Yasuoka Y .

             notochord development

Xtr Wt + chst1 MO (Fig. 3A r2c3 r2c3)
Xtr Wt + chst1 MO (Fig. 3B,C)
Xtr Wt + chst1 MO (Fig. S9 C c1r1)
Xtr Wt + chst1 MO (Fig. S9C c1r2)
Xtr Wt + chst1 MO (Fig. S9F c1r2)
Xtr Wt + chst3 CRISPR (Fig. 3 D)
Xtr Wt + chst3 MO (Fig. 3A r3c1 r3c2)
Xtr Wt + chst3 MO (Fig. 3A r3c3 r3c4)
Xtr Wt + chst3 MO (Fig. 3B,C)
Xtr Wt + chst3 MO (Fig. S9F c1r3)
Xtr Wt + chst6 CRISPR (Fig. 3. D)
Xtr Wt + chst6 MO (Fig. 3A r4c1 r4c2)
Xtr Wt + chst6 MO (Fig. 3A r4c3 r4c4)
Xtr Wt + chst6 MO (Fig. S9 F c1r3)
Xtr wt + chst1 CRISPR (Fig. 3D)

	FIGURE 1. Highly sulfated keratan sulfate (HSKS) is enriched in the notochord and otic vesicles of Xenopus embryos. (A) Molecular structure of HSKS and enzymes catalyzing its biosynthesis are schematically represented. Sulfation of D-GlcNAc is required for sulfation of D-Gal. (B–F) Immunostaining using 5D4 monoclonal antibody demonstrates that HSKS is specifically enriched in the notochord and otic vesicles in Xenopus tropicalis tadpoles. In late tadpole stage (F), notochord staining becomes weaker, possibly due to vacuole growth and cell death, which makes the extracellular space smaller. Tadpoles cleared in BB/BA solution (benzyl benzoate: benzyl alcohol = 2:1) are shown in lateral view. (C–E) A cross-section of a tadpole (st. 35/36) immunostained with 5D4 antibody revealed that HSKS is enriched inside otic vesicles and outside notochord, as designated by arrows in magnified images of an otic vesicle (D) and notochord (E). Br, brain; ov, otic vesicle; noto, notochord.
	FIGURE 2. Spatio-temporal expression patterns of HSKS catalytic genes are linked to HSKS synthesis during Xenopus development (A) Expression levels of HSKS biosynthetic genes along the developmental time-course of Xenopus embryos are visualized in Xenbase (https://www.xenbase.org/) using an available RNA-seq dataset (Owens et al., 2016). Most genes showed elevation of expression levels corresponding to enrichment of HSKS in the notochord and otic vesicles. (B) Spatial expression patterns of Xenopus HSKS biosynthetic genes are represented with whole mount in situ hybridization from neurula to tadpole stages (st. 15–42). To detect their expression in the notochord, some embryos were overstained, resulting in higher background, especially in the head region of tadpoles. Therefore, it is difficult to distinguish precise expression domains of genes strongly expressed in the brain such as chst1, chst2, and chst3. Arrowhead, notochord; open arrowhead, otic vesicles; cns, central nervous system; pnd, pronephric duct; pnt, pronephric tubules.
	FIGURE 3. Loss of function analysis revealed indispensable functions of Chst1, Chst3, and Chst5.1 for HSKS synthesis in Xenopus embryos. (A) Fluorescent immunostaining of HSKS of morphants at st. 35/36 and 42. Open arrowheads indicate lost or reduced enrichment of HSKS. See the text for a detailed explanation of phenotypes. (B) Smaller otic vesicles were observed in chst1 morphants at st. 45. White dashed lines indicate outlines of otic vesicles (C) Quantification of otic vesicle phenotypes. Box plots indicate length and width of otic vesicles on the left (L) or right (R) side in each sample. Each value (length of left vesicle, length of right vesicle, width of left vesicle, and width of right vesicle) was statistically analyzed with one-way ANOVA (p < 6.2 E−13, 9.9 E−11, 6.4 E−16, and 9.6 E−13, respectively), followed by Tukey’s honestly significant difference test with 95% confidence level (indicated with a, b, and c). The result demonstrated that otic vesicles of chst1 morphants are significantly smaller than those of chst3 morphants, control morphants, and uninjected controls (D) Genome editing experiments using the CRISPR-Cas9 system further demonstrated that chst1, chst3, and chst5.1 serve indispensable functions in HSKS in otic vesicles, notochord, and both, respectively (see Supplementary Figures S9–11 for more details). Numbers of embryos with observed phenotypes are indicated.
