Name: three and four-dimensional visualization and analysis approaches to study vertebrate axial elongation and segmentation
Uploaded: 2021-02-28T14:00:00
Description: Watch this Scientific Journal Video about Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation at JoVE.com

We would like to thank Olivier Pourqui&#233; 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&#39;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), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and Funda&#231;&#227;o para a Ci&#234;ncia e a Tecnologia (FCT, Portugal). Work described in this manuscript was supported by grants LISBOA-01-0145-FEDER-030254 (FCT, Portugal) and SCML-MC-60-2014 (Santa Casa da Miseric&#243;rdia, Portugal) to M.M., the research infrastructure Congento, project LISBOA-01-0145-FEDER-022170, and the PhD fellowship PD/BD/128426/2017 to A.D.

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 &#39;Instituto Gulbenkian de Ci&#234;ncia&#39; and by the Portuguese National Entity, &#39;Direc&#231;&#227;o Geral de Alimenta&#231;&#227;o e Veterin&#225;ria&#39; (license reference: 014308).
1. Sample preparation for 3D and 4D imaging
NOT...

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,<sup class="xr...

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 decision of differentiating into mesoderm or neuroectoderm5. Although we currently lack a precise molecular fingerprint for NMPs, these cells are generally thought to co-express T (Brachyury) and Sox25,6. The exact mechanisms regulating NMP fate decisions (i.e., whether they take neural or mesodermal routes) are only starting to be precisely defined. Tbx6 expression in the primitive streak region is an early marker of NMP fate decision, as this gene is involved in the induction and specification of mesoderm6,7. Interestingly, early mesoderm cells seem to express high levels of Epha18, and Wnt/&#946;-catenin signalling, as well as Msgn1 were also shown to play important roles in paraxial mesoderm differentiation and somite formation9,10. A complete spatial-temporal analysis of NMPs at a single-cell level will certainly be instrumental to fully understand the molecular mechanisms controlling mesoderm specification.
The formation of somites (vertebrae precursors) is a key feature of vertebrates. During axial elongation, the paraxial mesoderm becomes segmented in a series of bilateral repeating units known as somites. The number of somites and the time required for the formation of new segments varies among species11,12. Somitogenesis involve periodic signaling oscillations (known as the &#34;segmentation clock&#34;) that can be observed by the cyclic expression of several genes of the Notch, Wnt and Fgf signalling pathways in the presomitic mesoderm (e.g., Lfng)11,12. The current model of somitogenesis also postulates the existence of a &#34;maturation wavefront&#34;, a series of complex signalling gradients involving Fgf, Wnt and retinoic acid signaling that define the position of the posterior border of each new somite. A coordinated interaction between the &#34;segmentation clock&#34; and the &#34;maturation wavefront&#34; is therefore fundamental for the generation of these vertebrae precursor modules as perturbations in these key morphogenetic processes can result in embryonic lethality or in the formation of congenital malformations (e.g., scoliosis)13,14,15.
Despite substantial recent advances in imaging techniques, bioimage analysis methods and software, most studies of axial elongation and somitogenesis still rely on single/isolated two-dimensional image data (e.g., sections), which does not allow a full multidimensional tissue visualization and complicates clear differentiation between pathological malformations (i.e. due to mutations) vs normal morphological variation occurring during embryonic development16. Imaging in 3D has already uncovered novel morphogenetic movements, previously not identified by standard 2D methods17,18,19,20, highlighting the power of in toto imaging to understand the mechanisms of vertebrate somitogenesis and axial extension.
3D and 4D microscopy of mouse embryos, particularly live imaging, are technically challenging and require critical steps during sample preparation, image acquisition and data pre-processing in order to allow&#160;accurate and meaningful spatio-temporal analysis. Here, we describe a detailed protocol for live imaging and whole-mount immunofluorescence staining of mouse embryos, that can be used to study both NMPs and mesodermal cells during axial extension and segmentation. In addition, we also describe a protocol for optical projection tomography (OPT) of older embryos and fetuses, that allows 3D in toto visualization and quantification of pathological abnormalities that can result from problems during somitogenesis (e.g., bone fusion and scoliosis)13,21,22. Finally, we illustrate the power of 3D imaging reconstructions in the study and teaching of vertebrate segmentation and axial elongation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose low gelling temperature</td>
			<td>Sigma</td>
			<td>A9414</td>
			<td>Used to mounting embryos (e.g. for OPT)</td>
		</tr>
		<tr>
