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Fig. 1. katnal2is expressed in ciliated tissues and the developing nervous system. A-H) Whole mount RNA in situ hybridization for katnal2 during X. tropicalis development. A) katnal2 expression is enriched in the dorsal mesoderm (dm). A’) Transverse section of A. B) Dorsal view of katnal2 expression in the floor plate (fp) and epidermis (epi). B’) Lateral view of B, anterior to the left also showing notochord (nc) staining. C) Lateral view of katnal2 expression enriched in the nc, pronephric nephrostomes (pn), and pharyngeal arches (pa). C’) Higher magnification view of C highlighting pn and nc expression. D) Lateral view of katnal2 expression in the spinal cord (sc) and otic vesicle (ov). D’) Dorsal view of katnal2 expression in the brain (br), especially in the cells lining the ventricles. E) Sagittal section of stage 15 embryo showing expression in the gastroceol roof plate (grp), anterior to the blastopore (bp). F) Coronal section showing an interior dorsal view of a stage 15 embryo, with expression in the nc and grp. G) Transverse section of a stage 30 embryo showing expression in the neural tube (nt), floor plate (fp), and notochord (nc). H) Coronal section of stage 38 embryo showing high expression in the cells lining the telencephalic (tel) ventricle (vent). Abbreviations: dm = dorsal mesoderm; fp = floor plate; epi = epidermis; nc = notochord; pn = pronephric nephrostomes; pa = pharyngeal arches; ov = otic vesicle; sc = spinal cord, br = brain; grp = gastroceol roof plate; bp = blastopore; nt = neural tube; vent = ventricle; tel = telencephalon. Scale: 100 μm, except H where Scale: 10 μm.
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Fig. 2. Katnal2 localizes to epidermal cilia, basal bodies, and centrioles. A-B) Katnal2 rabbit antibody staining (green) of X. tropicalis multiciliated cells of the epidermis, co-stained with DAPI (nuclei, blue) and acetylated α-tubulin (Ac-α-tubulin; cilia, magenta). B) High-magnification image from the specimen shown in A. C) Katnal2 antibody staining (green) with Centrin4-CFP expression (magenta). D-H) KATNAL2-GFP (green) in the embryonic epidermis of X. laevis, co-stained for Centrin4-RFP (basal bodies/centrioles, magenta) and Ac-α-tubulin (cilia, blue). D-E) KATNAL2-GFP localizes to basal bodies in multiciliated cells. (E is close-up of D) F-H) KATNAL2-GFP localization in non-ciliated epidermal cells. G) KATNAL2-GFP signal in centrioles. H) KATNAL2-GFP signal in the midbody of dividing cells.
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Fig. 3. Katnal2 localizes to mitotic spindles and ciliary axonemes in the tadpole. A-F) Katnal2 rabbit antibody staining (green) in stage 45 X. tropicalis tadpoles, highlighting staining in ciliated and neural tissues. A) Low magnification view of Katnal2 staining in the cephalic region of the tadpole. B) Telencephalic (tel) region of the brain, showing expression throughout and especially within the first ventricle. B’) Transverse section through the telencephalon. B”) High-magnification image from specimen shown in B’, showing Katnal2 localization to a mitotic spindle. C) Olfactory epithelium (olf) stained for Katnal2 (green), poly-glutamylation (cilia, magenta) and DAPI (nuclei, blue). C’-C”’) High-magnification image of the specimen in C, showing localization of Katnal2 (green) to ciliary axonemes (poly-glutamylation, magenta). D) Eye. E) Multiciliated cells of the roof of the fourth ventricle of the rhombencephalon (rhomb). F) Katnal2 is expressed throughout the kidney. Abbreviations: tel = telecephalon; olf = olfactory epithelium; rhomb = rhombencephalon.
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Fig. 4. katnal2is required for blastopore closure, neural tube closure, and anterior-posterior axis elongation inX. tropicalis. A) katnal2 inhibition leads to incomplete blastopore closure. Shown are representative phenotypes (no effect, mild, and severe) with quantification by condition. MO control is injection of Centrin4-CFP mRNA. CRISPR control is targeting of slc45a2. B) katnal2 inhibition leads to defects in neural tube closure. Shown are representative phenotypes (no effect, mild, and severe) with quantification by condition. MO control is injection of Centrin4-CFP mRNA. CRISPR control is targeting of slc45a2. C) katnal2 inhibition leads to shortened anterior-posterior axis elongation. Embryo length from head to tail was measured and plotted by condition. MO control is injection of Centrin4-CFP mRNA. CRISPR control is targeting of slc45a2. ⁎: p < 0.05; ⁎⁎⁎⁎: p < 0.001.
