Reconstruction of zebrafish early embryonic development

by scanned light sheet microscopy


Philipp J. Keller1,2*, Annette D. Schmidt2, Joachim Wittbrodt1,2,3,4* and Ernst H.K. Stelzer1

 

1Cell Biology and Biophysics Unit and 2Developmental Biology Unit

European Molecular Biology Laboratory, Germany

3Institute of Zoology, Department for Developmental Physiology

University of Heidelberg, Germany

4Institute of Toxicology and Genetics

Karlsruhe Institute of Technology, Germany


*To whom correspondence should be addressed.

Email: keller(at)embl.de, wittbrod(at)embl.de

 

Science, 14 November 2008, vol. 322, no. 5904, pp. 1065-1069

 


Overview of the contents of this data repository

 

1)   Digital Embryo Movies (Movies 1-16)

2)   High-Quality Figures (Figures 1-6, S1-S9)

3)   Digital Embryo Databases (Embryos 1-7)

4)   Source Code of selected core modules of the Digital Embryo processing pipeline

 


1)   Digital Embryo Movies

 

Note: If you encounter problems during playback of the DivX movies, you most likely require the latest version of the DivX codec for your media player. The codec is freely available for download on the following websites:

 

Apple Mac:

http://www.divx.com/en/software/mac

Microsoft Windows:

http://www.divx.com/en/software/divx-plus

 

 

Movie 1: Schematic operation principle of DSLM

 

The movie shows a schematic illustration of specimen illumination and fluorescence light detection in the standard DSLM imaging mode. The laser beam illuminates the sample from the side and excites fluorophores along a single line inside the specimen. A pair of laser scanners moves the excitation laser line vertically and horizontally. An optically sectioned image is recorded by rapidly scanning an entire plane in the specimen and detecting the fluorescence at a right angle to the illumination axis. The top left inset shows a zoom-in on the illuminated fraction of the specimen, which is embedded in an agarose cylinder and placed inside the specimen chamber.


Primary FTP repository (EBI, Hinxton)

Download Movie 1 as a DivX AVI file (2 MB, DivX 6.8.2 codec).

Download Movie 1 as a QuickTime file (1 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 1 as a DivX AVI file (2 MB, DivX 6.8.2 codec).

Download Movie 1 as a QuickTime file (1 MB, H.264 codec).

 

 

Movie 2: DSLM multi-view time-lapse recording of zebrafish embryonic development

 

The movie shows maximum-intensity projections of a DSLM time-lapse multi-view recoding of zebrafish embryonic development, with a view on both the animal and vegetal hemispheres. The wild-type zebrafish embryo was injected with H2B-eGFP mRNA at the one cell stage. Imaging was started in the 64-cell stage. The entire data set consists of 1,226 time points, recorded in 90 s intervals. At each time point, 370 images with 2048 x 2048 pixels each were recorded with a z-spacing of 2.96 µm. The entire data set comprises 453,620 images (3.5 TBytes). The images were deconvolved with the Lucy-Richardson-algorithm (10 iterations) and normalized for constant overall intensity, thus compensating for the increasing intensity over time due to GFP expression. Detection lens: Carl Zeiss C-Apochromat 10x/0.45 W.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 2 as a DivX AVI file (30 MB, DivX 6.8.2 codec).

Download Movie 2 as a QuickTime file (41 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 2 as a DivX AVI file (30 MB, DivX 6.8.2 codec).

Download Movie 2 as a QuickTime file (41 MB, H.264 codec).

 

 

Movie 3: Reconstruction of zebrafish embryonic development

 

The movie shows an orthographic rendering of the digital embryo obtained from the microscopy data shown in Movie 2. In each frame, the reconstructions at ten time points, corresponding to a time interval of 15 min, are superimposed to indicate the nuclear movement directions. The darker shading corresponds to older time points. The colour-code indicates the current movement speed of each nucleus. A bright orange corresponds to an average movement speed of 1.2 µm/min or more, while a bright cyan highlights effectively non-migratory nuclei. Intermediate speeds are shown by linear interpolation along the colour gradient.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 3 as a DivX AVI file (28 MB, DivX 6.8.2 codec).

Download Movie 3 as a QuickTime file (41 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 3 as a DivX AVI file (28 MB, DivX 6.8.2 codec).

Download Movie 3 as a QuickTime file (41 MB, H.264 codec).

