The authors thank Robert Kelsh for kindly sharing the zebrafish sox10 promoter reagent.

1. Embryo Collection for Photoconversion
<ol>
	<li>Set up at least ten pairs of sox10: kaede transgenic zebrafish between 5 and 6pm in the evening.</li>
	<li>The next morning, pull dividers and collect eggs around noon. Transfer them to Petri dishes and put them into 28.5 &deg;C incubator.</li>
	<li>At around 24 hr post fertilization, clean the Petri dishes by removing dead embryos. Check the developmental stage of embryos 13 using light field microscopy and any fluorescent microscope with enhanced green fluorescent protein (EGFP) filter. Kaede will be visible through this filter.</li>
	<li>When embryos reach 60 hpf transfer five brightly fluorescent embryos into a separate Petri dish and dechorionate them if necessary.</li>
</ol>
2. Mounting of Embryo for Photoconversion
<ol>
	<li>Mount the embryo using a normal fluorescent microscope. Mounting of embryos on confocal microscope is extremely challenging.</li>
	<li>Use standard E3 water or prepare fresh E3 using the following recipe: 
	Add the following per one liter of distilled water dH2O add the following to make 1x E3L: 
	0.292 g 5.0 mM NaCl 
	0.013 g 0.17mM KCl 
	0.044 g 0.33mM CaCl2 (desiccant, 96%, A.C.S. reagent) 
	0.081 g .33mM MgSO4 (anhydrated) 
	Optional: add 200 &#956;l of 0.05% methylene blue to 1X E3 as a fungicide and stir. 
	Medium keeps at room temperature for 1 week.</li>
	<li>Use standard tricaine or prepare tricaine using following recipe: 
	For 5 L of 0.2% tricaine mix the following ingredients: 
	45 ml tris-Cl (1M pH9.5) 
	5 L H2O 
	10 g tricaine</li>
	<li>Prepare 'movie juice' (50 ml E3, 0.015% tricaine solution), by adding 3.75 ml 0.2% tricaine to 46.25 ml E3.</li>
	<li>Prepare agarose by mixing 46.25 ml E3 water with 46.35 mg low melting point (LMP) agarose in a 50 ml glass flask and dissolve solid particles by microwaving. Then add 3.73 ml 0.2% tricaine to agarose and put glass flask with agarose into 50 &deg;C water bath until usage.</li>
</ol>
3. Preparation and Mounting of Embryo
<ol>
	<li>Anesthetize embryos with E3/0.015% tricaine solution made in step 1.1.2 and transfer embryo to chamber slide.</li>
	<li>Soak up all excess E3 with paper tissue and the put agarose onto embryo.</li>
	<li>Position embryo perfectly dorsal with microloader tips. Make sure the whole embryo does not float on top of the agarose, but push the whole body down. Then pour E3/0.015% tricaine solution over agarose embedded embryo until chamber is fully filled with fluid.</li>
</ol>
4. Adjusting Software Settings on Confocal Microscope
<ol>
	<li>Use 20X air immersible objective (numerical aperture 0.75; pinhole size 20) for focusing the embryo. Then press the L100 light button on the microscope and select channels in software. Open the icons view/acquisition controls/A1settings and click the confocal setup button. Next click &#39;auto&#39; and select following channels before closing the window again: 
	Ch1: DAPI channel- 403.4 nm (wave lengths between 350-410 nm can be used) 
	Ch2: 488.0 nm 
	Ch3: 561.9 nm</li>
</ol>
5. Photoconversion
<ol>
	<li>Turn off Ch1 and 3 and turn on Ch2 before clicking the remove interlock button and scanning.</li>
	<li>Focus on cells of interest for photoconversion, which is best done by starting with 1frame/sec and high voltage (HV) 200 and then going up in the frames/sec and lowering the HV accordingly until reaching 1/32 frames/sec and HV around 100-120, depending on the background noise.</li>
	<li>When the embryo is in focus, look on the right edge of screen, grab the green rim and make it smaller so it fits the area of interest for photoconversion and right click with the mouse. Smaller is better because the photoconversion is irreversible.</li>
	<li>Turn frames/sec to 1 and HV to 200 and hit scan. A zoomed in image of the area of photoconversion is now visible.</li>
	<li>Photoconvert cells by turning on Ch1 (403.4 nm). Expose to the photoconverting laser anywhere from 5-60 seconds, depending on size of the region until green kaede as seen through the GFP channel is nearly absent.</li>
	<li>When the green kaede signal is nearly absent, turn Ch1 off. Turn Ch2 and 3 on. Then click right onto window on A1 scan area field and hit &#39;return to original size&#39; to check if desired cells were photoconverted.</li>
</ol>
6. Making a Z-stack
<ol>
	<li>Find the icon &#39;capture Z-series&#39; in the menu bar and set upper and lower limits of Z-stack before starting it. Set LUTs.</li>
</ol>
7. Software- Settings Time-lapse
<ol>
	<li>Go to A1Simple GUI box and tick following settings: 
	1/32 frames/sec 
	size 1024 
	average 4 or 8X depending on the number of Z-stacks that has to be taken in one loop</li>
	<li>Go to: view/ acquisition controls/ ND sequence acquisition and set time frame (8-30min), click continuous and click Z-stack. Set borders as tested before and start movie.</li>
	<li>Keep adding E3/0.015% tricaine solution chamber slide throughout the day. At night, fill up chamber completely. That will last until the next morning.</li>
	<li>The next morning, refill E3/0.015% tricaine solution and check if embryo is still alive. Loss of fluorescence indicates death. Continue refills and checks throughout the day.</li>
	<li>When the structure has finished forming, or the embryo has died, finish the movie.</li>
</ol>
8. Post-production Processing
<ol>
	<li>Click &#39;show maximum intensity projection&#39; on tool bar, then right click on the image and create new document. The video is now visible in the best possible quality. Go on to delete frames that are not needed and adjust LUTs. Finally save as .avi. This format will be fine for Windows. For Mac download software to convert to .mov or .wmv.</li>
</ol>

