Choi B et al. (2022), Development and Degeneration of Retinal Ganglio...

XB-ART-59470

Mol Cells 2022 Nov 30;4511:846-854. doi: 10.14348/molcells.2022.0081.

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Development and Degeneration of Retinal Ganglion Cell Axons in Xenopus tropicalis.

Choi B , Kim H , Jang J , Park S , Jung H .

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Neurons make long-distance connections via their axons, and the accuracy and stability of these connections are crucial for brain function. Research using various animal models showed that the molecular and cellular mechanisms underlying the assembly and maintenance of neuronal circuitry are highly conserved in vertebrates. Therefore, to gain a deeper understanding of brain development and maintenance, an efficient vertebrate model is required, where the axons of a defined neuronal cell type can be genetically manipulated and selectively visualized in vivo. Placental mammals pose an experimental challenge, as time-consuming breeding of genetically modified animals is required due to their in utero development. Xenopus laevis, the most commonly used amphibian model, offers comparative advantages, since their embryos ex utero during which embryological manipulations can be performed. However, the tetraploidy of the X. laevis genome makes them not ideal for genetic studies. Here, we use Xenopus tropicalis, a diploid amphibian species, to visualize axonal pathfinding and degeneration of a single central nervous system neuronal cell type, the retinal ganglion cell (RGC). First, we show that RGC axons follow the developmental trajectory previously described in X. laevis with a slightly different timeline. Second, we demonstrate that co-electroporation of DNA and/or oligonucleotides enables the visualization of gene function-altered RGC axons in an intact brain. Finally, using this method, we show that the axon-autonomous, Sarm1-dependent axon destruction program operates in X. tropicalis. Taken together, the present study demonstrates that the visual system of X. tropicalis is a highly efficient model to identify new molecular mechanisms underlying axon guidance and survival.

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Species referenced: Xenopus tropicalis
Genes referenced: nmnat1 tec
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	Fig. 1. Fig. 1. Development of the retinotectal pathway in X. tropicalis. (A) Retinotectal pathway. (B) Coronal section of a stage 45 embryo. Anti-acetylated(ac)-Tubulin immunoreactivity mostly visualizes axons, with nuclear counterstaining (DAPI). ON, optic nerve; ONH, optic nerve head; RGC, retinal ganglion cell. Scale bar = 200 µm. (C) Photographs of embryos at key developmental stages. st, stage. Scale bar = 1 mm. (D) DiI-labeling of the retinotectal pathway originating from the right eye. (E) “Open book” imaging strategy. OC, optic chiasm; Tec, optic tectum. (F) DiI-labeled retinal axons in the contralateral (left) brain hemisphere viewed from the side. Arrows, the distal tip of the retinotectal pathway. Scale bar = 100 µm. (G) Summary of retinotectal pathway development in X. tropicalis.
	Fig. 2. Visualization of retinotectal pathway development by electroporation. (A) Electroporation of nucleotides into the right retinal primordium by targeted electroporation (left) using a custom-made Sylgard chamber (right). (B) Right eye-specific expression of mCherry encoded in the electroporated plasmid. Scale bar = 1 mm. (C and D) Coronal sections of electroporated right eyes. (E-H) “Open book” visualization of retinal axons growing in or terminating at the contralateral (left) brain hemisphere. st, stage; ONH, optic nerve head; OC, optic chiasm; ON, optic nerve; Tec, optic tectum; OT, optic tract; Telen, telencephalon; Dien, diencephalon; arrows, growth cones at the tip of growing axons; arrowhead, terminal branches of target-arrived axons; hollow arrow, contralateral optic track. Scale bars = 100 µm and 10 µm (inset). (I) Live imaging of electroporated retinal axons growing in an intact brain using “open brain” preparation. (J) Time-lapse photographs of a single retinal axon taken from shortly after midline crossing at the OC for approximately 40 h. Arrows, growth cone; hollow arrowhead, collapsed growth cone; filled arrowhead, extensively branched axon terminal. Scale bar = 50 µm.
	Fig. 3. Fig. 3. Assessment of co-electroporation efficiency. (A) mCherry DNA (axon tracer) was mixed with another DNA (EGFP) or fluorescein-tagged morpholino (MO) oligonucleotides, and electroporated into the right retinal primordium. st, stage. (B) Modeling of gain- or loss-of-function by co-electroporation of DNA or MO with a tracer. (C and D) Coronal section (C) of the retina after DNA-DNA co-electroporation and cell counting (D). Scale bar = 50 µm. (E and F) Coronal section (E) of the retina after DNA-MO co-electroporation and cell counting (F). Scale bar = 50 µm. White arrows, overlay; Green arrows, fluorescein-tagged antisense morpholino (MO); Red arrows, mCherry.
	Fig. 4. Fig. 4. Wallerian degeneration of retinal axons in vivo. (A) Strategy to visualize Wallerian degeneration of retinal axons. st, stage. (B) Open book imaging of electroporated, fully mature retinal axons. Telen, telencephalon; Dien, diencephalon; Tec, optic tectum; Rhomben, rhombencephalon; ON, optic nerve; OC, optic chiasm. Scale bar = 100 µm. (C-F) Time-course of Wallerian degeneration of retinal axons in vivo. Green and red arrows indicate healthy and degenerating axons, respectively. OT, optic tract. Scale bar = 100 µm. (G and H) Retinal axon before (G) and 48 h after axotomy (H and H'). Green and red arrows indicate healthy and degenerating axons, respectively. Note the beaded morphology of degenerating axons. Scale bars = 100 µm.
	Fig. 5. Axon-autonomous function of Wlds and Sarm1 in Wallerian degeneration. The same experiments were performed as described in Fig. 4 , except that Wlds-encoding plasmid or Sarm1 translation-blocking antisense morpholino (MO) oligonucleotide was co-electroporated with mCherry tracer. (A and B) Control axons without gene alteration. Axotomy induces near-synchronous degeneration of distal axons (red arrow) in all animals tested (0/26). A' and B' are enlarged image of the boxed area in A and B, respectively. Red and green arrows indicate degenerating and healthy axons, respectively. OC, optic chiasm; Tec, optic tectum. (C and D) Wlds protects severed axons from degeneration in all animals tested (green arrow, 16/16). P = 6.0 × 10–12, Fisher’s exact test versus control. (E and F) Sarm1 MO protects severed axons in 81% of animals tested (green arrow, 17/21). P = 2.2 × 10–9, Fisher’s exact test versus control. Scale bar = 100 µm.
	Still from Supplementary Movie S1. Time-lapse movie of a single retinal axon in Fig. 2J.

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Coleman, Programmed axon degeneration: from mouse to mechanism to medicine. 2020, Pubmed