Ttc30a affects tubulin modifications in a model for ciliary chondrodysplasia with polycystic kidney disease

CRISPR/Cas9 Targeting of ift80 and ift172 Leads to Chondrodysplasia and Cystic Kidneys in Xenopus tropicalis.

We chose Xenopus to establish a model for ciliary chondrodysplasia because it is an aquatic organism that allows for analysis of limb development, has a high sequence conservation to humans, is an established model in cilia research, and provides good experimental access (50⇓⇓⇓–54). We targeted two well characterized SC genes (ift80 and ift172) for loss-of-function analysis using CRISPR/Cas9–based genome editing. Indel frequency and knockout scores were used to identify highly effective single guide RNAs (sgRNAs) against both targets in X. tropicalis embryos (SI Appendix, Fig. 1 A–C). Injection of F0 embryos with Cas9/sgRNA ribonucleoprotein (RNP) resulted in mosaic gene disruption and has previously been shown to elicit specific phenotypes at high frequency and penetrance (55⇓⇓⇓⇓–60).

Phenotypic analysis of 3- to 4-d-old embryos (stage 42) injected at the one-cell stage showed prominent generalized edema in both ift80– and ift172-targeted embryos. Importantly, this phenotype was rescued by coinjecting ift80 or ift172 messenger RNA (mRNA), demonstrating the specificity of the sgRNA-mediated loss of function (Fig. 1 A and B). Morpholino oligonucleotide knockdown in X. tropicalis and CRISPR/Cas9 targeting elicited the same phenotype in Xenopus laevis, confirming the specificity of the phenotype in a separate species (SI Appendix, Fig. 1 D and E).

Fig. 1.

CRISPR targeting of ift80 and ift172 leads to skeletal defects in X. tropicalis. (A and B) Analysis of edema formation in ift80 and ift172 crispants at stage 42. Note the smaller eyes in ift80-targeted embryos. (C) Limb phenotypes in postmetamorphic froglets (stage 62/63). MicroCT scans of representative crispant froglets of slc45a2 (negative control), ift80, and ift172 revealed cartilage accumulations in CRISPR-targeted froglets (green arrowheads). Injections of RNPs were performed unilaterally (Left) at the 2-cell stage. Polydactyly is indicated by orange arrowheads that mark black nailed digits. In the corresponding 3-dimensional reconstructions, limb muscles are shown in red, eyes in yellow, and red arrowheads point to the affected limbs. sgR L: left-sided sgRNA injection, R: right, L: left. (D) Histological sections of hindlimbs were stained with hematoxylin-eosin. R: resting zone; Pr: proliferating zone; PH: prehypertrophic zone; and H: hypertrophic zone. (E) The length of uninjected (green dots) versus injected (red dots) limbs of the same individual are plotted (gray lines). ns: (not significant); *P < 0.05; **P < 0.01; ***P < 0.001. [Scale bars, (A) 0.5 mm, (C) 5 mm, and (D) 200 µm.]

The most prominent phenotypes in chondrodysplasia patients are malformations of the axial skeleton and limbs [i.e., shortened ribs, long bone dysplasia, and polydactyly (4)]. Both ift80 and ift172 were expressed in limb buds (SI Appendix, Fig. 1F). Unilateral CRISPR targeting of F0 animals has been shown to be effective to disrupt genes essential for postmetamorphic limb development in Xenopus (61⇓–63). To test if Ift80 and Ift172 act in limb development in Xenopus, we unilaterally injected two-cell–stage embryos, which limits gene editing to only one half of the body while retaining the unaffected half as an internal control. We targeted slc45a2 as a negative control, which only results in a loss of pigmentation (59, 64). Apart from the expected loss of skin pigmentation, metamorphic froglets were phenotypically normal, demonstrating that sgRNA/Cas9 injection per se did not have an impact on development (Fig. 1C).

In contrast to control edited at the ift80 or ift172 locus showed severe limb defects. Both fore- and hindlimbs were substantially shorter than in control slc45a2-targeted froglets, as quantified in contrast enhanced micro-computed tomography (microCT) scans of mosaic mutant froglets (Fig. 1 C–E, green and red arrowheads). Despite unilateral RNP injections, we observed bilateral limb malformation in some animals, suggesting that in these cases, the first cell cleavage had not fully separated at the time of injection, allowing for gene editing to occur across the midline.

