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

The overall goal of the following experiment is to visualize chondrocyte inter collation and extension via zebra bow clonal cell analysis. This is achieved by generating a SOX 10 ERT two Cree zebra bow M line to achieve tissue specific recombination in the cranial facial cartilage and embryos for clonal analysis. Next, the embryos are pulsed with tamoxifen at the 10, so mite stage for three hours in order to induce the recombination process.

Then embryos are selected for the expression of CRE activity and strong red fluorescent or RFP expression, and the embryos are mounted for imaging of C craniofacial cartilage. The results show multispectral cell analysis of the cranio facial chondrocytes based on confocal imaging. The main advantage of this technique over existing methods like S in blue staining or transgenic lines such as so and GFP or SOX and kde, is that zebra bow allows us to specifically observe and follow clonal cell populations involved in an organ formation.

To prepare for embryo collection on day one, set up several male and female pairs of sox, 10 ERT two Cree zebra bow M transgenic zebra fish in divided mating tanks between five and 6:00 PM the following day after verifying under a light microscope that the embryos have reached the 10 cellulite stage under a fluorescent microscope with an RFP filter. Isolate the bright red embryos and transfer them to a new Petri dish with fresh E three medium to induce Cree recombination. Add 10 micromolar hydroxy tamoxifen solution to the Petri dish and incubate the embryos at 28.5 degrees Celsius for three hours.

Then decant the Tamoxifen solution into a biological waste container and use E three to wash the embryos several times after incubating the embryos overnight to the 28 to 29, so mite stage, transfer the embryos to E three PTU solution and return them to the incubator. To visualize the fully developed C craniofacial structures, select embryos at four DPF with an intense red signal and yellow Cree expression in the retina. After anesthetizing an embryo in Trica, according to the text protocol, transfer it into a Petri dish containing a drop of Methylcellulose on a clean confocal slide.

Place a drop of fresh Methylcellulose and use a micro loader tip to mount the embryo positioned dorsally while taking care not to directly touch it with a 25 by 25 cover slip. Cover the embryo and use the cover slip to manipulate the embryo if necessary. To image the embryo, use a 20 x dry lens objective.

To focus on the subject, press the L 100 button on the microscope, then select the confocal setup button. Click auto and select the CFP for channel one GFP for channel two and RFP or M cherry for channel three. Then close the window.

Turn on channels two and three before clicking the remove interlock button. Then click play to begin scanning. After adjusting the settings, capture the desired images and save in the confocal software format as well as in TIF format for image processing by channel.

To carry out CFP imaging turn off channels two and three and turn on channel one without changing the focus area. Readjust the scan settings before capturing images, save images as just described, and carry out image processing as described by pan and colleagues as shown here. Traditional cartilage visualization by whole Mount Sian blue staining has been invaluable in observing the developing mekel cartilage and commonly used to visualize final cellular organization to further analyze the developing chondrocytes over time lineage tracing.

Using SOX 10 kda transgenic lines has been used to study cell migration and convergence and extension in live embryos using live imaging of the zebra bow m transgenic line. To further study the organization of chondrocytes in the C craniofacial skeletal structure reveals the process of chondrocyte inter collation and elongation that mediates the extension and growth of meckel's cartilage. This figure shows the lower jaw from UI zebra bow M Sox 10 ERT two Cree transgenic embryos between 3D PF and five days post fertilization.

The lower jaw is best pictured ventrally to allow clear views of cell organization in the meles cartilage as shown here. The stably maintained and inherited colors allow easy lineage tracing and cranial neural crest cell fate mapping. The single blue cell appears to migrate and interpolate with its neighboring cells, then elongates along the anterior posterior axis to form a uniformly shaped chondra site, thus maintaining the global shape of the meles cartilage as it extends anteriorly.

This technique paves a way for researcher in the field of cat development to examine chondro in zebrafish skeletal formation.