	FIGURE 4. Putative roles of HSKS in early Xenopus development.The contribution of HSKS to tissue hydration in Xenopus tadpoles is schematically represented. Accumulated HSKS in extra cellular matrix retains water, which may help otic vesicles and notochord vacuoles to swell. HSKS is enriched inside otic vesicles (Figure 1D), but outside of the notochord (Figure 1E), implying that aquaporin transports water from the extracellular space to intracellular vacuoles. Our results demonstrate that tissue-specifically expressed chst genes drive HSKS biosynthesis for normal development.
	FIGURE 5. A chst gene is expressed in amphioxus notochord, suggesting evolutionary conservation of HSKS functions in chordates. (A) Whole-mount in situ hybridization using amphioxus embryos showed that expression of a putative Chst gene LOC118425790 is restricted to the notochord and a part of the neural tube. (B) An evolutionary scenario related to Chst genes in chordates. In this scenario, Chst expression is an ancestral feature of the notochord, and in association with the acquisition of otic vesicles, chst1 and chst3 (and others, possibly) are subfunctionalized to catalyze biosynthesis of HSKS in various tissues.
	Figure S1. Genome browser representations around chst4 in Xenopus (A) In X. tropicalis v10, chst4 was annotated as LOC100496630. (B) In X. tropicalis v9.1, chst4 was annotated as chst5. (C) In X. laevis v9.2, chst4.L was annotated as chst5.L. (D) In X. laevis v9.2, chst4.S was annotated as chst5.S.
	Figure S1. Genome browser representations around chst4 in Xenopus continued (C) In X. laevis v9.2, chst4.L was annotated as chst5.L. (D) In X. laevis v9.2, chst4.S was annotated as chst5.S.
	Figure S2 Genome browser representations around chst5.1 in Xenopus In X. tropicalis v10, chst5.1 was annotated as chst6. In X. laevis v9.2, chst5.1.L was annotated as chst6.L. In X. laevis v9.2, chst5.1.S was annotated as chst6.S.
	Figure S3 Genome browser representations around chst5.2 in Xenopus In X. tropicalis v10, chst5.2 was not annotated. In X. tropicalis v9.1, chst5.2 was annotated as LOC100485834. In X. laevis v9.2, chst5.2.L was annotated as LOC108714029. In X. laevis v9.2, chst5.2.S was annotated as LOC108715076.
	Figure S5 Genome browser representations around chst16 in Xenopus In X. tropicalis v10, chst16 was annotated as LOC100485856. In X. laevis v9.2, chst16.L was annotated as LOC108710801. Genes in the vicinity of chst16.L (tmem263.L and cry1.L) showed strong expression in otic vesicles. An ohnolog of cry1.L, cry2.L, is located near chst1.L and is also expressed strongly in otic vesicles. Forming a genomic regulatory block with these genes, chst16 may be expressed in otic vesicles. In X. laevis v9.2, chst16.S was not annotated in the genome.
	Figure S4 Genome browser representations around chst4/5-like in Xenopus In X. tropicalis v10, chst4/5-like was annotated as chst4. In X. laevis v9.2, chst4/5-like.S was annotated as chst4.S. chst4/5-like.L was not found in the genome.
	Figure S7 Validation of translational block by chst1 and chst5.1 MOs Sequence alignments of chst1 wild-type sequence (WT) and chst1 mutated sequence (mt) with chst1 MO1 and chst1 MO2 sequence. The start codon (ATG) is underlined. Western blot of FLAG-tagged Chst1 protein produced by an in vitro translation system (Promega L2081). WT or mt constructs of chst1 were mixed with control or chst1 MOs to achieve an MO concentration of 100 μM. The result indicates that chst1 MOs inhibits translation of Chst1 in a sequence-specific manner. HSKS immunostaining of chst1 morphants demonstrated that both chst1 MO1 and chst1 MO2 inhibited HSKS synthesis in otic vesicles (open arrowheads), but not in the notochord. Numbers of embryos with observed phenotypes are indicated. (D,E) Translational block by chst5.1 MO1 was validated as well as that by chst1 MOs (A, B). A bent axis phenotype was observed in chst5.1 morphants. Numbers of embryos with observed phenotypes are indicated. All embryos injected with chst5.1 MO2 died during gastrulation, even when small amount (0.25 pmol) was injected, possibly due to its high cytotoxicity.