			<td>Amira software</td>
			<td>Thermofisher</td>
			<td>-</td>
			<td>Commerial software tool</td>
		</tr>
		<tr>
			<td>Anti-Brachyury (Goat polyclonal)</td>
			<td>R and D Systems</td>
			<td>AF2085 RRID:AB_2200235</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr>
			<td>Anti-Sox2 (Rabbit monoclonal)</td>
			<td>Abcam</td>
			<td>ab92494 RRID:AB_10585428</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr>
			<td>Anti-Tbx6 (Goat polyclonal)</td>
			<td>R and D Systems</td>
			<td>AF4744 RRID:AB_2200834</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr>
			<td>Anti-Laminin111 (Rabbit polyclonal)</td>
			<td>Sigma</td>
			<td>L9393 RRID:AB_477163</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr>
			<td>Anti-goat 488 (Donkey polyclonal)</td>
			<td>Molecular Probes</td>
			<td>A11055 RRID:AB_2534102</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr>
			<td>Anti-rabbit 568 (Donkey polyclonal)</td>
			<td>ThermoFisher &#160; Scientific</td>
			<td>A10042 RRID:AB_2534017</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr>
			<td>Benzyl Alcohol (99+%)</td>
			<td>(any)</td>
			<td>-</td>
			<td>Used to clear embryos (component of BABB)</td>
		</tr>
		<tr>
			<td>Benzyl Benzoate (99+%)</td>
			<td>(any)</td>
			<td>-</td>
			<td>Used to clear embryos (component of BABB)</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Biowest</td>
			<td>P6154</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr>
			<td>Coverglass 20x20 mm #0</td>
			<td>(any)</td>
			<td>-</td>
			<td>100um thick</td>
		</tr>
		<tr>
			<td>Coverglass 20x20 mm #1</td>
			<td>(any)</td>
			<td>-</td>
			<td>170um thick</td>
		</tr>
		<tr>
			<td>Coverglass 20x60 mm #1.5</td>
			<td>(any)</td>
			<td>-</td>
			<td>To use as &#8220;slides&#8221;</td>
		</tr>
		<tr>
			<td>DAPI (4&#8217;,6-Diamidino-2- Phenylindole Dihydrochloride)</td>
			<td>Life Technologies</td>
			<td>D3571</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr>
			<td>Drishti software</td>
			<td>(open source)</td>
			<td>-</td>
			<td>Free software tool</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>ED2SS</td>
			<td>For demineralization</td>
		</tr>
		<tr>
			<td>Fiji/ImageJ software</td>
			<td>(open source)</td>
			<td>-</td>
			<td>Free software tool</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>NZYtech</td>
			<td>MB01401</td>
			<td>For immunofluorescence</td>
		</tr>
		<tr>
			<td>Huygens software</td>
			<td>Scientific Volume Imaging</td>
			<td>-</td>
			<td>Commerial software tool</td>
		</tr>
		<tr>
			<td>HyClone defined fetal bovine serum</td>
			<td>GE Healthcare</td>
			<td>#HYCLSH30070.03</td>
			<td>For live imaging</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution 30 %</td>
			<td>Milipore</td>
			<td>1085971000</td>
			<td>For clearing</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane / Oxford instruments</td>
			<td>-</td>
			<td>Commerial software tool</td>
		</tr>
		<tr>
			<td>iSpacers</td>
			<td>SunJin Lab</td>
			<td>(varies)</td>
			<td>Use as spacers for preparations</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>#25030&#8211;024</td>
			<td>For live imaging medium</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>Gibco</td>
			<td>11054020</td>
			<td>For live imaging medium</td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>Sigma</td>
			<td>M7167</td>
			<td>To dissect embryos</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>VWRC20847.307</td>
			<td>For dehydration and rehydration steps</td>
		</tr>
		<tr>
			<td>Methyl salicylate</td>
			<td>Sigma</td>
			<td>M6752</td>
			<td>Used to clear embryos</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>Used in solution to fix embryos</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>#P0781</td>
			<td>For live imaging medium</td>
		</tr>
		<tr>
			<td>PBS (Phosphate-buffered saline solution)</td>
			<td>Biowest</td>
			<td>L0615-500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>RapiClear</td>
			<td>SunJin Laboratory</td>
			<td>RapiClear 1.52</td>
			<td>Used to clear embryos</td>
		</tr>
		<tr>
			<td>Secure-Sea hybridization chambers</td>
			<td>Sigma</td>
			<td>C5474</td>
			<td>Use as spacers for preparations</td>
		</tr>
		<tr>
			<td>simLab software</td>
			<td>SimLab soft</td>
			<td>-</td>
			<td>Commerial software tool</td>
		</tr>
		<tr>
			<td>Slide, depression concave glass - 75x25 mm</td>
			<td>(any)</td>
			<td>-</td>
			<td>To mount thick embryos.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>For immunofluorescence</td>
		</tr>
	</tbody>
</table>

three and four-dimensional visualization and analysis approaches to study vertebrate axial elongation and segmentation

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.

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 &#215; 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&#160;et al. (2013)28.
Live imaging (4D analysis)<b...

Watch this Scientific Journal Video about Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation at JoVE.com

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

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 ...

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