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	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+at+Birth+of+Cleft+Lip+With+or+Without+Cleft+Palate:+Data+From+the+International+Perinatal+Database+of+Typical+Oral+Clefts+(IPDTOC).">Prevalence at Birth of Cleft Lip With or Without Cleft Palate: Data From the International Perinatal Database of Typical Oral Clefts (IPDTOC).</a> Cleft Palate Craniofac. J. 48 (1), 66-81 (2011).</li><li>Dixon, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleft+lip+and+palate:+understanding+genetic+and+environmental+influences.">Cleft lip and palate: understanding genetic and environmental influences.</a> Nat. Rev. Genet. 12 (3), 167-178 (2011).</li><li>Mangold, E., Ludwig, K. U., Nothen, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakthroughs+in+the+genetics+of+orofacial+clefting.">Breakthroughs in the genetics of orofacial clefting.</a> Trends Mol. Med. 17 (12), 725-733 (2011).</li><li>Kimmel, C. B., Miller, C. T., Moens, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+and+morphogenesis+of+the+zebrafish+larval+head+skeleton.">Specification and morphogenesis of the zebrafish larval head skeleton.</a> Dev. Biol. 233 (2), 239-257 (2001).</li><li>Dougherty, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Fate+Map+of+First+Pharyngeal+Arch+Structures+in+the+sox10:+kaede+Zebrafish+Transgenic+Model.">Embryonic Fate Map of First Pharyngeal Arch Structures in the sox10: kaede Zebrafish Transgenic Model.</a> J. Craniofac. Surg. 23 (5), 1333-1337 (2012).</li><li>Trainor, P. A., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hox+genes,+neural+crest+cells+and+branchial+arch+patterning.">Hox genes, neural crest cells and branchial arch patterning.</a> Curr. Opin. Cell Biol. 13 (6), 698-705 (2001).</li><li>Schilling, T. F., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segment+and+cell+type+lineage+restrictions+during+pharyngeal+arch+development+in+the+zebrafish+embryo.">Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo.</a> Development. 120 (3), 483-494 (1994).</li><li>Swartz, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+a+palatogenic+gene+program+in+zebrafish.">Examination of a palatogenic gene program in zebrafish.</a> Dev. Dyn. 240 (9), 2204-2220 (2011).</li><li>McCollum, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+toxicity+screening+in+zebrafish.+Birth+Defects+Res.">Developmental toxicity screening in zebrafish. Birth Defects Res.</a> C. Embryo Today. 93 (2), 67-114 (2011).</li><li>Wada, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132 (17), 3977-3988 (2005).</li><li>Eberhart, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Hedgehog+signaling+from+neural+to+oral+epithelium+organizes+anterior+craniofacial+development.">Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development.</a> Development. 133 (6), 1069-1077 (2006).</li><li>Ando, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc. Natl. Acad. Sci. U S A. 99 (20), 12651-12656 (2002).</li><li>Kimmel, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203 (3), 253-310 (1993).</li><li>Kawakami, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish-secreted+matrix+protein+you/scube2+is+implicated+in+long-range+regulation+of+hedgehog+signaling.">The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling.</a> Curr. Biol. 15 (5), 480-488 (2005).</li><li>Lombardo, V. 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The authors declare that they have no competing financial interests.

Here a new method for visualization of craniofacial development in the zebrafish model is shown. The sox10: kaede transgenic zebrafish line has been successfully used to describe the migratory pattern of CNCC in detail is used as a model organism 5.
Previous studies have used gross landmarks such as the eye to target cells and have relied on kaede mRNA injection, photoconversion assays or caged fluorescein dextran for photoconversion 10, 11, 14, 15 .The sox:10 kaede trans...

