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.

The authors declare no conflicts of interest.

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><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 &amp;nbsp; 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 &amp;ldquo;slides&amp;rdquo;</td></tr><tr><td>DAPI (4&amp;rsquo;,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&amp;ndash;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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Developmental Biology

3.6K Views.  Instituto Gulbenkian de Ciência. De organogenesestadia in de muizenembryo's blijven slecht bestudeerd, deels als gevolg van de moeilijke visualisatie van de embryocomplexstructuren. Dit protocol beschrijft technieken die 3D- en 4D-visualisatie en analyse van muizenembryo's mogelijk maken tijdens axiale extensie en segmentatie. Het grote voordeel van deze techniek is dat het onderzoekers in staat stelt om de complexe structuren van het muizenembryo te bestuderen en te tonen als 3D-dynamische objecten.Dit protocol bevat...

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

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Neuroscience

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid fissure closure. Recently, the identification of individuals with coloboma in the superior aspect of the iris, retina, and lens led to the discovery of a novel structure, referred to as the superior fissure or superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during vertebrate eye development. Although this structure is conserved across mice, chick, fish, and newt, our current understanding of the SOS is limited. In order to elucidate factors that contribute to its formation and closure, it is imperative to be able to observe it and identify abnormalities, such as delay in the closure of the SOS. Here, we set out to create a standardized series of protocols that can be used to efficiently visualize the SOS by combining widely available microscopy techniques with common molecular biology techniques such as immunofluorescent staining and mRNA overexpression. While this set of protocols focuses on the ability to observe SOS closure delay, it is adaptable to the experimenter&#39;s needs and can be easily modified. Overall, we hope to create an approachable method through which our understanding of the SOS can be advanced to expand the current knowledge of vertebrate eye development.

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Here, we present a standardized series of protocols to observe the superior ocular sulcus, a recently-identified, evolutionarily-conserved structure in the vertebrate eye. Using zebrafish larvae, we demonstrate techniques necessary to identify factors that contribute to the formation and closure of the superior ocular sulcus.

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from ...

Preparation of Single-Somite Explants from Zebrafish Embryos

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular prepatterns determine the initial length-scale of somites, mechanical processes have been implicated in setting their final size and shape. To better understand the intrinsic material properties of somites, a method is developed to culture single-somite explant from zebrafish embryos. Single somites are isolated by first removing the skin of embryos, followed by yolk removal and sequential excision of neighboring tissues. Using transgenic embryos, the distribution of various sub-cellular structures can be observed by fluorescent time-lapse microscopy. Dynamics of explanted somites can be followed for several hours, thus providing an experimental framework for studying tissue-scale shape changes at single-cell resolution. This approach enables direct mechanical manipulation of somites, allowing for dissection of the material properties of the tissue. Finally, the technique outlined here can be readily extended for explanting other tissues such as the notochord, neural plate, and lateral plate mesoderm.

We present a protocol for isolating single somites from zebrafish embryos, the dynamics of which can be followed in culture for several hours by fluorescence time-lapse microscopy, thus providing a methodology to quantify tissue-scale shape changes at single-cell resolution.

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Biology

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

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

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