In most animals, the fingers and toes were shortened and less developed, sometimes not even identifiable (Fig. 1C). The number and size of clawed digits of the hindlimbs varied substantially from wild-types, in particular in ift80-targeted froglets (SI Appendix, Fig. 1G). X. tropicalis usually possess six digits at the hindlimb, four of which form claws (65). In ift80-targeted froglets, the number of clawed toes were consistently either reduced to less than three (syndactyly) or increased to six or seven (polydactyly) (Fig. 1C). In conclusion, this phenotype is highly reminiscent of the polydactyly phenotype observed in mouse models and human patients (24, 37, 40, 49, 66).

MicroCT images suggested that the shortened, not-yet-ossified limbs consisted mainly of cartilage accumulations (Fig. 1C, green arrowheads), consistent with the phenotype in humans and mouse models (48, 50, 67). Histological sections confirmed that the resting and proliferation zones of cartilage dominated clearly in ift80– and ift172-targeted animals at the expense of the hypertrophic and ossification zones visible in slc45a2-targeted control animals (Fig. 1D). In general, the chondrocytes were less well organized in ift80– and ift172-depleted cartilage than in limbs of controls. However, cartilage differentiation was unaffected in vertebrae, suggesting a limb-specific defect (SI Appendix, Fig. 2A).

Similar phenotypes have been described for conditional ift80 knockout mice and other chondrodysplasia models (24, 26, 68⇓–70).

Contrast-enhanced microCT was also able to resolve the structure of the mesonephric kidneys in the postmetamorphic froglets at high resolution and revealed polycystic kidneys in most of the mutant animals (Fig. 2 A–C). Quantification and volumetric analysis showed that both ift80 and ift172 disruption resulted in multiple cysts, particularly on the injected side (Fig. 2D). The occurrence of cysts on the uninjected side in ift80 mosaic mutants likely reflects the incomplete segregation of gene editing to one half, as observed for limb measurements. The total cyst volume was significantly larger on the injected side of ift80-edited froglets (Fig. 2E), while the volume of noncystic renal parenchyma was not significantly different (Fig. 2F).

Fig. 2.

Cystic kidney disease and cilia defects in ift80 and ift172 CRISPR–targeted X. tropicalis. (A–C) CRISPR/Cas9 targeting of slc45a2, ift80, and ift172 were performed unilaterally in two-cell–stage embryos. Mesonephroi of stage 61 to 63 froglets were analyzed by microCT scans and kidneys and cysts (red arrowheads in B and C) were segmented for 3D volumetric analysis; red: cysts on the injected side; green: uninjected side). (D) The number of cysts (>0.2mm), (E) the ratio of total cyst volume to kidney volume, and (F) the kidney volume (excluding cysts) was calculated and compared with kidneys of control injected animals. (G) Whole-mount in situ hybridization detects ift80 and ift172 in the multiciliated nephrostomes of the pronephros in stage 36 to 38 X. laevis. (H) Schematic depiction of the embryonic renal system of Xenopus. (I and J) Excretion assay with fluorescein-dextran at stage 38 to 40. Blue arrowheads point to the proximal part of the pronephros. Yellow arrowheads indicate fluorescence signal in the distal tubule, lacking on the injected side (J). A: anterior, P: posterior; excr: excreting; and emb: embryos. (K) Confocal images of multiciliated epidermal cells (MCCs) stained against acetylated tubulin (cyan). Centrin-RFP fusion protein served as a lineage marker (red arrowheads) and indicates CRISPR-targeted MCCs. Blue arrowheads point to nontargeted (wild type) cells. (L) The ciliated area was determined for each cell. Error bars indicate SEM. P > 0.05 ns (not significant); *P < 0.05; **P < 0.01; ***P < 0.001. [Scale bars, (A–C) 1 mm, (G and I) 0.5 mm, and (L) 10 µm.]

As ciliopathies can lead to retinal disease, we sectioned both embryo and froglet eyes for histological analysis. Apart from a mildly disorganized outer nuclear layer in ift80-targeted embryos, which may be due to the accompanying edema, we did not observe any obvious photoreceptor defects (SI Appendix, Fig. 2B). Likewise, no laterality defects were found (SI Appendix, Fig. 2 C and D).

We conclude that froglets with mutations in ift80 and ift172 displayed shortened extremities with polydactyly and cystic kidney disease, confirming that chondrodysplasia can be modeled in Xenopus. This supplements the available mouse and zebrafish lines and offers additional opportunities to analyze chondrodysplasia genes and their functions (49, 50, 67, 71).

