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BACKGROUND: In a high-throughput RNA sequencing analysis, comparing the transcriptional response between Xenopus laevis regenerative and non-regenerative stages to spinal cord injury, cornifelin was found among the most highly differentially expressed genes. Cornifelin is mainly expressed in stratified squamous epithelia, but its expression in the spinal cord and other central nervous structures has only been described during early development.
RESULTS: Here, we report cornifelin expression in the spinal cord, retina, and cornea throughout metamorphosis and in the spinal cord after injury. Cornifelin was detected in the grey matter and meninges of the spinal cord from NF-50 to NF-66, with decreased expression in the grey matter during metamorphosis. In the retina, cornifelin was expressed in the ganglion cell layer, the inner and outer nuclear layer, and the outer segment from NF-50 to NF-66. After spinal cord injury, we only observed cornifelin upregulation in NF-66 but no significant changes in NF-50. However, we found cornifelin positive cells in NF-50 meninges closing the spinal cord stumps 1 day after injury and delineating the borders of the spinal cord following the continuity of tissue regeneration in the following days after injury. Instead, in NF-66, cornifelin positive cells were distributed to the ventral side of the spinal cord at 6 days after injury, and at the injury gap at 10 days after injury.
CONCLUSIONS: Cornifelin is expressed in the Xenopus laevis spinal cord and eye during metamorphosis and plays a role in the meningeal response to spinal cord injury.
Fig. 1. Cornifelin expression in the spinal cord during metamorphosis.
A. Cornifelin mRNA levels detected by RT-qPCR were normalized against the housekeeping gene eef1a1. Total mRNA was extracted from spinal cords from stages 50, 54, 58, and 66. Data indicate the mean ± SEM, from four biological replicates. Inset: Comparison of cornifelin levels between stages 50 and 66. A double-tailed t-test between these stages indicated a statistical difference, *P < 0.05 (p = 0.0138). B. In situ hybridization in longitudinal sections from stage 50 animals with the anti-sense probes. C-D-E show magnifications of the boxed areas in B, showing the meninges, grey matter, and ependymal layer, respectively. Black arrows and arrowheads indicate neurons and meninges, respectively. F. In situ hybridization with the sense probe. G-J-L-O-R-U. Representative images of immunohistochemistry in transverse sections from NF-50 (G-J-L), and NF-66 animals (O-R-U). H–I–K-M-N-P-Q-S-T-V-W. Magnification of the boxed areas. Black arrows and arrowheads indicate neurons and meninges, respectively. Panels are representative of three biological replicates for each stage.
Fig. 2. Cornifelin expression in the eye during metamorphosis.
A. Cornifelin mRNA levels detected by RT-qPCR were normalized against the housekeeping gene eef1a1. Total mRNA was extracted from the spinal cord from stages 50, 54, 58, and 66. Data indicate the mean ± SEM, from three biological replicates. A double-tailed t-test between these stages found a statistical difference, *P < 0.05 (p = 0,0092). B–I. Immunofluorescence in Xenopus retina paraffin sections at the four different stages during metamorphosis, nuclei (cyan), and cornifelin (magenta) are shown. Magnifications of the insets are shown at the lower right of each image. Scale bar = 15 μm. J. In situ hybridization in longitudinal sections from NF-50 animals for the anti-sense probe. K-M. Immunohistochemistry in transversal sections from NF-50 (K) and NF-66 (L) retina, and NF-50 cornea (M). GCL: Ganglion cell layer. INL: inner nuclear layer. ONL: outer nuclear layer. OS: Outer segment. RPE: Retinal pigmented epithelium. IC: Inner cornea. Str: Stroma. OC: Outer cornea. The panels represent three biological replicates for each stage.
Fig. 3. Cornifelin expression in regenerative animals in response to spinal cord injury.
A. Cornifelin mRNA levels after spinal cord transection in NF-50. mRNA levels were first normalized against eef1a1 and then with their sham counterparts. Log2 was calculated and graphed. Data indicate the mean ± SEM, from three independent experiments. A one-sample T and Wilcoxon test against hypothetical value 0, were performed for statistical analysis. ns: not significant. B-E-H-K-N. Cornifelin immunohistochemistry on transversal sections after 6 and 12 h, and 1, 6, and 10 days post tran section. C-D-F-G-I-J-L-M-O-P. Magnification of the boxed areas. The black arrows and arrowheads indicate the expression of cornifelin positive cells in the meninges or the injury gap, respectively. The panels represent two or three biological replicates for each temporal point.
Fig. 4. Cornifelin expression in non-regenerative animals in response to spinal cord injury.
A. Cornifelin mRNA levels after spinal cord transection in NF-66. mRNA levels were first normalized against eef1a1 and then with their sham counterparts. Log2 was calculated and graphed. Data indicate the mean ± SEM, from three independent experiments. A one-sample T and Wilcoxon test against hypothetical value 0, were performed. *P < 0.05. B-D-F-H. Cornifelin immunohistochemistry on transversal sections after 2, 6, and 10 days post spinal cord transection injury. C-E-G-I. Magnification of the boxed areas. The black arrows and arrowheads indicate the expression of cornifelin positive cells in the meninges and motor neurons, respectively. The panels represent three biological replicates for each stage. J. Cornifelin immunohistochemistry on longitudinal sections 10 days after transection injury. K-L. Magnification of the boxed areas. Arrows show the meningeal cells. The panels represent three biological replicates.
Supplemental Figure 1: A. Homology sequence between cornifelin a and b (111 amino acids). Underline letters show the homologs amino acids, which reach 91% of identity. In yellow are shown the 15 cysteine residues of cornifelin. The blue square shows the sequence of the antibody design for Xenopus cornifelin. B. Western blot results from spinal cord (SC) and eye from NF-50 animals. a, b, c, and d depict the four bands that the cornifelin antibody detects with each band migrating differently. L: Protein ladder. C-D. Negative controls from NF-50 and NF-66 slides. E-F. Immunohistochemistry on transversal sections from NF-50 animals showing the neuronal marker NeuN on the grey matter of the spinal cord.
Supplemental Figure 2: Negative controls from eye slides. A. Sense control from NF-50 retina. B. Negative control from NF-50 retina slice without primary antibody. C-D. Negative control from retina slices of NF-50 and NF-66, respectively.
Supplemental Figure 3: Negative controls from spinal cord slides after spinal cord injury from NF-50 and NF-66 animals. A-B. Longitudinal sections from NF-50 animals 6 hours after injury without primary antibody. C-F. Transversal sections from NF-66 animals uncut and 2, 6, and 10 days after injury without primary antibody. NF-50 retina slice without primary antibody. G. Longitudinal section from NF-66 animals 10 days after injury without primary antibody.
