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Graphical abstract |
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Figure 1. Hemimosaic genetic manipulation through blastomere injection
(A) Injecting mRNA or morpholino oligonucleotide into one blastomere of two-cell stage tadpole embryos results in mosaic expression of protein or morpholino restricted to one lateral half of the body.
(B) Schematic of the tadpole retinotectal system. The tadpole tectum can be visually separated into two regions: the cell body layer mainly consisting of somata of tectal cells, and the neuropil layer mainly consisting of the dendrites of tectal neurons and the axon terminals of RGCs projecting in from the contralateral eye. Due to the crossing over of RGC axons, tadpoles with hemimosaic expression of fluorescent protein or morpholino will display fluorescent protein expression or morpholino knockdown in RGC axon terminals in one tectal hemisphere, and in tectal neurons in the opposite hemisphere. |
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Figure 2. 3D-printed injection chamber placed inside a 60 cm diameter Petri dish
The outer diameter (OD), inner diameter (ID) and height (H) of the injection chamber is 5 cm, 4 cm and 1.2 cm, respectively. The grid spacing of the bottom mesh is 1.2 mm. |
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Figure 3. Marking and calibrating a glass pipette for blastomere injection
(A) Draw 1 mm spaced marks on the barrel of the pipette with a fine-tip permanent marker to measure fluid volume.
(B) Front load the pipette with RNase-free distilled water at least up to the 3 mm mark for calibration.
(C) Closeup image of the tip of a calibrated pipette. |
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Figure 4. Priming injection for female frog
(A) Restrain the frog with an aquarium fish net, and deliver priming hormone through subcutaneous injection into the dorsal lymph sac.
(B) Diagram of injection locations in the dorsal side of the frog. Dotted blue line shows the approximate caudal limit of the dorsal lymph sac. Blue shaded areas are ideal locations for injections. Insert the syringe needle at a shallow angle, in the direction indicated by the black arrow. |
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Figure 5. Extraction of testes from male frog
(A) Testes can be found attached to fat tissue located in the abdomen of the frog.
(B) Once a testis is located, carefully cut it out with a piece of fat tissue attached, then rinse and store the extracted testis in chilled 1× MBSH. |
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Figure 6. Restraining and squeezing a female frog to collect eggs for in vitro fertilization
(A) Hold the frog with your dominant hand, using your other hand to help support the frog’s belly. Spread the frog’s hind legs with your index and middle finger to expose the cloaca, and massage the frog’s back and belly from the sacrum towards the cloaca to facilitate egg release.
(B) Alternatively, you can also spread the frog’s hind legs using your index fingers from both hands, which will give better access to the frog’s back for massaging.
(C) Healthy eggs should naturally form clumps and stick to the bottom of the Petri dish, each egg surrounded in jelly coat and well separated from each other. |
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Figure 7. In vitro fertilization
(A) After mincing a piece of testis and mixing it with the eggs, adding 0.1× MBSH will activate the sperm and initiate fertilization. Eggs are spread out into a single layer at the bottom of the dish.
(B) At 22°C ambient temperature, the first cell division will start at 75–90 min post-fertilization. Healthy eggs will start to form cleavage, while unhealthy or unfertilized eggs will not divide, and will appear bloated, deflated or start to disintegrate. |
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Figure 8. Blastomere injection
(A) Equipment setup for blastomere injection. ①Microinjector ②Dissecting scope ③Pantographic micromanipulator ④Injection chamber ⑤Blastomere injection medium (2% Ficoll in 1× MBSH) ⑥6-well plate to store injected embryos ⑦Transfer pipette to move embryos ⑧Calibrated injection pipettes.
(B) Close-up of injection pipette in position for blastomere injection into a 4-cell stage embryo (white arrow). A shallow dent can be seen forming on the surface of the blastomere where the pipette tip is pressing down. The dent will disappear once the pipette tip penetrates the cell membrane. Note that the embryo indicated with a red arrow has been damaged and contents can be seen leaking out. This embryo should be discarded. |
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Figure 9. Screening animals for hemimosaicism
(A) Stage 43 tadpoles anesthetized and lined up in a 60 mm Petri dish for screening.