Ift80 and ift172 Targeting Affects MCCs, Resulting in Pronephric Excretion Defects.

Next, we explored the effect of ift80 and ift172 disruption on a cellular level. The most prominent phenotype of ift80 and ift172 mutant tadpoles was generalized edema, suggesting a deficiency in water or ion homeostasis due to pronephric, lymphatic, vascular, or cardiac insufficiency. In situ hybridization of established marker genes (aplnr, vasculature; nkx2.5, heart; prox1, lymphatic system) did not reveal an obvious developmental disruption of any of these organ systems in Xenopus embryos targeted for ift80 and ift172 disruption (SI Appendix, Fig. 3 C and D). Given the prominent renal phenotype in froglets, the edema phenotype in embryos, and because Jeune syndrome patients and mouse models frequently suffer from kidney disease, we focused on the pronephros of X. tropicalis embryos (47). We did not find structural tubular defects when staining for the tubular epithelium (SI Appendix, Fig. 3 E and F) nor were the expression of segment-specific marker genes (nkcc2, distal tubule; sglt1 proximal tubule) affected (SI Appendix, Fig. 3 C and D), suggesting a normal differentiation of tubular tissue.

Expression analysis of ift80 and ift172 by in situ hybridization revealed both genes to be prominently expressed in MCCs of the epidermis and nephrostomes (Fig. 2G and SI Appendix, Fig. 3 A and B). Nephrostomes are the most proximal tubular segments that facilitate uptake of coelomic fluid into the pronephric tubule (Fig. 2H). To test whether impaired fluid uptake into the tubule could explain the observed edema phenotype in ift80 and ift172 bilaterally disrupted embryos, we employed a dextran excretion assay. Embryos were injected with sgRNA/Cas9 at the two- to four-cell stage, and fluorescently labeled dextran was injected into the coelom at stage 39 to 40 when the pronephros is fully functional. Timelapse movies of slc45a2 sgRNA–injected (negative control) embryos showed rapid uptake and acceleration of dextran through the pronephric tubule and excretion at the cloaca on both sides in 84.6% of embryos (Fig. 2 I and J). In contrast, entry into the pronephric tubule was observed in only 12.5 and 13.8% of embryos within 3 min on the ift80– and ift172-targeted side (Fig. 2 I and J), respectively. Dextran uptake was comparable to wild type and much faster on the nontargeted side of unilaterally gene-edited embryos. Normal excretion on the uninjected side could also be indirectly observed in the timelapse movies where dextran entered the medium of ift80– and ift172-targeted embryos (Movie 1).

Motile cilia in the nephrostomes actively propel fluid into the renal tubules. Thus, structural defects of ciliated cells may explain the observed loss of excretion in CRISPR-targeted tadpoles (45, 71). To analyze the role of ift80 and ift172 in motile cilia, we turned to the epidermal MCCs, a well-established and accessible model for ciliogenesis.

Centrin-red fluorescent protein mRNA was used as an injection marker to identify and label basal bodies of ciliated cells that had received the CRISPR RNPs, allowing us to analyze CRISPR-targeted and wild-type cells in the same images. Cilia appeared to be fewer and shorter in length, which was confirmed by quantification of cilia in ift80 and ift172 knockout cells (Fig. 2 K and L). In contrast, basal body number and apical docking was not altered in crispant MCCs when compared with controls (SI Appendix, Fig. 3G). Thus, the ciliogenesis defect is consistent with findings in other model organisms (45, 71) and a likely cause for impaired tubular uptake.

To gain more detailed insights into the molecular signals affected by a loss of ift80 or ift172, we investigated the transcriptional profile of embryos targeted for ift80 and ift172. Because embryos were in vitro fertilized and developed synchronously, stage-dependent variability was minimized between control and CRISPR-targeted embryos. We isolated DNA and confirmed a high indel frequency in the same samples subjected to RNA-Sequencing analysis at stage 22 (neurula) (SI Appendix, Fig. 4 A and B). The top-most down-regulated genes were ift80 and ift172, respectively (SI Appendix, Fig. 4 C–E and Dataset S1), confirming the specificity of our targeting strategy and a direct effect on mRNA levels likely due to nonsense-mediated decay. We found 15 (ift80) and 5 (ift172) genes to be differentially regulated in either condition (SI Appendix, Fig. 4 D and E) and three genes (abcb1 and two uncharacterized transcripts) to be significantly up-regulated in both conditions. The ATP-dependent multidrug-resistance transporter Abcb1 is transcriptionally regulated by Gli1 and β-catenin (72, 73). Interestingly, an additional five genes that only reached significance in one condition were also associated with Hh signaling (ccng1, ulk1, riok3, olfm4, and col1a1) and four with Wnt signaling (c3ar1, col1a1, olfm4, and ulk1) (SI Appendix, Fig. 4F) (72, 74⇓⇓⇓⇓⇓⇓⇓–82). Thus, we detect Hh- and Wnt-dependent gene regulation to be altered upon depletion of ift80 and ift172 in Xenopus embryos.