Orofacial clefts represent the most prevalent craniofacial deformity, with 1/700-1,000 deliveries affected 1. Disruption of early embryological craniofacial development can lead to formation of cleft lip and palate (CL/P). While causes for syndromic cleft have been largely shown, the genetic and epigenetic bases of nonsyndromic forms of orofacial clefting still need to be uncovered 2-4. In order to understand the etiology and pathogenesis of these malformations, it is necessary to elucidate the development of craniofacial structures on a cellular basis.
In all vertebrate species cranial neural crest cells (CNCC) migrate from the dorsal neural tube to populate the pharyngeal arches, which will contribute to formation of orofacial structures. Disruption of early embryological neural crest development can lead to formation of craniofacial malformations including CL/P 5-7.
In addition to structural similarities between zebrafish and mammalian craniofacial development (CNCCs reside in homologous regions), the gene regulatory network is highly conserved. It has also been shown that CNCCs develop in the same fashion between amniote species and zebrafish 8, making the zebrafish a powerful organism for the study of developmental and genetic basis of CL/P. It has many advantages, including small size, rapid and ex-utero embryonic development, and high breeding rates. Moreover, the embryo is optically transparent, making it amenable to observation of complex developmental events under the microscope 9. It is an ideal animal model for the study of migration and differentiation of cranial neural crest cells.
Expanding on previously published work 8, 10, 11, the migratory pattern of CNCC was described in detail using the sox10: kaede transgenic model 5. Kaede is a photo- convertible protein that turns from green to red after photo activation and makes it possible to trace CNCCs precisely. During this transformation the peptide backbone is cleaved, suggesting that the conversion is stable, meaning the cells can be tracked to their final destination 12. Transgenic lines labeled with kaede under transcriptional control of sox10 showed that the amniote palate and the ethmoid plate of zebrafish are formed homologously by fusion of bilateral maxillary prominences (MXP) with the frontonasal prominence (FNP) and that the Y shaped fusion seam is analogous between species.
Among other applications, the sox10: kaede transgenic zebrafish model was used to generate videos of zebrafish embryos at different developmental stages to show formation of normal and abnormal craniofacial structures. Photoconversion of specific groups of cells makes it possible to track their development. With this method an approach to create live imaging of developing craniofacial structures in zebrafish is introduced, making it easy to visually demonstrate this complex developmental process.
This protocol is aimed at sharing the experience of generating these videos using the normal development of the ethmoid plate in sox10: kaede transgenic zebrafish as an example. This protocol can further be applied to making time-lapse videos of any structure derived from cranial neural crest cells in zebrafish.

<table><tbody><tr><td>Sox10:kaede transgenic zebrafish line </td><td>MGH</td><td></td><td>Available via the Liao lab</td></tr><tr><td>Petri dishes 100x15 mm</td><td>BD Falcon</td><td>351029</td><td></td></tr><tr><td>Petri dishes 35 mmx10 mm</td><td>BD Falcon</td><td>351008</td><td></td></tr><tr><td>Ultrapure Low melting point (LMP) Agarose</td><td>Invitrogen</td><td>15517022</td><td></td></tr><tr><td>Lab Tek 2 Chamber SlideSystem </td><td>LabTek</td><td>154453</td><td></td></tr><tr><td>Microloaders 200/pk</td><td>Fisher</td><td>E5242956003</td><td></td></tr><tr><td>Nikon A1R Si Confocal Ti series</td><td>Nikon</td><td>No Catalog number</td><td></td></tr><tr><td>NIS Elements Software AR3.2 64-bit</td><td>Nikon</td><td>No Catalog number</td><td></td></tr></tbody></table>

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.

In the sox10: kaede transgenic line, migratory and post migratory CNCCs are labeled fluorescently green. The CNCC cells labeled with green fluorescent kaede recapitulate endogenous sox10 mRNA expression 5.
Among other applications, this animal model was used to better visualize the development of CNCC dependent craniofacial structures. Normal development of specific structures and also pathologic development of craniofacial malformations, especially cleft lip and palate have been ca...

Watch this Scientific Journal Video about Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy at JoVE.com

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

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, ...

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Research

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Biology

12.8K Views.  Massachusetts General Hospital. 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.

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

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. ...

Developmental Biology

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

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 ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, proliferation, and differentiation. Time-lapse imagining offers unique insight to the behavior of cells during eye development because it allows us to visualize oculogenesis in vivo. Zebrafish are an excellent model to visualize this process due to their highly conserved vertebrate eye and their ability to develop rapidly and externally while remaining optically transparent. Time-lapse imaging studies of zebrafish eye development are greatly facilitated by use of the transgenic zebrafish line Tg(rx3:GFP). In the developing forebrain, rx3:GFP expression marks the cells of the single eye field, and GFP continues to be expressed as the eye field evaginates to form an optic vesicle, which then invaginates to form an optic cup. High resolution time lapse imaging of rx3:GFP expression, therefore, allows us to track the eye primordium through time as it develops into the retina. Lightsheet microscopy is an ideal method to image ocular morphogenesis over time due to its ability to penetrate thicker samples for fluorescent imaging, minimize photobleaching and phototoxicity, and image at a high speed. Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software. The resulting dataset can provide valuable insights into the process of ocular morphogenesis, as well as perturbations to this process as a result of genetic mutation, exposure to pharmacological agents, or other experimental manipulations.

Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software.&#160;

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, ...

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 ...

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

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Chapters in this video

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel’s Cartilage

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

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

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy