1/9/2024 0 Comments Movi pro brainboHyperspectral imaging is highly sought after in many fields including mineralogy and geology, environment and agriculture, astronomy and, importantly, biomedical imaging and biological fluorescence. (L-N) Knockdown of Vsx1 results in loss of most Vsx1+ BCs, but there is almost a complete compensation in BC numbers via an increase in Vsx2+ BCs. Most BCs are green, indicating that they are Vsx1+. (I-K) Cross sections of uninjected Vsx1:cytGFP Vsx2:cyt dsRed retinas. (G,H) Knockdown of Vsx1 alone leads to a small decrease in BCs because of compensation from Vsx2, whereas simultaneous knockdown of Vsx1 and Atoh7 leads to increases in ACs. (D,F) Atoh7/Ptf1a double morphants, showing a retina devoid of RGCs and HCs, with a significant reduction in the number of dAC and ACs (red only), a significant increase in the number of PRs (purple) and BCs (blue), and an increased amount of transfating. In these morphants, we see reductions in the generation of ACs, dACs and HCs, and an increase in the number of RGCs (red), BCs (blue) and PRs (purple). Ptf1a MO1 (E) is a splice-blocking morpholino, which consequently knocks down the translation of Ptf1a, but not ptf1a:cytGFP, so cells that would have been ACs and HCs can be seen in their transfated states. Ptf1a MO2 (C) is a more potent translation-blocking morpholino that interferes with both Ptf1a and Ptf1a:cytGFP expression. (C,E) Ptf1a MO1 and MO2 morphant retinas. (B) Atoh7 morphant retina in which there is a decrease in RGCs and a clear increase in the number of dACs and ACs (red and green cells), BCs (blue cells) and PRs (purple cells). Images show examples of loss-of-function analysis performed using SoFa. (K) A SoFa2 clone: PRs (cyan and red) BCs (cyan) AC/dAC/HC (red and yellow) RGCs (red) 3D projection, z stack=40 μm, step size=2 μm.įate switching. (J) SoFa1: PRs (purple) BCs (blue) AC/dAC/HC (red and green) RGCs (red) single confocal image. (J,K) Examples of SoFa clones at 76 hpf, when all cell types have differentiated and show their characteristic laminar position in the retina. Crx+ BCs did not yet arise through the duration of the movie: ∼12 h time-lapse movie extended focus confocal image, z stack=25.5 μm step size=1.5 μm. At the onset of differentiation, it is possible to identify the final fate of these cells before they reach their final position: RGCs (red cells expressing increasing levels of gapRFP, arrow), inhibitory neurons (red and green cells), PRs (purple cells). Some Atoh7+ cells differentiate into RGCs, whereas others divide and start expressing gapCFP (Crx+) or cytGFP (Ptf1a+). Imaging started at ∼45 hpf, corresponding to the time of the appearance of the first gapRFP-expressing cells (Atoh7+) and image stacks were collected every hour. We presume this clone arose from a single progenitor cell due to its final size. (A-I) Time lapse series of a developing SoFa1 clone in an unlabelled wild-type host. SoFa lines allow precise identification of cell fate within developing clones. The grey line marks the absolute minimum in each graph. (D-F) The Index of Interdigitation varies across the maturation wave for the basal side of the PR layer (D), the apical side of the IN layer (E) and the apical side of the RGC layer (F). (C) A schematic showing how the Index of Interdigitation is calculated. (B) Traces of the apical surface (white), the basal side of the photoreceptor (PR) layer (blue), the apical side of the inhibitory neuron (IN) layer (green) and apical side of the retinal ganglion cell (RGC) layer (red) from when they first appear continuous. The timescale, which references time in hours to the first appearance of the proto-IPL (0 h*), is based on matching the degree of lamination along the maturation wave to what we observe in our timelapse movies, where retinal lamination of a small section of the retina is followed over time (e.g. (A) Snapshot of a 52 hpf retina showing various degrees of interdigitation of the different cell types along the differentiation wave.
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