mRNA expression analysis in wild-type embryos of differentially regulated genes identified by RNA-Sequencing (ccng1, ulk1, and riok3) found an overlapping expression with chondrodysplasia genes in neuronal tissue and neural crest (SI Appendix, Fig. 4G). However, we did not find a change in the Sonic Hedgehog (Shh) marker nkx2.2 after unilateral ift80 or ift172 targeting, suggesting that the differences in mRNA abundance are below the detection level of in situ hybridization (SI Appendix, Fig. 4 H and I).

Data Mining Identifies Skeletal Ciliopathy–Associated Genes.

Next, we aimed to identify novel proteins related to the pathogenesis of SC. We reasoned that proteins with similar molecular properties, such as subcellular localization, common protein interactors, or differential regulation in loss-of-function models, could be unrecognized components of the pathogenetic molecular mechanism. Therefore, we assembled lists of genes from 90 published genome-wide screens or unbiased analyses (“resource lists”) (Dataset S1) based on literature curation. We identified 16 resource lists to be significantly enriched for genetically validated chondrodysplasia genes in comparison with 10 million random draws of an equal number of query genes (Fig. 3A). Within these 16 lists, 65 unique genes not yet linked to chondrodysplasia were found to be significantly enriched (Dataset S1). Among these potential candidate genes were three that have been identified as bona fide chondrodysplasia genes in the meantime [i.e., ift122, ift43, and traf3ip1 (70, 83, 84)]. Ift22 was described to be mutated in a SRPS type IV patient, ift43 and traf3ip1 in SRPS type II patients, and traf3ip1 additionally in a case of JATD (70, 83, 84). This confirmed that our approach was able to predict valid genetic associations.

Fig. 3.

In silico screening for candidates with similar properties to SC genes. (A) Schematic of the in silico screen. Known SC disease genes served as an input list. Resource lists represent datasets from various published screening approaches and were tested for enrichment of genes contained in the input list genes. Genes in significantly enriched resource lists were then scored based on membership or rank. The gene scores were statistically validated by an empirical P value based on 10 million random drawings of input list. (B) Whole-mount in situ hybridizations for four candidate genes (c11orf74, cep192, cfap20, and ttc30a) that were experimentally followed up are shown for tadpoles at stage 31 to 35 (nephrostome expression enlarged) and limb buds (stage 55 to 57). (Scale bars, 0.5 mm.) p, posterior; a, anterior; pr, proximal; and d, distal.

Among the 65 potential chondrodysplasia-related candidate genes, we focused on the top four genes with the lowest P value that had not been linked to any ciliopathies nor characterized in a knockout mouse model and were targetable in X. tropicalis for functional analysis (C11ORF74, CEP192, CFAP20, and TTC30B; Fig. 3B and SI Appendix, Fig. 4K). All proteins encoded by these four candidate genes had previously been associated with cilia. CFAP20 (cilia- and flagella-associated protein 20) regulates cilia length and morphology (85, 86). Entering of the BBSome into the cilium via interaction with the IFT-A complex is controlled by C11ORF74 (87). CEP192 (centrosomal protein of 192 kDa) is required for mitotic centrosome and spindle assembly (88, 89). TTC30B (tetratricopeptide repeat domain 30b, also known as IFT70) is essential for polyglutamylation and polyglycylation of axonemal tubulin and is part of the IFT-B complex (90⇓–92). TTC30B has one closely related homolog in humans (TTC30A) and three in mice (Ttc30a1, Ttc30a2, and Ttc30b) but only one ortholog in X. tropicalis and Danio rerio (ttc30a/fleer) (Fig. 4A) (91).

