No abstract

Determination of fate maps and cell lineage tracing have previously been carried out in the zebrafish embryo by following the progeny of individual cells injected with fluorescent dyes. We review the information obtained from these experiments and then present an approach to fate mapping and cell movement tracing, utilizing the activation of caged fluorescein-dextran. This method has several advantages over single-cell injections in that it is rapid, allows cells at all depths in the embryo to be marked, can be used to follow cells starting at any time during development, and allows an appreciation of the movements of cells located in a coherent group at the time of uncaging. We demonstrate that the approach is effective in providing additional and complementary information on prospective mesoderm and brain tissues studied previously. We also present, for the first time, a fate map of placodal tissues including the otic vesicle, lateral line, cranial ganglia, lens, and olfactory epithelium. The prospective placodal cells are oriented at the 50% epiboly stage on the ventral side of the embryo with anterior structures close to the animal pole, and posterior structures nearer to the germ ring.

ST and BPTB autografts were able to reproduce the native size of the ACL mid-substance cross-sectional area. The ST-G graft was significantly larger than the ACL cross-sectional area. For clinical relevance, ST and BPTB grafts are recommended in order to reproduce the native size of the ACL in anatomical ACL reconstruction with autograft.

S U M M A R YFading is one of the major obstacles to reliable observation in fluorescence microscopy. Using a confocal laser scanning microscope (CLSM) coupled to a computer, we quantitatively measured fading of fluorescence to formulate an equation, evaluated the anti-fading ability of several anti-fading media, and restored the faded images to the original level according to this equation. NIH 3T3 cells were stained with fluorescein isothiocyanate (FITC)-phalloidin, mounted with several commercial and homemade anti-fade media, and observed with CLSM under repeated illumination. With any mounting medium, attenuation of fluorescence intensity at a certain pixel occurred stepwise and the decrease was proportional to the intensity of the previous scan. From these results, we formulated an equation that has three coefficients: anti-fading factor ( A ), indicating the ability to retard fading; fluorescent intensity at the first scan ( EM 1 ); and background fluorescence ( B ). The fluorescent intensity at a certain point following n th scan is given as EM n ϭ EM 1 • A (n Ϫ 1) . This equation was available for restoring faded images to their original states, even after the image had faded to only 60% of its original intensity.