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Fig. 5. katnal2is required for eye, pigmentation, and neural crest development inX. tropicalis. A) Representative embryos at stage 40 following injection of a katnal2 translation-blocking morpholino into half of the embryo. Morpholino dose indicated on the left. Asterisk (⁎) indicates injected side of the embryo. The injected side shows defects in axis elongation, eye formation, cranial cartilage development, and pigmentation. Control embryos were injected with Centrin4-CFP mRNA. B) Representative embryos at stage 44 following injection of katnal2 morpholino. Asterisk (⁎) indicates injected side of the embryo. Injected animals show defects in axis elongation, eye formation, and cranial cartilage formation. Control embryos were injected with Centrin4-CFP mRNA. C) katnal2 morpholino-injected animals show defects in pigmentation (white arrows), especially migration of melanocytes, and defects in eye formation (red arrow). Control embryos were injected with Centrin4-CFP mRNA. D) Representative embryos at stage 44 following genome targeting of katnal2 by CRISPR/Cas9 into half of the embryo. Asterisk (⁎) indicates injected side. katnal2-targeted embryos show defects in eye formation, cranial cartilage formation, and pigmentation on the injected side. Control embryos were injected with Cas9 protein and a scrambled sgRNA. E) Stage 18 embryos following RNA in situ hybridization against snail2, labeling neural crest. Asterisk (⁎) indicates injected side. katnal2 loss-of-function inhibits neural crest migration. Control embryos were injected with Centrin4-CFP mRNA.
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Fig. 6. katnal2is required for ciliogenesis. A-J) Multiciliated X. tropicalis embryonic epidermis following injection of katnal2 morpholino at two doses or CRISPR/Cas9 RNP, showing Centrin4-CFP (blue), Actin (phalloidin, green) and acetylated α-tubulin (Ac-α-tubulin, cilia, magenta). A) Control embryos (injected with Centrin4-CFP alone) show normal ciliogenesis. B-C) Embryos injected with 2–4 pmol of katnal2 morpholino show reduced number and length of cilia. D-E) Embryos injected with katnal2 CRISPR/Cas9 RNPs also show reduced number and length of cilia. F-J) High magnification view of each condition. Asterisks (⁎) mark cells that did not undergo katnal2 disruption (Centrin4-CFP negative).
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Fig. 7. katnal2is required for telencephalon development inX. tropicalis. A) Representative stage 46 X. tropicalis tadpole, following 1 pmol katnal2 morpholino injection into half of the embryo, stained for β-tubulin to visualize the nervous system. B) Close-up of the telencephalic region in A. C) Quantification of telencephalon size, comparing the uninjected side of the tadpole to the injected side, reveals reduced size on the morpholino-injected side (p < 0.0001; n = 37). D) Representative stage 46 X. tropicalis tadpole following 1 pmol katnal2 morpholino injection into half of the embryo, stained for proliferating cell nuclear antigen (PCNA) to visualize the proliferative cells of the nervous system, shows a reduction of area on the morpholino injected side. E) Close up of the telencephalic region (first ventricle) from D. F) Quantification of PCNA positive area in the telencephalic region (first ventricle), comparing the uninjected side to the injected side (p < 0.0001; n = 25). G) Transverse section of telencephalic region, stained for PCNA (ventricular zone progenitors, green) and vGLUT1 (glutamatergic neurons, magenta).
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Fig. S1. X. tropicalis katnal2cloning and early embryonic expression. A) Full-length katnal2 cDNAs, generated by RT-PCR, migrate as 2 distinct bands above 1.5 kb. B) Nucleotide and protein sequences of the 2 alternatively spliced X. tropicalis katnal2 isoforms, bearing the typical Walker motif A (boxed) and an N-terminal LiSH domain (underlined). The grey shaded area (32 amino acids) corresponds to sequence encoded by exon 6 that is absent from the shorter isoform. X. tropicalis katnal2 sequences were submitted to Genbank with accession numbers MH036373 (larger isoform) and MH036374 (shorter isoform). C) Semi-quantitative RT-PCR of katnal2 throughout X. tropicalis development, compared to drosha and smn2. Note that two isoforms of katnal2 are present in the two-celled embryo and at all later stages sampled (lower expanded panel). D-G) Whole mount RNA in situ hybridization for katnal2 in early X. tropicalis embryos. D) Lateral view of katnal2 expression in the animal hemisphere. E-G) Animal view of maternal katnal2 expression in developing embryos (stages 6–9), compared to sense negative control probe control at stage 9 (H).
