Suzuki S et al. (2022), Xenopus laevis il11ra.L is an experimentally pr...

XB-ART-58868

Sci Rep 2022 Feb 03;121:1903. doi: 10.1038/s41598-022-05954-w.

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Xenopus laevis il11ra.L is an experimentally proven interleukin-11 receptor component that is required for tadpole tail regeneration.

Suzuki S , Sasaki K , Fukazawa T , Kubo T .

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Xenopus laevis tadpoles possess high regenerative ability and can regenerate functional tails after amputation. An early event in regeneration is the induction of undifferentiated cells that form the regenerated tail. We previously reported that interleukin-11 (il11) is upregulated immediately after tail amputation to induce undifferentiated cells of different cell lineages, indicating a key role of il11 in initiating tail regeneration. As Il11 is a secretory factor, Il11 receptor-expressing cells are thought to mediate its function. X. laevis has a gene annotated as interleukin 11 receptor subunit alpha on chromosome 1L (il11ra.L), a putative subunit of the Il11 receptor complex, but its function has not been investigated. Here, we show that nuclear localization of phosphorylated Stat3 induced by Il11 is abolished in il11ra.L knocked-out culture cells, strongly suggesting that il11ra.L encodes an Il11 receptor component. Moreover, knockdown of il11ra.L impaired tadpole tail regeneration, suggesting its indispensable role in tail regeneration. We also provide a model showing that Il11 functions via il11ra.L-expressing cells in a non-cell autonomous manner. These results highlight the importance of il11ra.L-expressing cells in tail regeneration.

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Species referenced: Xenopus laevis
Genes referenced: dcbld2 eef1a2 fgfrl1 hpse il11ra il6st jak1 jak2 lif lifr pam stat3.2 wnt5a
GO keywords: interleukin-11 receptor activity [+]
???displayArticle.antibodies??? pStat3 (Tyr705) Ab1
gRNAs referenced: il11ra gRNA1 il11ra gRNA2

Phenotypes: Xla Wt + il11ra CRISPR (Fig. 2 A r1c3, BCDEFG)