	Figure S8 Validation of splicing and translational block by chst3 MOs Schematic representation of PCR primers and chst3 MO on the exon-intron structure of chst3 gene. Blue and orange boxes indicate UTR and protein coding sequences. RT-PCR of chst3 mRNA with ex1-F and ex2-R primers using control morphants, chst3 morphants, and an uninjected control embryo (U). Equivalent sizes and amounts of PCR products were detected in all embryos, indicating that chst3 MO does not inhibit transcription and splicing of the first intron of chst3. RT-PCR of chst3 mRNA with ex1-F and ex3-R primers using control morphants, chst3 morphants, and an uninjected control embryo (U). 1-kb larger PCR products were detected from chst3 morphants than those from control samples, indicating that chst3 MO inhibits splicing of the second intron of chst3, leading to malfunction of Chst3. (D,E) Translational block by chst3 MO2 and chst3 MO3 was validated as well as that by chst1 MOs (Fig. S7A, B). HSKS immunostaining of chst3 morphants demonstrated that both chst3 MO1 and chst3 MO3 eliminated HSKS in the notochord (open arrowheads), but not in otic vesicles by the later stage (st42) although embryos injected with chst3 MO3 retained moderate HSKS (grey arrowhead) at the earlier stage (st35/36). Embryos injected with chst3 MO2 died during gastrulation, even when a small amount (0.5 pmol) was injected, possibly due to its high cytotoxicity. Embryos injected with chst3 MO3 at 1 pmol/embryo also showed severe developmental defects during organogenesis, whereas those at 0.5 pmol/embryo appeared to develop normally. The smaller injection amount of chst3 MO3 might have resulted in incomplete inhibition of HSKS synthesis. Numbers of embryos with observed phenotypes are indicated.
	Figure S9 Genome editing of the chst1 locus reproduced loss of HSKS phenotypes in Xenopus embryos. (A) The protein coding sequence of chst1 is represented together with its translated amino acid sequence. Forward (5’) and reverse (3’) primer sequences, which were used to amplify target DNA by PCR in the following assays, are highlighted with light blue. Target sequences of sgRNAs (chst1-sgRNA1-4) are highlighted with yellow and their corresponding PAM sequences are highlighted with green. Magenta and blue letters of the translated amino acid sequence indicate 5’-PSB and 3’-PB, respectively (see Figure S14). (B) An in vitro cleavage assay validated enzymatic activity of the Cas9-sgRNA complex to digest target DNA. Digested DNA fragments were detected as expected. C, control (w/o sgRNA); 1, chst1-sgRNA1; 2, chst1-sgRNA2; 1+2, chst1-sgRNA1+2; 3, chst1-sgRNA3; 4, chst1-sgRNA4; and M, 100 bp ladder. (C-E) T7E1 assays validated the genome editing efficiency in embryos injected with Cas9 protein preincubated with chst1-sgRNAs. Five embryos were examined separately in each experiment. PCR fragments digested by T7E1 enzyme at positions of sgRNA2 and sgRNA4 were detected in chst1 CRISPRants, but not in uninjected embryos. This indicates that these CRISPRants harbor mutations in the coding sequence of chst1. F, F-primer; R, R-primer; 2, sgRNA2; and 4, sgRNA4. (F) HSKS staining of chst1 CRISPRants demonstrated that loss of function of chst1 by mutagenesis led to loss of HSKS in otic vesicles, in consistent with chst1 morphants (Fig. S7C). Due to the mosaicism of mutated cells in CRISPRants, some chst1 CRISPRants retained HSKS in otic vesicles to some extent. (G) Otic vesicle sizes in CRISPRants were quantified as well as in Fig. 3C. phenotypes. Each value (length of left vesicle, length of right vesicle, width of left vesicle, and width of right vesicle) was statistically analyzed with one-way ANOVA (P < 7.4E-8, 1.15E-5, 4.56E-4, and 9.77E-5, respectively), followed by Tukey’s honestly significant difference test with 95% confidence level (indicated with a and b). This result further demonstrates that Chst1 regulates otic vesicle morphogenesis via HSKS synthesis in Xenopus embryos.