 

 

Movie 4: DSLM multi-view time-lapse recording of MZoep mutant embryogenesis

 

The movie shows maximum-intensity projections of a DSLM time-lapse multi-view recording of zebrafish MZoep mutant embryonic development, with a view on the dorsal and ventral hemispheres. Preparation and microscopy settings as in Movie 2.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 4 as a DivX AVI file (32 MB, DivX 6.8.2 codec).

Download Movie 4 as a QuickTime file (42 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 4 as a DivX AVI file (32 MB, DivX 6.8.2 codec).

Download Movie 4 as a QuickTime file (42 MB, H.264 codec).

 

 

Movie 5: Reconstruction of MZoep mutant embryogenesis

 

The movie shows an orthographic rendering of the MZoep mutant digital embryo obtained from the microscopy data shown in Movie 4, with a dorsal view on the left and a lateral view on the right. Visualization settings as in Movie 3.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 5 as a DivX AVI file (29 MB, DivX 6.8.2 codec).

Download Movie 5 as a QuickTime file (56 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 5 as a DivX AVI file (29 MB, DivX 6.8.2 codec).

Download Movie 5 as a QuickTime file (56 MB, H.264 codec).

 

 

Movie 6: Digital visualization of the MZoep mutant defect in mesendoderm formation

 

The movie shows cell tracking in a slice through the shield for the microscopy time-lapse data set presented in Movie 4 (left: speed colour-code, right: directional colour-code). The 100-µm-thick slices are shown from a lateral view, with the dorsal side to the right. Speed colour-code as in Movie 3. The nuclear movements were analyzed for 30 min intervals and colour-encoded as follows (see also sphere at the bottom): anterior movements (green), posterior movements (cyan), movements away from the body axis (yellow), movements towards the body axis (red) and movements toward the yolk cell (pink). The corresponding colours were assigned, if a movement of at least 20 µm along the respective direction was determined within a time interval of 30 min.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 6 as a DivX AVI file (13 MB, DivX 6.8.2 codec).

Download Movie 6 as a QuickTime file (14 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 6 as a DivX AVI file (13 MB, DivX 6.8.2 codec).

Download Movie 6 as a QuickTime file (14 MB, H.264 codec).

 

 

Movie 7: DSLM time-lapse recording of zebrafish embryogenesis in the animal hemisphere

 

The movie shows maximum-intensity projections of a DSLM time-lapse recoding of zebrafish embryonic development, with a view on the animal hemisphere. The wild-type zebrafish embryo was injected with H2B-eGFP mRNA at the one cell stage. Imaging was started at 1.6 hours post fertilization, during the transition from the 32- to the 64-cell stage. The entire data set consists of 1,470 time points, recorded in 60 s intervals. For each time point, 205 images with 2048 x 2048 pixels each were recorded within 30 s with a z-spacing of 2.96 µm (i.e. a 600 µm deep volume). The entire data set comprises 301,350 images (2.3 TBytes). The images were deconvolved with the Lucy-Richardson-algorithm (20 iterations) and normalized for constant overall intensity, thus compensating for the increasing intensity over time due to GFP-production. Since the illumination laser beam has to travel through the yolk in order to excite fluorophores on the far left side of the animal hemisphere, the average nuclear brightness is lower in this part of the embryo. However, because of the excellent signal-to-noise ratio and the dynamic range of 14 bit of the DSLM images, the corresponding nuclei can also be robustly identified (see Figure S5). Detection lens: Carl Zeiss C-Apochromat 10x/0.45 W.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 7 as a DivX AVI file (30 MB, DivX 6.8.2 codec).

Download Movie 7 as a QuickTime file (39 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 7 as a DivX AVI file (30 MB, DivX 6.8.2 codec).

Download Movie 7 as a QuickTime file (39 MB, H.264 codec).

 

 

Movie 8: Reconstruction of zebrafish embryogenesis in the animal hemisphere

 

The movie shows a perspective rendering of the reconstruction as well as nuclei population statistics for the zebrafish wild-type embryo in Movie 7. Left: view on the animal pole from the centre of the yolk cell. Right: lateral view. In each frame, the reconstructions at ten time points, corresponding to a time interval of 10 min, are superimposed to indicate the nuclear movement directions. The darker shading corresponds to older time points.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 8 as a DivX AVI file (30 MB, DivX 6.8.2 codec).

Download Movie 8 as a QuickTime file (32 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 8 as a DivX AVI file (30 MB, DivX 6.8.2 codec).

Download Movie 8 as a QuickTime file (32 MB, H.264 codec).

