No abstract

Zebrafish embryos can attain a stable cryogenic state by microinjection of cryoprotectants followed by rapid cooling, but the massive size of the embryo has consistently led to failure during the convective warming process. Here we address this zebrafish cryopreservation problem by using gold nanorods (GNRs) to assist in the warming process. Specifically, we microinjected the cryoprotectant propylene glycol into zebrafish embryos along with GNRs, and the samples were cooled at a rate of 90 000 °C/min in liquid nitrogen. We demonstrated the ability to unfreeze the zebrafish rapidly (1.4 × 10 °C/min) by irradiating the sample with a 1064 nm laser pulse for 1 ms due to the excitation of GNRs. This rapid warming process led to the outrunning of ice formation, which can damage the embryos. The results from 14 trials (n = 223) demonstrated viable embryos with consistent structure at 1 h (31%) and continuing development at 3 h (17%) and movement at 24 h (10%) postwarming. This compares starkly with 0% viability, structure, or movement at all time points in convectively warmed controls (n = 50, p < 0.001, ANOVA). Our nanoparticle-based warming process could be applied to the storage of fish, and with proper modification, can potentially be used for other vertebrate embryos.

Neurogenesis of the developing embryonic retina is described for the African cichlid fish, Haplochromis burtoni, from 4 days post fertilization until all cell phenotypes are generated (day 7). Cell addition and differentiation both begin at the same absolute location which later becomes the central retina. As observed in most other vertebrates, cones and ganglion cells differentiate first, followed by amacrine and bipolar cells. Rod photoreceptors, which are added late, differentiate last. Changes in retinal thickness, retinal stretching, cell size, and cell density were measured during development. From day 4 through 7, there is an increase in retinal thickness largely due to the expansion of the inner plexiform layer (IPL) and outer nuclear layer (ONL). The inner nuclear layer (INL) decreases in thickness and there is a transient decrease in the density of cells in the scleral portion of the INL. Cells increase in size in the ganglion cell layer (GCL) and the vitread INL, decrease in size in the sclerad INL, and remain the same in the ONL. Changes in the density of the cell layers were observed: the density of ONL cells increased, the density of GCL cells decreased, and INL cells increased then decreased. From day 4 to day 6, eye growth is entirely due to cell addition because no retinal stretching was observed in the ONL or the horizontal layer. During this same developmental period, the pattern and rate of neurogenesis were measured in the differentiated portion of the retina by means of 3H-thymidine labeling. A small number of cell divisions within the differentiated INL precede the onset of cell divisions in the ONL. The number of 3H-thymidine labeled cells within the INL increases at a low rate consistent with an asymmetric pattern of cell division characteristic of stem cells. In contrast, cell divisions in the ONL increase exponentially, consistent with a symmetric pattern of cell division characteristic of progenitor cells. Double-label experiments (3H-thymidine and a rod specific opsin antibody) show that some of the symmetrically dividing cells in the ONL express the rod specific opsin within 2 days, suggesting that these dividing cells are rod progenitors. Although we do not hae conclusive evidence, these developmental processes support the hypothesis that stem cells within the INL could be the source of rod precursors in the embryonic teleost retina.