Bolkhovitinov L et al. (2022), Tissue Rotation of the Xenopus Anterior-Posteri...

XB-ART-59357

J Dev Biol 2022 Sep 10;103:. doi: 10.3390/jdb10030038.

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Tissue Rotation of the Xenopus Anterior-Posterior Neural Axis Reveals Profound but Transient Plasticity at the Mid-Gastrula Stage.

Bolkhovitinov L , Weselman BT , Shaw GA , Dong C , Giribhattanavar J , Saha MS .

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The establishment of anterior-posterior (AP) regional identity is an essential step in the appropriate development of the vertebrate central nervous system. An important aspect of AP neural axis formation is the inherent plasticity that allows developing cells to respond to and recover from the various perturbations that embryos continually face during the course of development. While the mechanisms governing the regionalization of the nervous system have been extensively studied, relatively less is known about the nature and limits of early neural plasticity of the anterior-posterior neural axis. This study aims to characterize the degree of neural axis plasticity in Xenopus laevis by investigating the response of embryos to a 180-degree rotation of their AP neural axis during gastrula stages by assessing the expression of regional marker genes using in situ hybridization. Our results reveal the presence of a narrow window of time between the mid- and late gastrula stage, during which embryos are able undergo significant recovery following a 180-degree rotation of their neural axis and eventually express appropriate regional marker genes including Otx, Engrailed, and Krox. By the late gastrula stage, embryos show misregulation of regional marker genes following neural axis rotation, suggesting that this profound axial plasticity is a transient phenomenon that is lost by late gastrula stages.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: egr2 en2 muc2 otx2
GO keywords: central nervous system development

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	Figure 1. Schematic of experimental design for experiments. Fluorescein-injected donor embryo is on the left and uninjected host embryo is on the right. Dorsal view, with anterior up and posterior down. The precise tissue removed was determined from preliminary fate mapping experiments and published work [54].
	Figure 2. Examples of normal versus abnormal embryos. Significant deviations from embryos shown in Nieuwkoop and Faber [53] were regarded as abnormal. Scale bars = 1 mm.
	Figure 3. Images showing criteria for discerning between co-localization of gene expression with integrated transplant tissue and endogenous expression. Light blue coloring indicates transplanted tissue and purple coloring indicates Otx2 expression via in situ hybridization. These specific examples are sections from the mid-gastrula sham surgery showing Otx2 expression (left) and the late gastrula sham surgery showing Otx2 expression (right).
	Figure 4. Morphology of embryos with transplanted tissues at late neurula (St. 18) (A) and hatching stages (St. 30) (B).
	Figure 5. Representative examples of expression of regional marker genes in transplants and sibling controls at late neurula stage (St. 18). With a lateral view with dorsal facing up for XCG-1 and Otx2, and dorsal view for En-2 and Krox20, anterior is to the right in all images. Purple stain marked with a solid arrowhead indicates gene expression and aqua blue stain was used to indicate transplant incorporation. Scale bar represents 1 mm.
	Figure 6. Representative histology for transplants at late neurula stage with sibling control comparisons. Dorsal is facing up. The four regional marker genes XCG-1, Otx2, En-2, and Krox20 are expressed at the cement gland, eye and forebrain, midbrain, and rhombomeres 3 and 5 of the hindbrain, respectively. Purple stain marked with a solid arrowhead indicates gene expression colocalized with transplant tissue, purple stain marked with an empty arrowhead indicates endogenous gene expression, and blue stain indicates transplant incorporation. Late gastrula rotated embryos show decreased level and more restricted area of marker gene expression at correct locations compared with mid-gastrula sham and rotated embryos. Scale bar represents 250 µm.
	Figure 7. Histology scores for transplants at late neurula stage. Histological sections were scored for expression levels of regional marker genes, both correct “New On” expression (A) and ectopic “Old Off” expression (B). Horizontal black lines represent the mean, and error bars represent the SEM. ** p < 0.01. N for each gene and condition is indicated along the x-axis in gray. Late gastrula rotated embryos have significantly lower levels of correct marker gene expression than mid-gastrula sham embryos (p = 0.0077) and mid-gastrula rotated embryos (p = 0.0018).
	Figure 8. Representative examples of expression of regional marker genes in transplants and sibling controls at hatching stage. Lateral view of all embryos with dorsal facing up; anterior is to the right. Purple stain marked with a solid arrowhead indicates gene expression and blue stain indicates transplant incorporation. Scale bar represents 1 mm.
	Figure 9. Representative histology for transplants at hatching stage, with sibling control comparisons. Dorsal is facing up. Purple stain marked with a solid arrowhead indicates gene expression colocalized with transplant tissue, purple stain marked with an empty arrowhead indicates endogenous gene expression, and blue stain indicates transplant incorporation. Late gastrula rotated embryos show decreased levels of and more restricted area of marker gene expression at correct places compared with mid-gastrula sham and rotated embryos. Late gastrula rotated embryos also present more ectopic gene expression than mid-gastrula rotated embryos. Scale bar represents 250 µm.
	Figure 10. Histology scores for transplants at hatching stage. Histological sections were scored for expression levels of regional marker genes, both correct “New On” expression (A) and ectopic “Old Off” expression (B). Horizontal black lines represent the mean, and error bars represent the SEM. ** p < 0.01. N for each gene and condition is indicated along the x-axis in gray. Late gastrula rotated embryos have significantly less appropriate marker gene expression than mid-gastrula sham (p = 0.0064) and Rotated embryos (p = 0.0012). Late gastrula rotated embryos also have significantly more ectopic marker gene expression (p = 0.0022) than mid-gastrula rotated embryos.
	Figure 11. Representative examples of expression of regional marker genes in (A) sibling control embryos, (B) mid-gastrula rotated transplants, and (C) late gastrula rotated embryos at stages 14 to 16. Dorsal view for all embryos, anterior is to the right. Purple stain marked with a solid arrowhead indicates gene expression and blue stain indicates transplant incorporation. Scale bar represents 250 µm.
	Figure 12. Representative histology images for sibling control embryos (A), mid-gastrula rotated transplants (B), and late gastrula rotated transplants (C) at stages 14–16. Dorsal is facing up. Purple stain marked with a solid arrowhead indicates gene expression colocalized with transplant tissue, purple stain marked with an empty arrowhead indicates endogenous gene expression, and blue stain indicates transplant incorporation. As embryos develop from stage 14–16, sections showed progressive activation of correct marker gene expression and repression of ectopic marker gene expression. Scale bar represents 250 µm.
	Figure 13. Histology scores for mid- and late gastrula rotated transplants at stage 14 to 16. Histological sections were scored for expression levels of regional marker genes, both correct “New On” expression (A) and ectopic “Old Off” expression (B). Horizontal black lines represent the mean, and error bars represent the SEM. * p < 0.05, * p < 0.001, ** p < 0.0001. n = 3 for each stage and gene. For mid-gastrula rotated embryos, stage 15 embryos have significantly higher levels of correct marker gene expression than stage 14 embryos (p = 0.0100), and stage 16 embryos have significantly higher levels of expression than both stage 14 (p < 0.0001) and stage 15 (p = 0.0007) embryos. Stage 16 mid-gastrula rotated embryos also have significantly higher levels of correct marker gene expression than stage 16 late gastrula rotated embryos (p < 0.0001).

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