 

 

Movie 9: Global cell tracking and colour-indexing of morphogenetic domains

 

We tracked all nuclei reconstructed from the microscopy time-lapse data set shown in Movie 7. The digital embryo is visualized as a perspective rendering with a view on the animal pole from the centre of the yolk cell. The nuclear movements were analyzed for 30 min intervals and colour-encoded as follows (see also sphere to bottom left): anterior movements (green), posterior movements (cyan), movements away from the body axis (yellow), movements towards the body axis (red) and movements toward the yolk cell (pink). The corresponding colours were assigned, if a movement of at least 20 µm along the respective direction was determined within a time interval of 30 min.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 9 as a DivX AVI file (26 MB, DivX 6.8.2 codec).

Download Movie 9 as a QuickTime file (38 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 9 as a DivX AVI file (26 MB, DivX 6.8.2 codec).

Download Movie 9 as a QuickTime file (38 MB, H.264 codec).

 

 

Movie 10: Global three-dimensional pattern of cell divisions

 

The movie shows 4,500 cell division events in the data set presented in Movie 7, detected by the automated processing pipeline at an efficiency of over 90 % for the time interval 100-400 mpf (minutes post fertilization at 26.5 °C). Dividing cells are marked in red and their corresponding daughter cells are shown in cyan. The movie illustrates the initial radial division waves, followed by the symmetry break and by slow peripheral waves in division cycles 10-13. Beyond that time interval, cell divisions appear in a complex pattern of asynchronous patches. The statistical analysis of the division axes of these 4,500 cell divisions is shown in Figure S6b.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 10 as a DivX AVI file (14 MB, DivX 6.8.2 codec).

Download Movie 10 as a QuickTime file (14 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 10 as a DivX AVI file (14 MB, DivX 6.8.2 codec).

Download Movie 10 as a QuickTime file (14 MB, H.264 codec).

 

 

Movie 11: Reverse engineering of migratory tracks of retinal progenitor cells

 

Visualization of the tracking of retinal progenitor cells (red nuclei) in the digital embryo (gray nuclei). The cells were identified by their spatial position in the last time point, i.e. when the optic vesicle had already formed. A reverse engineering of their migratory tracks over a time interval of six hours provides information about the morphogenetic blueprint of eye organogenesis. The automatically computed tracks are in good agreement with the manual reconstruction presented in Rembold et al. (2006). The movie illustrates the principle of organ lineaging using our digital embryos.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 11 as a DivX AVI file (14 MB, DivX 6.8.2 codec).

Download Movie 11 as a QuickTime file (16 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 11 as a DivX AVI file (14 MB, DivX 6.8.2 codec).

Download Movie 11 as a QuickTime file (16 MB, H.264 codec).

 

 

Movie 12: DSLM time-lapse recording of zebrafish embryogenesis in the dorsal hemisphere

 

The movie shows maximum-intensity projections of a DSLM time-lapse recoding of zebrafish embryonic development, with a view on the embryonic shield. The wild-type zebrafish embryo was injected with H2B-eGFP mRNA at the one cell stage. Imaging was started 5 hours post fertilization at approximately 35 % epiboly. The entire data set consists of 1,000 time points, recorded in 60 s intervals. At each time point, 205 images with 2048 x 2048 pixels each were recorded within 30 s with a z-spacing of 2.96 µm (i.e. a 600 µm deep volume). The images were deconvolved with the Lucy-Richardson-algorithm (10 iterations) and normalized for constant overall intensity, thus compensating for the increasing intensity over time due to GFP-production. Detection lens: Carl Zeiss C-Apochromat 10x/0.45 W.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 12 as a DivX AVI file (24 MB, DivX 6.8.2 codec).

Download Movie 12 as a QuickTime file (38 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 12 as a DivX AVI file (24 MB, DivX 6.8.2 codec).

Download Movie 12 as a QuickTime file (38 MB, H.264 codec).

 

 

Movie 13: Reconstruction of zebrafish embryogenesis in the dorsal hemisphere

 

Reconstruction and nuclei population statistics for the embryo shown in Movie 12. Settings as in Movie 8.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 13 as a DivX AVI file (30 MB, DivX 6.8.2 codec).

Download Movie 13 as a QuickTime file (37 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 13 as a DivX AVI file (30 MB, DivX 6.8.2 codec).

Download Movie 13 as a QuickTime file (37 MB, H.264 codec).

 

 

Movie 14: Global cell tracking in the dorsal hemisphere

 

The movie shows global cell tracking for the microscopy time-lapse data set presented in Movie 12. The parameter settings are identical to those used in Movie 9. The nuclear movements are colour-encoded as follows: towards the vegetal pole (cyan), towards the animal pole (yellow), towards the body axis (green) and towards the yolk cell (pink).