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Fig. S2. Validation of Katnal2 rabbit antibody. A-B) X. tropicalis embryonic epidermis stained for Katnal2 (green) and acetylated α-tubulin (Ac-α-tubulin, cilia, magenta) in the presence (A) or absence (B) of Katnal2 primary rabbit antibody.
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Fig. S3. Katnal2 localizes to mitotic spindles, basal bodies, and cilia in theX. laeviscell line XL177. A-D) Examples of Katnal2 antibody staining (green) in mitotic subphases of cycling cells, co-stained for DAPI (DNA, blue) and α-tubulin (α-tub, red). A-B) Katnal2 antibody raised in goat. C-D) Katnal2 antibody raised in rabbit. E-F) Examples of Katnal2 antibody staining (green) in serum-starved cells, co-stained for DAPI (DNA, blue) and acetylated α-tubulin (ac-tub, ciliary axonemes, red). Primary cilia are indicated with white arrowheads. E) Katnal2 antibody raised in goat. F) Katnal2 antibody raised in rabbit. Accompanying magnified detailed views of one of the cilia (bottom) from each image are given. Scale: 10 μm.
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Fig. S4. Targeting ofkatnal2by CRISPR/Cas9 genome editing using IDT Alt-R sgRNAs. A) Nucleotide and protein sequences of X. tropicalis katnal2, indicating the target sequences for CRISPR/Cas9 targeting (sgRNA CRISPR#1 target sequence in magenta and sgRNA CRISPR#2 in cyan). B) Schematic representation of PCR amplicon used as a template for the CRISPR/Cas9 in vitro assay. Arrows show predicted fragment sizes resulting from the endonucleolytic activity of CRISPR#1 or CRISPR#2 sgRNA/Cas9 RNP. C) DNA gel electrophoresis confirming the expected fragment sizes, following the in vitro activity of the CRISPR#1 and CRISPR#2 RNPs on the synthetic katnal2 template (upper gel). Repetition of the assay including a scrambled CRISPR#1 negative control RNP leaves the template uncut. D) Representative traces from Fragment Length Analysis on control and injected embryos. Multiple peaks are present for genome-targeted embryos. E) Right and left side view of a X. tropicalis embryo, injected in one blastomere at the two-cell stage embryo with RNPs targeting the pigmentation gene slc45a2. Red arrows highlight the reduction in eye pigmentation on the left (injected) side of the embryo. F) Representative examples of embryo phenotypes following katnal2 genome targeting with CRISPR#1-targeting RNP, displaying shortened anterior-posterior axis and eye pigmentation defects, as seen in Fig. 5 with morpholino and in vitro transcribed sgRNA injections.
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Fig. S5. katnal2loss-of-function phenotypes inX. laevis. A) Representative X. laevis embryos at stage 40 following katnal2 morpholino injection. Asterisk (⁎) indicates injected side. B) Representative X. laevis embryos at stage 44 following katnal2 morpholino injection. Asterisk (⁎) indicates injected side. C-E) Multiciliated embryonic epidermis following injection of katnal2 morpholino at two doses in X. laevis, showing Centrin4-CFP (blue), Actin (phalloidin, green) and acetylated α-tubulin (Ac-α-tubulin, cilia, magenta). C) Control embryos. D) Embryos injected with 2 pmol of katnal2 morpholino. E) Embryos injected with 4 pmol of katnal2 morpholino. Injected embryos show abnormal ciliogenesis. Asterisks (⁎) mark cells that were not targeted (Centrin4-CFP negative).
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Fig. S6. katnal2loss disrupts basal body distribution and actin network formation in theX. tropicalisembryonic epidermis. A) Control embryos showing actin (phalloidin, green), Centrin4-CFP (blue, basal bodies), and cilia (Ac-α-tubulin, magenta). B) Embryos injected with 4 pmol katnal2 morpholino show abnormal actin network formation (B), basal body distribution (B’), and cilia length and number (B”). C) Embryos injected with 6 pmol katnal2 morpholino show abnormal actin network formation (C), basal body distribution (C’), and cilia length and number (C”).
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katnal2 (katanin p60 subunit A-like 2 ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 1, horizontal view, animal up.
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katnal2 (katanin p60 subunit A-like 2 ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 9, animal view.
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katnal2 (katanin p60 subunit A-like 2 ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 10, vegetal view, dorsal up.
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katnal2 (katanin p60 subunit A-like 2 ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior up.
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katnal2 (katanin p60 subunit A-like 2 ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.
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katnal2 (katanin p60 subunit A-like 2 ) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 38, lateral view, anterior left, dorsal up.
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