(B) GCaMP6s fluorescence in two stage 43 tadpoles. Bottom tadpole is an example of a “good” animal, showing bright fluorescence in both the eye and spinal cord. Top tadpole does not show noticeable fluorescence in any parts other than the stomach. The top tadpole would be safe to discard if the other side of the animal does not show good fluorescence as well.
(C) Examples of GCaMP6s fluorescence in two more stage 43 tadpoles. Bottom tadpole shows good fluorescence in the eye and brain. In comparison, although parts of the head and the heart of the top tadpole are fluorescent, there is no noticeable fluorescence present in the central nervous system, making it less likely to be a good hemimosaic animal.
(D) Example of a custom screening chamber for stage 44 and older animals. The impression of a tadpole is carved into a block of Sylgard elastomer. The shape and size of the impression should be adjusted to snugly fit a tadpole and a drop of medium and allow a cover slip to be placed over the tadpole without compressing it.
(E) GCaMP6s fluorescence in a stage 48 tadpole, showing bilateral hemimosaic distribution of fluorescent protein. The left half of the animal shows bright green fluorescence in most regions, whereas the right half is only fluorescent in parts of the tectum.
(F) Close-up image of tadpole in (E), focusing on the tectum. GCaMP fluorescence can be seen in the entire left tectal hemisphere, as well as the neuropil region of the right tectal hemisphere (white arrow).
(G–I) Widefield images of a stage 45 GluN1 MO tadpole, adapted from Kesner et al.1 (G) Brightfield, (H) MO-lissamine fluorescence, (I) merged image. |
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Figure 10. Example results
(A) Two-photon optical section from a bilateral hemimosaic GCaMP6s animal. GCaMP florescence can be seen restricted to the left tectum and the neuropil region of the right tectum.
(B) Two-photon optical section from the left tectal hemisphere of a bilateral hemimosaic GCaMP6s animal with GCaMP florescence in postsynaptic tectal neurons on the left side. Yellow box marks a region of interest (ROI) in the neuropil area.
(C) Two-photon optical section from the right tectal hemisphere of the same animal as shown in (B), displaying GCaMP fluorescence in RGC axon terminals distributed in the neuropil area. Yellow box marks an ROI in the neuropil area.
(D) Example visual stimulus-evoked tectal cell calcium response trace, averaged over the ROI marked in (B). Grey triangles under the trace mark timepoints when a stimulus was shown to the right eye (a luminance step from white to various shades of grey, displayed on a small LED screen positioned next to the eye. The colour of the triangles represents the intensity of the luminance step – darker colours represent larger luminance steps).
(E) Example visual stimulus-evoked RGC axonal calcium response trace averaged over the ROI marked in (C). The visual stimulus is the same as in (D) but presented to the left eye.
(F) Cell body spontaneous calcium activity recorded from a bilateral hemimosaic GCaMP6s tadpole (different animal from B-E). (Left) Two-photon optical section from the left tectal hemisphere. Color overlays: ROIs of spontaneously active tectal neurons, identified by Suite2P software.23 Each ROI contains the cell body of a single tectal neuron that showed spontaneous activity. (Right) Spontaneous calcium activity traces extracted by Suite2P, each trace corresponding to a different ROI in the left panel.
(G) (Left) Same two-photon optical section as shown in (F), overlaid with ROIs generated from Suite2P based on responses to a repeated looming stimulus (dark circle on a bright background expanding from small to large, displayed on a small LED screen positioned next to the right eye). (Right) Stimulus-evoked response traces extracted by Suite2P, each trace corresponding to a different ROI in the left panel. Top orange ticks denote the onset times of stimulus presentations (once every 6 seconds).
(H) Confocal immunofluorescent section of a bilateral hemimosaic GluN1-MO animal, adapted from Kesner et al.1 Left: MO-lissamine fluorescence (magenta); middle: immunofluorescence staining for GluN1 (green); right: merged image.
(I) Widefield epifluorescence image of a GCaMP6s transgenic tadpole with hemimosaic expression of jRGECO from mRNA blastomere injection. Left: GCaMP fluorescence (green); middle: jRGECO fluorescence (red); right: merged image. |