 

Primary FTP repository (EBI, Hinxton)

Download Movie 14 as a DivX AVI file (20 MB, DivX 6.8.2 codec).

Download Movie 14 as a QuickTime file (30 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 14 as a DivX AVI file (20 MB, DivX 6.8.2 codec).

Download Movie 14 as a QuickTime file (30 MB, H.264 codec).

 

 

Movie 15: Global cell tracking in the ventral hemisphere

 

Global cell tracking with parameter settings identical to those used in Movie 9. The nuclear movements are colour-encoded as follows: towards the vegetal pole (cyan), towards the animal pole (yellow), towards the body axis (green) and towards the yolk cell (pink).

 

Primary FTP repository (EBI, Hinxton)

Download Movie 15 as a DivX AVI file (29 MB, DivX 6.8.2 codec).

Download Movie 15 as a QuickTime file (27 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 15 as a DivX AVI file (29 MB, DivX 6.8.2 codec).

Download Movie 15 as a QuickTime file (27 MB, H.264 codec).

 

 

Movie 16: Side-by-side comparison of hypoblast formation in the dorsal/ventral hemispheres

 

Frontal and lateral view on embryonic slices on the dorsal and ventral hemispheres of the digital embryo (right: slice close to the shield, left: slice opposite of the shield; complete reconstructions are shown in Movies 14 and 15). The montage visualizes cell internalization in the form of a wave of mesendodermal progenitor cells folding under cell layers of ectodermal progenitors (emboly) between 40 and 50 % epiboly. In the reconstruction, four cell populations were tracked: green nuclei at the tip of the early wave, yellow nuclei in the late wave, blue nuclei at the leading edge moving towards the vegetal pole and pink nuclei close to the animal pole moving towards the vegetal pole. Internalized cells move towards the animal pole on the dorsal hemisphere. In contrast, on the ventral hemisphere, internalized cells move only briefly towards the animal pole and then change their migration direction back towards the vegetal pole. A comprehensive morphogenetic model of hypoblast formation is provided in Figure 6.

 

Primary FTP repository (EBI, Hinxton)

Download Movie 16 as a DivX AVI file (15 MB, DivX 6.8.2 codec).

Download Movie 16 as a QuickTime file (18 MB, H.264 codec).

 

HTTP mirror (EMBL, Heidelberg)

Download Movie 16 as a DivX AVI file (15 MB, DivX 6.8.2 codec).

Download Movie 16 as a QuickTime file (18 MB, H.264 codec).

 


2)   Figures 1-6 and S1-S9 in high quality

 

Figures included in the PDF document (requires Adobe Acrobat Reader 6.0 or higher):

Figure 1: Digital Scanned Light Sheet Microscopy

Figure 2: Imaging and reconstruction of zebrafish embryogenesis

Figure 3: Cell tracking and detection of cell divisions in the digital embryo

Figure 4: Symmetry breaking of the global cell division pattern

Figure 5: Mesendoderm internalization and migration in dorsal and ventral hemispheres

Figure 6: A model of mesendoderm formation in zebrafish

 

Figure S1: Blueprint of the Digital Scanned Laser Light Sheet Fluorescence Microscope

Figure S2: DSLM multi-view imaging of large specimen

Figure S3: Statistical analysis of nuclear properties in the animal hemisphere

Figure S4: The image processing pipeline

Figure S5: Pre-processing/visualization of DSLM recordings of zebrafish embryogenesis

Figure S6: Global statistical analysis of cell divisions

Figure S7: Embryo-to-embryo variability of morphogenetic key parameters

Figure S8: Quantification of the properties of the peripheral cell division waves

Figure S9: Analysis of the patterns of mesendoderm internalization on the dorsal and ventral hemispheres


Download high-quality Figures 1-6 and S1-S9 (27 MB, ZIP archive).

 

 

3)   Digital Embryo Databases

Each digital embryo database is provided as a Matlab MAT-file, containing a multi-cell array (with each cell corresponding to one time point) and a two-dimensional parameter table in each cell characterizing the nuclei segmented at the respective time point. Each table has n rows (n being the number of nuclei at the respective time point) and 17 columns listing the following parameters:

Columns 1-3:

x-y-z coordinates of the centre-of-mass of the nucleus

Columns 4/5:

Average (column 4) and centre-of-mass (column 5) fluorescence intensity (in grey levels) of the nucleus. The average is formed for the central 10-200 voxels of the nucleus (scaling linearly with total nucleus size).

Columns 6-11:

x-y-z start and end coordinates of the bounding box of the segmented nucleus core (from 6 to 11: x-start, y-start, z-start, x-end, y-end, z-end).