Fig. 4.

ttc30 loss of function resembles the SC-phenotype. (A) Phylogenetic tree of protein sequences of TTC30A/B orthologs in various species. Genetic duplication events are marked by red squares. (B) CRISPR targeting of ttc30a at the one-cell stage led to edema formation in stage 41 tadpoles, quantified in (C). Coinjection of ttc30a mRNA partially rescued the phenotype. (D) Unilaterally CRISPR-targeted ttc30a froglets developed shortened limbs on the injected side (green arrowhead). MicroCT analysis, 3D reconstructions, and histological sections stained with hematoxylin-eosin demonstrated accumulation of cartilage (green arrowhead). Quantification of limb length reduction of ttc30a targeted compared with nontargeted side. (E) MicroCT scans show cystic kidneys in ttc30a-targeted animals (red arrowhead). 3D reconstruction of the kidneys and cysts (red) were used for volumetric analysis. Quantifications of cyst number, the ratio of total cyst volume to kidney volume, and total kidney volume (excluding cysts). [Scale bars, (B) 0.5 mm, (D and E, 3D reconstruction) 2 mm, (D, histological section) 200 µm.] R, right; L, left; P, proximal; D, distal. P > 0.05 ns (not significant); *P < 0.05; **P < 0.01; ****P < 0.0001.

CRISPR/Cas9 Targeting ttc30a Replicates the Phenotype of ift80– and ift172-Targeted Embryos.

We designed sgRNAs targeting all Xenopus orthologs of the four candidates for CRISPR/Cas9–mediated loss-of-function experiments. Interestingly, cfap20 and ttc30a crispants formed edema suggestive of embryonic renal excretion defects. This effect was specific, as coinjection with corresponding mRNAs significantly rescued the edema phenotype (Fig. 4 B and C and SI Appendix, Fig. 5 A–E).

In order to evaluate a potential effect on limb development, we raised cfap20, ttc30a, c11orf74, and cep192 CRISPR–targeted tadpoles to metamorphosis. Limb formation of cep192, c11orf74, and cfap20 crispants was normal (SI Appendix, Fig. 5 G and H). Froglets with ttc30a mutations, however, developed severe limb defects (Fig. 4D, green arrowheads and SI Appendix, Fig. 5F). Extremities were generally shorter with polydactyly (Fig. 4D and SI Appendix, Fig. 5 F and H). MicroCT scans found large depositions of cartilage. Histology revealed a chondrocyte differentiation defect, as previously observed for ift80– and ift172-targeted animals. Ttc30a-targeted embryos had reduced fore- and hindlimb length (Fig. 4D), and analysis of the kidneys revealed a significantly higher number of cysts on the injected side of the froglets (Fig. 4E). However, total cyst and kidney volume did not change significantly (Fig. 4E). Excretion assays confirmed that renal tubular uptake of dextran was drastically reduced (Fig. 5 A and B and Movie 2), and epidermal cilia were shorter or absent in embryos injected with sgRNA and RNPs targeting ttc30a (Fig. 5 C and D). In conclusion, CRISPR targeting ttc30a in Xenopus replicated all phenotypes observed when targeting the chondrodysplasia genes ift80 and ift172.

Fig. 5.

Single cell RNA-Sequencing analysis of Ttc30a/b-positive cells in developing mouse limbs. (A and B) Excretion assay of slc45a2 sgRNA–injected controls and ttc30a-targeted embryos. Blue arrowheads point to the proximal tubule, and yellow arrowheads highlight fluorescent dextran in the distal tubules as a measure of excretion. (C) Confocal images of epidermal MMCs of stage-36 embryos. centrin-RFP (red) marks cells targeted for ttc30a (red arrowheads). Blue arrowheads label wild-type cells. Cilia are stained with acetylated gamma-tubulin (cyan). (D) Quantification of ciliated area per cell in slc45a2 and ttc30a CRISPR–targeted cells. Error bars indicate SEM. P > 0.05 ns (not significant); *P < 0.05; **P < 0.01; and ***P < 0.001. (E) Clustering of scRNA-Seq data from E15.5 mouse limbs into 10 different cell clusters using typical marker genes. (F) Detection of Ttc30b expression in respective cell clusters. (G) Comparison of percentage of cells per cluster expressing Ttc30a1, Ttc30a2, Ttc30b, Ift80, and Ift172. (H) Expression level of Ttc30b, Ift80, and Ift172 in respective tissue clusters. [Scale bars, (A) 0.5 mm and (C) 10 µm.] GP, growth plate.