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	Figure 1. X. laevis Il11ra.L functions as an Il11 receptor component. (A) Gene expression involved in Il11 signaling in the XTC-YF cell line. (BD) Oocyte culture supernatants were subjected to SDS-PAGE, followed by (B) CBB staining and (C) Western blotting using anti-FLAG antibody and (D) mouse IgG2b isotype control. Size of the synthesized proteins estimated by their amino acid sequences was: Il11, 22.2 kDa; Lif, 25.5 kDa; and GST, 28.4 kDa. Arrowheads in (B) indicate the synthesized proteins. The 66.4-kDa bands (asterisk) in (B) the CBB staining represent BSA contained in the culture medium. (EQ) Detection of XTC-YF P-Stat3 nuclear translocation by immunostaining. (Top) P-Stat3 signal is shown in red. (Middle) Nuclei were stained with DAPI. (Bottom) Merged images of P-Stat3 and DAPI staining. Data shown are representative of 3 independent experiments (except the experimental condition of Il11 50 ng/mL; n=2). Scale bars: 50 m. Raw images of the gel and blots are shown in Fig. S8.
	Figure 2. il11ra.L is required for X. laevis tadpole tail regeneration. (A) Typical examples of regenerated tails at 7 dpa of tadpoles injected with either (left) cas9 mRNA only, (center) cas9 mRNA and guide RNA #1, or (right) cas9 mRNA, and guide RNA #1 and #2. Dashed lines indicate the amputation site. Scale bars: 500 m. (B) Regeneration outcomes of il11ra.L KD tadpoles. Tadpoles at 7 dpa were classified into 4 groups depending on their tail regeneration: Excellent, completely regenerated with all tissues; Good, with all tissues but short or bent tail; Partial, with tissue(s) defect; None, no regeneration. Numbers shown in the bar chart are numbers of tadpoles classified into each group. Statistical significance was assessed by Fishers exact test with the BenjaminiHochberg method, P<0.05, P<0.01, P<0.001. (CJ) Measurement of regenerated tails. (C) Regeneration outcomes of the KD experiment used for measurement. The same classification criteria as in (B) were used. cas9 group, n=25; #1 KD group, n=32. P<0.001 by Fishers exact test. (D) Schematic figure of the measurement. (E) Length of the regenerated tails. Bars indicate means. P<11010 by Welch's t-test. (F) Axis angle of the regenerated tails. The dispersion in the 2 groups was compared by F-test, P<11010. (G) Combined plot of length and axis angle of the regenerated tails. (HJ) Measured area of (H) whole regenerated tail, (I) muscle and (J) notochord in the regenerated tail. Bars indicate means. *P<1109 by Welch's t-test.
	Figure 3. il11ra.L is widely and constitutively expressed in the intact tadpole and regenerating tail. (A) In situ hybridization of il11ra.L on a section of intact tadpole (upper) with an antisense probe, and (lower) with a sense probe. Anterior is left and dorsal is up. Scale bars: 500 m. n=4. (BD) In situ hybridization of il11ra.L in a section of a regenerating tail. Anterior is left and dorsal is up. Arrowheads indicate notable signals. Scale bars: 500 m. High magnification images of boxed areas in (C) and (D) are shown below. Scale bars: 100 m. 1 dpa, n=5; 3 dpa, n=4; 5 dpa, n=5.
	Figure 4. il11ra.L KO cells can contribute to form the regenerated tail. Estimated mutation ratio at the guide RNA #1 target site of the developmental and regenerated tail of each individual is shown. Linked points represent the same individual. n=24. P=0.44, paired t-test. Representative result of 2 experiments is shown. The result of the second experiment is shown in Fig. S7.
	Supplementary Fig. S1 Expression of Il11 signaling molecules in XTC-YF culture cell line and establishment of XTC-YF il11ra.L KO cell lines. (A) Expression levels of genes related to Il11 signaling measured by quantitative RT-PCR using 3 lots of RNA extracted from XTC-YF. Relative expression levels of the genes normalized by those of ef1a are shown in logarithmic scale. Bars indicate the mean value. (B and C) Genomic il11ra.L sequences of established XTC-YF il11ra.L KO cell lines. (B) KO1 and KO2 were established with guide RNA #1, (C) KO3 and KO4 were established with guide RNA #2. PAM, protospacer adjacent motif.
	Supplementary Fig. S2 Construction of mRNAs of (left) N-terminal 3×flag-tagged il11.L, (center) N-terminal 3×flag-tagged gst, and (right) C-terminal flag-tagged lif.L.
	Supplementary Fig. S3 Heteroduplex mobility assay (HMA) for detection of gene editing. (A) Schematic figure of HMA. (B and C) HMA on amputated tails. Genomic loci including the target site of (B) guide RNA #1 or (C) #2 were amplified by PCR. Smeared bands representing multiple heteroduplexes are shown from the PCR products derived from embryos injected with guide RNA and cas9 mRNA (KD), whereas single bands are shown from control embryos injected with only cas9 mRNA (cas9). Numbers represent the serial numbers of samples. M, Marker lane. Full-length gel images are presented in Fig. S9.
	Supplementary Fig. S4 Expression of genes that are significantly upregulated in cas9 24 hpa tail stumps compared with cas9 0 hpa tail, and 0 and 24 hpa il11ra.L KD tail stumps. TPM, transcriptions per million.
	Supplementary Fig. S5 il11ra.L expression in single cell RNA-seq of regenerating tails reported by Aztekin et al.1 is visualized with the web platform (Xenopus tail regeneration at single cell resolution; https://marionilab.cruk.cam.ac.uk/XenopusRegeneration/).
	Supplementary Fig. S6 Schematic of the putative mechanism of Il11 function. (A) Putative models of the mechanism of Il11 function. Direct model; Il11 directly activates stem cells or precursors of each tissue in the tail. Indirect model; Il11 triggers downstream events that activate stem cells or precursors of each tissue in the tail. Il11R, Il11 receptor complex. (B) Experimental procedure. (C) Putative behaviors of il11ra.L KO cells in both models and results of the mutation ratio comparison.
	Supplementary Fig. S7 Second result of the mutation ratio estimation of developmental and regenerated tails. n=16. P=0.79, paired t-test.

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