	Figure S10 Genome editing of the chst3 locus reproduced loss of HSKS phenotypes in Xenopus embryos. (A) The protein coding sequence of chst3 is represented together with its translated amino acid sequence, as in Fig. S9. An intron inserted position is designated by an arrow (see Fig. S7). (B) An in vitro cleavage assay validated enzymatic activity of the Cas9-sgRNA complex to digest target DNA. Digested DNA fragments were detected as expected. C, control (w/o sgRNA); 1, chst3-sgRNA1; 2, chst3-sgRNA2; 1+2, chst3-sgRNA1+2; 3, chst3-sgRNA3; 4, chst3-sgRNA4; and M, 100 bp ladder. (C-E) T7E1 assays validated the genome editing efficiency in embryos injected with Cas9 protein preincubated with chst3-sgRNAs. Five embryos were examined separately in each experiment. As a result, PCR fragments were significantly digested by T7E1 enzyme only at the position of chst3-sgRNA3. This indicates that CRISPRants with chst3-sgRNA3 harbor mutations in the coding sequence of chst3. F, F-primer; R, R-primer; and 3, sgRNA3. (F) HSKS staining of chst3 CRISPRants revealed that notochordal HSKS was eliminated from CRISPRants with chst3-sgRNA3, in consistent with chst3 morphants (Fig. S8F).
	Figure S11 Genome editing further confirmed that chst5.1 functions in HSKS synthesis in Xenopus embryos. (A) The protein coding sequence of chst5.1 is represented together with its translated amino acid sequence, as in Fig. S8. (B) An in vitro cleavage assay validated enzymatic activity of the Cas9-sgRNA complex to digest target DNA. Both chst5.1-sgRNA1 and chst5.1-sgRNA2 showed sufficient cleavage activity. (C-E) A T7E1 assay validated the genome editing efficiency in embryos injected with Cas9 protein preincubated with chst5.1-sgRNA1 (C), chst5.1-sgRNA2 (D), or both (E). Five embryos were examined separately in each experiment. PCR fragments digested by T7E1 enzyme at each sgRNA position were detected in CRISPRants, but not in uninjected embryos, indicating that these CRISPRants harbor mutations in the coding sequence of chst5.1. F, F-primer; R, R-primer; 1, sgRNA1; and 2, sgRNA2. (E) HSKS staining of chst5.1 CRISPRants validated that chst5.1 is required for HSKS synthesis in the notochord and otic vesicles. CRISPRants with chst5.1-sgRNA2 showed relatively weaker phenotypes than those with chst5.1-sgRNA1, although their enzymatic activity and genome editing efficiencies are equivalent (B-E). A part of CRISPRants with chst5.1-sgRNA1+2 exhibited almost complete loss of HSKS in otic vesicles, but others did not. These variable phenotypes suggest that HSKS can be synthesized by small number of normal cells/enzymes in otic vesicles. Open arrowheads indicate lost or reduced enrichment of HSKS.
	Figure S10 Genome editing of the chst3 locus reproduced loss of HSKS phenotypes in Xenopus embryos. (A) The protein coding sequence of chst3 is represented together with its translated amino acid sequence, as in Fig. S9. An intron inserted position is designated by an arrow (see Fig. S7). (B) An in vitro cleavage assay validated enzymatic activity of the Cas9-sgRNA complex to digest target DNA. Digested DNA fragments were detected as expected. C, control (w/o sgRNA); 1, chst3-sgRNA1; 2, chst3-sgRNA2; 1+2, chst3-sgRNA1+2; 3, chst3-sgRNA3; 4, chst3-sgRNA4; and M, 100 bp ladder. (C-E) T7E1 assays validated the genome editing efficiency in embryos injected with Cas9 protein preincubated with chst3-sgRNAs. Five embryos were examined separately in each experiment. As a result, PCR fragments were significantly digested by T7E1 enzyme only at the position of chst3-sgRNA3. This indicates that CRISPRants with chst3-sgRNA3 harbor mutations in the coding sequence of chst3. F, F-primer; R, R-primer; and 3, sgRNA3. (F) HSKS staining of chst3 CRISPRants revealed that notochordal HSKS was eliminated from CRISPRants with chst3-sgRNA3, in consistent with chst3 morphants (Fig. S8F).
