析的3D重建。我们将此协议的重点放在使用Imaris上,因为它具有分割感兴趣区域和3D重建单个细胞的能力(图5)。

食谱

  1. 胚胎培养基

致谢

我们感谢Sharleen Buel提供技术支持和斑马鱼护理,Madeline Clark用于视频演示,感谢Peu Santra提供数据演示方面的帮助。 Tg(ubi:Zebrabow-M) a131 菌株是来自Alex Schier Lab(哈佛大学)的礼物。该方案对应于(Dasgupta 等人,2018)中提出的实验。这项工作得到NIH拨款R01HL095690和R01GM117598的支持。

利益争夺

作者没有利益冲突申报。

参考

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Copyright Dasgupta et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Dasgupta, A., Jacob, A. E. and Amack, J. D. (2018). Mosaic Labeling and 3-Dimensional Morphological Analysis of Single Cells in the Zebrafish Left-right Organizer. Bio-protocol 8(22): e3090. DOI: 10.21769/BioProtoc.3090.
  2. Dasgupta, A., Merkel, M., Clark, M. J., Jacob, A. E., Dawson, J. E., Manning, M. L. and Amack, J. D. (2018). Cell volume changes contribute to epithelial morphogenesis in zebrafish Kupffer's vesicle. Elife 7: e30963.
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