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Developmental Biology

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Published: February 28th, 2021



1Instituto Gulbenkian de Ciência, 2Faculdade de Ciências da Universidade de Lisboa, 3NOVA School of Science and Technology

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Vertebrate body axis formation is a highly complex and dynamic process occurring during embryonic development. At the end of gastrulation [in the mouse, around embryonic day (E) 8.0], a group of epiblast progenitor cells known as neuromesodermal progenitors (NMPs) become a key driver of axial extension in a head to tail sequence, generating the neural tube and paraxial mesodermal tissues during neck, trunk and tail formation1,2,3,4. Interestingly, the position that these NMPs occupy in the caudal epiblast seems to play a key role in the deci....

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Experiments involving animals followed the Portuguese (Portaria 1005/92) and European (Directive 2010/63/EU) legislations concerning housing, husbandry, and welfare. The project was reviewed and approved by the Ethics Committee of 'Instituto Gulbenkian de Ciência' and by the Portuguese National Entity, 'Direcção Geral de Alimentação e Veterinária' (license reference: 014308).

1. Sample preparation for 3D and 4D imaging


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The representative results shown in this paper for both the live and the immunofluorescence imaging, were obtained using a two-photon system, with a 20 × 1.0 NA water objective, the excitation laser tuned to 960 nm, and GaAsP photodetectors (as described in Dias et al. (2020)43. Optical projection tomography was done using a custom built OPenT scanner (as described in Gualda et al. (2013)28.

Live imaging (4D analysis)

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Axial elongation and segmentation are two of the most complex and dynamic processes occurring during vertebrate embryonic development. The use of 3D and 4D imaging with single-cell tracking has been applied, for some time, to study these processes in both zebrafish and chicken embryos, for which accessibility and culture conditions facilitate complex imaging19,44,45,46,

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We would like to thank Olivier Pourquié and Alexander Aulehla for the LuVeLu reporter strain, the SunJin laboratory for the RapiClear test sample, Hugo Pereira for the help using BigStitcher, Nuno Granjeiro for helping to set up the live imaging apparatus, the IGC animal facility and past and present members of the Mallo lab for useful comments and support during the course of this work.

We thank the technical support of IGC's Advanced Imaging Facility, which is supported by Portuguese funding ref# PPBI-POCI-01-0145-FEDER-022122 and ref# PTDC/BII-BTI/32375/2017, co-financed by Lisboa Regional Operational Programme (Lisboa 2020), un....

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Name Company Catalog Number Comments
Agarose low gelling temperature Sigma A9414 Used to mounting embryos (e.g. for OPT)
Amira software Thermofisher - Commerial software tool
Anti-Brachyury (Goat polyclonal) R and D Systems AF2085 RRID:AB_2200235 For immunofluorescence
Anti-Sox2 (Rabbit monoclonal) Abcam ab92494 RRID:AB_10585428 For immunofluorescence
Anti-Tbx6 (Goat polyclonal) R and D Systems AF4744 RRID:AB_2200834 For immunofluorescence
Anti-Laminin111 (Rabbit polyclonal) Sigma L9393 RRID:AB_477163 For immunofluorescence
Anti-goat 488 (Donkey polyclonal) Molecular Probes A11055 RRID:AB_2534102 For immunofluorescence
Anti-rabbit 568 (Donkey polyclonal) ThermoFisher   Scientific A10042 RRID:AB_2534017 For immunofluorescence
Benzyl Alcohol (99+%) (any) - Used to clear embryos (component of BABB)
Benzyl Benzoate (99+%) (any) - Used to clear embryos (component of BABB)
Bovine serum albumin Biowest P6154 For immunofluorescence
Coverglass 20x20 mm #0 (any) - 100um thick
Coverglass 20x20 mm #1 (any) - 170um thick
Coverglass 20x60 mm #1.5 (any) - To use as “slides”
DAPI (4’,6-Diamidino-2- Phenylindole Dihydrochloride) Life Technologies D3571 For immunofluorescence
Drishti software (open source) - Free software tool
EDTA Sigma ED2SS For demineralization
Fiji/ImageJ software (open source) - Free software tool
Glycine NZYtech MB01401 For immunofluorescence
Huygens software Scientific Volume Imaging - Commerial software tool
HyClone defined fetal bovine serum GE Healthcare #HYCLSH30070.03 For live imaging
Hydrogen peroxide solution 30 % Milipore 1085971000 For clearing
Imaris software Bitplane / Oxford instruments - Commerial software tool
iSpacers SunJin Lab (varies) Use as spacers for preparations
L-glutamine Gibco #25030–024 For live imaging medium
Low glucose DMEM Gibco 11054020 For live imaging medium
M2 medium Sigma M7167 To dissect embryos
Methanol VWR VWRC20847.307 For dehydration and rehydration steps
Methyl salicylate Sigma M6752 Used to clear embryos
Paraformaldehyde Sigma P6148 Used in solution to fix embryos
Penicillin-streptomycin Sigma #P0781 For live imaging medium
PBS (Phosphate-buffered saline solution) Biowest L0615-500 -
RapiClear SunJin Laboratory RapiClear 1.52 Used to clear embryos
Secure-Sea hybridization chambers Sigma C5474 Use as spacers for preparations
simLab software SimLab soft - Commerial software tool
Slide, depression concave glass - 75x25 mm (any) - To mount thick embryos.
Triton X-100 Sigma T8787 For immunofluorescence

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