Method Article

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

DOI:

10.3791/62086

⸱

February 28th, 2021

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

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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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Protocol

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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

NOTE: Here we provide a detailed description on how to dissect and prepare mouse E8.25 to E10.5 embryos for live imaging (1.1), E7.5 to E11.5 embryos for....

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Results

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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)
A repre.......

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Discussion

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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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Disclosures

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The authors declare no conflicts of interest.

Acknowledgements

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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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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Agarose low gelling temperatureSigmaA9414Used to mounting embryos (e.g. for OPT)
Amira softwareThermofisher-Commerial software tool
Anti-Brachyury (Goat polyclonal)R and D SystemsAF2085 RRID:AB_2200235For immunofluorescence
Anti-Sox2 (Rabbit monoclonal)Abcamab92494 RRID:AB_10585428For immunofluorescence
Anti-Tbx6 (Goat polyclonal)R and D SystemsAF4744 RRID:AB_2200834For immunofluorescence
Anti-Laminin111 (Rabbit polyclonal)SigmaL9393 RRID:AB_477163For immunofluorescence
Anti-goat 488 (Donkey polyclonal)Molecular ProbesA11055 RRID:AB_2534102For immunofluorescence
Anti-rabbit 568 (Donkey polyclonal)ThermoFisher   ScientificA10042 RRID:AB_2534017For 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 albuminBiowestP6154For 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 TechnologiesD3571For immunofluorescence
Drishti software(open source)-Free software tool
EDTASigmaED2SSFor demineralization
Fiji/ImageJ software(open source)-Free software tool
GlycineNZYtechMB01401For immunofluorescence
Huygens softwareScientific Volume Imaging-Commerial software tool
HyClone defined fetal bovine serumGE Healthcare#HYCLSH30070.03For live imaging
Hydrogen peroxide solution 30 %Milipore1085971000For clearing
Imaris softwareBitplane / Oxford instruments-Commerial software tool
iSpacersSunJin Lab(varies)Use as spacers for preparations
L-glutamineGibco#25030–024For live imaging medium
Low glucose DMEMGibco11054020For live imaging medium
M2 mediumSigmaM7167To dissect embryos
MethanolVWRVWRC20847.307For dehydration and rehydration steps
Methyl salicylateSigmaM6752Used to clear embryos
ParaformaldehydeSigmaP6148Used in solution to fix embryos
Penicillin-streptomycinSigma#P0781For live imaging medium
PBS (Phosphate-buffered saline solution)BiowestL0615-500-
RapiClearSunJin LaboratoryRapiClear 1.52Used to clear embryos
Secure-Sea hybridization chambersSigmaC5474Use as spacers for preparations
simLab softwareSimLab soft-Commerial software tool
Slide, depression concave glass - 75x25 mm(any)-To mount thick embryos.
Triton X-100SigmaT8787For immunofluorescence

References

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  1. Wilson, V., Olivera-Martinez, I., Storey, K. G. Stem cells, signals and vertebrate body axis extension. Development. 136 (12), 2133(2009).
  2. Dias, A., Aires, R. Axial Stem Cells and the Formation of the Vertebrate Body. Concepts and Applications of Stem Cell Biolog....

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Tags

Vertebrate SomitogenesisAxial Elongation3D Visualization4D ImagingMouse EmbryosOptical Projection TomographyWhole Mount ImmunofluorescenceNeuromesodermal ProgenitorsTissue Clearing3D Reconstruction

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