Columns 12-17:

x-y-z FWHM coordinates of the three-dimensional intensity distribution around the centre-of-mass of the nucleus (considering the background level). These parameters indicate at which pixel positions along all three dimensions the fluorescence intensity first drops below 50 % of the level at the centre-of-mass position. For more than 97 % of all nuclei, these parameters yield a good approximation of the nucleus shape and the total volume. It should be noted, however, that at very late time points, some nuclei at the centre of the body axis are too densely packed to allow for a decrease in fluorescence of 50 % in between neighbouring nuclei. If the intensity spread could not be determined along one of the dimensions, the table indicates +Inf or –Inf values for the corresponding parameter.

Total number of nucleus entries for all developmental blueprints: 55,707,328

 

Note: Raw DSLM data sets can be provided upon request. The cropped and compressed DSLM image databases are two terabytes in size and require shipping on hard disks. If you are interested in this option, please send an email to keller(at)embl.de for further instructions. The microscopy data sets are helpful e.g. for manual verification of the segmentation data. An example of such an overlay of DSLM data and the segmentation centres in the digital embryo can be downloaded here (30 MB). The overlay represents data set 2 at time point 100 (see below). The DSLM microscopy data was cropped, gamma-corrected and reduced to an 8 bit look-up-table in order to simplify the comparison with the segmentation data.

 


Data set 1: In toto reconstruction of zebrafish wild-type development


Specification of data set 1:
Number of time points and temporal sampling:
901 time points (100-1,450 mpf), 90 sec intervals
Total number of nucleus entries: 9,613,526
Database size: 416 MB
Related database items: Movie 2, Movie 3

 

Download the Matlab database for data set 1.

    

    

Data set 2: Reconstruction of the zebrafish animal hemisphere (A)


Specification of data set 2:
Number of time points and temporal sampling:
1,456 time points (100-1,555 mpf), 60 sec intervals
Total number of nucleus entries: 8,736,014
Database size: 431 MB
Related database items: Movie 7, Movie 8, Movie 9, Movie 10, Movie 11

 

Download the Matlab database for data set 2.

 

 

Data set 3: Reconstruction of the zebrafish animal hemisphere (B)


Specification of data set 3:
Number of time points and temporal sampling:
1,501 time points (200-1,700 mpf), 60 sec intervals
Total number of nucleus entries: 10,622,019
Database size: 518 MB

 

Download the Matlab database for data set 3.

 

 

Data set 4: Reconstruction of the zebrafish animal hemisphere (C)


Specification of data set 4:
Number of time points and temporal sampling:
821 time points (120-1,350 mpf), 90 sec intervals
Total number of nucleus entries: 3,447,269
Database size: 177 MB

 

Download the Matlab database for data set 4.

 

 

Data set 5: Reconstruction of the zebrafish dorsal hemisphere


Specification of data set 5:
Number of time points and temporal sampling:
1,001 time points (300-1,300 mpf), 60 sec intervals
Total number of nucleus entries: 5,629,925
Database size: 283 MB
Related database items: Movie 12, Movie 13, Movie 14, Movie 16

 

Download the Matlab database for data set 5.

 

 

Data set 6: Reconstruction of the zebrafish ventral hemisphere


Specification of data set 6:
Number of time points and temporal sampling:
1,381 time points (230-1,610 mpf), 60 sec intervals
Total number of nucleus entries: 6,981,065
Database size: 344 MB
Related database items: Movie 15, Movie 16

 

Download the Matlab database for data set 6.

 

 

Data set 7: In toto reconstruction of zebrafish MZoep mutant development


Specification of data set 7:
Number of time points and temporal sampling:
597 time points (230-1,274 mpf), 90 sec intervals
Total number of nucleus entries: 10,677,510
Database size: 554 MB
Related database items: Movie 4, Movie 5, Movie 6

 

Download the Matlab database for data set 7.

 


4)   Source code of 16 core modules of the Digital Embryo processing pipeline

 

Modules included in the package:

Segmentation pipeline:

clusterNuclei, clusterCollect
clusterFilter, clusterCorrelate, clusterSaturate
clusterCombine

Data analysis pipeline:

clusterDivisions
clusterMaps, clusterVectorize
clusterIngression, clusterMovement
clusterReduce, clusterRendering
clusterStatistics
clusterTrace, clusterTracking


Download the compressed Matlab source code (65 kB, ZIP archive).

 

 

 (c) Philipp J. Keller, EMBL Heidelberg; September 2008