	Figure S11 Genome editing further confirmed that chst5.1 functions in HSKS synthesis in Xenopus embryos. (A) The protein coding sequence of chst5.1 is represented together with its translated amino acid sequence, as in Fig. S8. (B) An in vitro cleavage assay validated enzymatic activity of the Cas9-sgRNA complex to digest target DNA. Both chst5.1-sgRNA1 and chst5.1-sgRNA2 showed sufficient cleavage activity. (C-E) A T7E1 assay validated the genome editing efficiency in embryos injected with Cas9 protein preincubated with chst5.1-sgRNA1 (C), chst5.1-sgRNA2 (D), or both (E). Five embryos were examined separately in each experiment. PCR fragments digested by T7E1 enzyme at each sgRNA position were detected in CRISPRants, but not in uninjected embryos, indicating that these CRISPRants harbor mutations in the coding sequence of chst5.1. F, F-primer; R, R-primer; 1, sgRNA1; and 2, sgRNA2. (E) HSKS staining of chst5.1 CRISPRants validated that chst5.1 is required for HSKS synthesis in the notochord and otic vesicles. CRISPRants with chst5.1-sgRNA2 showed relatively weaker phenotypes than those with chst5.1-sgRNA1, although their enzymatic activity and genome editing efficiencies are equivalent (B-E). A part of CRISPRants with chst5.1-sgRNA1+2 exhibited almost complete loss of HSKS in otic vesicles, but others did not. These variable phenotypes suggest that HSKS can be synthesized by small number of normal cells/enzymes in otic vesicles. Open arrowheads indicate lost or reduced enrichment of HSKS.
	Figure S14 Adenosine 3’-phosphate 5’-phosphosulfate binding motifs are widely conserved among chordate Chst proteins Sequence alignments of chordate Chst proteins indicate conservation of 5’-phosphosulfate binding motifs (5’-PSB) and 3’-phosphate binding motifs (3’-PB). Core motifs, RS/TGSSF in 5’-PSB and RDPR in 3’-PB, are widely conserved among chordate Chst proteins, whereas some residues of 5’-PSB and 3’-PB have changed specifically to each ohnolog, which may have caused differentiation of substrates. Xtr-Chst5.2 sequence was obtained from the record XP_012817703, which was now removed as a result of standard genome annotation processing. Mutations in the core motif of 5’-PSB and longer branch length in the phylogenetic tree (see Figs. S11 and S12) imply that chst5.2 is a dying gene. Hsa, Homo sapiens; Xtr, Xenopus tropicalis; Dre, Danio rerio; Cin, Ciona intestinalis; Bfl, Branchiostoma floridae.
	Figure S13 ML tree of putative deuterostome Chst genes An ML tree was constructed using the same set of sequences and color codes for gene names as in Fig. S6. Drosophila genes were used as the outgroup. Genes with notochordal expression are highlighted in light blue for Xenopus, Branchiostoma, and Ciona genes.
	Figure S14 Adenosine 3-phosphate 5-phosphosulfate binding motifs are widely conserved among chordate Chst proteins Sequence alignments of chordate Chst proteins indicate conservation of 5-phosphosulfate binding motifs (5-PSB) and 3-phosphate binding motifs (3-PB). Core motifs, RS/TGSSF in 5-PSB and RDPR in 3-PB, are widely conserved among chordate Chst proteins, whereas some residues of 5-PSB and 3-PB have changed specifically to each ohnolog, which may have caused differentiation of substrates. Xtr-Chst5.2 sequence was obtained from the record XP_012817703, which was now removed as a result of standard genome annotation processing. Mutations in the core motif of 5-PSB and longer branch length in the phylogenetic tree (see Figs. S11 and S12) imply that chst5.2 is a dying gene. Hsa, Homo sapiens; Xtr, Xenopus tropicalis; Dre, Danio rerio; Cin, Ciona intestinalis; Bfl, Branchiostoma floridae.

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