To elucidate the expression pattern of mammalian orthologs of ttc30a, we analyzed publicly available single-cell sequencing data of E13.5 mouse limb buds (SI Appendix, Fig. 6 A–E) (93). We found that among the three orthologs, Ttc30b and to a lesser degree Ttc30a1 were most prominently expressed in cartilage, bone, and growth plate clusters (Fig. 5 E–G). The distribution of cell proportions expressing Ttc30b was similar to that of Ift80 and Ift172 (Fig. 5 G and H). Thus, the expression pattern of Ttc30b and Ttc30a1 suggest a potential role in cartilage formation of mammals. We screened for TTC30A/B mutations in a cohort of 24 unresolved severe ciliary chondrodysplasia cases by exome sequencing, but no likely pathogenic variants were identified. Screening of whole-exome sequencing data in a worldwide cohort of more than 500 patients with nephronophthisis related ciliopathies and cystic kidney disease also did not reveal potentially causative recessive mutations in either TTC30A or TTC30B.

Ciliary Compartmentalization Is Altered in ttc30a- and ift172-Depleted Cilia.

Ttc30a mediates tubulin modifications such as glycylation and glutamylation to be deposited within the ciliary axoneme (15, 91, 92).

Immunostaining with polyglutamylation- and monoglycylation-specific antibodies revealed that MCCs of the epidermis and in the nephrostomes were acetylated, glutamylated, and glycylated (Fig. 6 A–D). In contrast, ependymal MCCs positioned in the ventral neural tube were only glycylated but neither acetylated nor glutamylated (Fig. 6 A and B). All three analyzed modifications were also detected in primary cilia of the embryonic renal tubule cells. Closer inspection of the epidermal cilia revealed acetylated tubulin to be present in the entire axoneme and enhanced at the ciliary tip. Tubulin glycylation was present in the axoneme but spared the ciliary tip, while glutamylation was detected in a short proximal part of the axoneme (Fig. 6 C and D). Thus, posttranslational tubulin modifications distinguish cilia of various tissue cell types and compartmentalize the axoneme of individual cilia.

Fig. 6.

Ttc30a affects posttranslational tubulin modifications and ciliary compartmentalization. (A) Glycylation (cyan) in combination with acetylation (red) and (B) glutamylation (cyan) of pronephric and neural tube cilia were detected by immunostaining in wild-type embryos at stage 39. Cells of the pronephros and neural tube were visualized by Lectin staining (white/gray). White arrowhead points to ventral cilia of the neural tube, and red arrowhead to primary cilia of the pronephric tubular system. Stainings of the nephrostomes and tubular system are also shown as single channels. L, lateral; M, medial; D, dorsal; V, ventral; gly, glycylation; lec, lectin; glu, glutamylation; and acet, acetylation. Influence of targeting ift80, ift172, and ttc30a by CRISPR/Cas9 on glycylation (C) and glutamylation (D) was analyzed by immunostainings of epidermal cilia at stage 36 (glutamylation/glycylation, cyan; acetylation, red). (E and F) A schematic model for the role of ttc30a during bone and kidney development. (E) Ttc30a interacts with Kif17 in the cilium and is crucial for tubulin modifications (blue, glycylation; red, glutamylation; and yellow, acetylation) (15, 92, 112). Mutations of ttc30a in Xenopus reduce acetylation and glycylation, negatively impact cartilage differentiation, and result in kidney cysts (F). Green, intraflagellar transport (IFT)-B proteins; red circled, known SC genes; gray, ciliary tubulin. Error bars indicate SEM. P > 0.05 ns (not significant); *P < 0.05; **P < 0.01; and ***P < 0.001. (Scale bars, 10 µm.)

Targeting ttc30a by CRISPR/Cas9 injections revealed a reduction in the absolute and relative intensity of tubulin glycylation in relation to acetylation, used for normalization and segmentation of cilia (SI Appendix, Fig. 7 A–C). However, an increased glutamylation to acetylation index found in ttc30a– and ift172-depleted cilia was due to reduced axonemal acetylation, while glutamylation intensity was not changed (SI Appendix, Fig. 7 D–F). Closer inspection found that in ttc30a-, ift80-, and ift172-depleted cilia, the prominent acetylated tip was no longer present (Fig. 6 C and D), indicating defective axoneme compartmentalization.