Abstract
Although zebrafish embryos have been utilized to study ciliogenesis and to model polycystic kidney disease (PKD), adult zebrafish remain unexplored. Here, we report the generation and characterization of a zebrafish mutant of tmem67, a homologue of the mammalian causative gene for Meckel syndrome type 3 (MKS3). Although a small population of homozygous embryos exhibited pronephric cysts, all mutants were able to survive to adulthood and developed progressive mesonephric cysts with full penetrance. The cysts in the adult zebrafish kidneys manifested features of mammalian PKD, including switching of cyst origin from the proximal tubules to the collecting ducts, increased proliferation of cyst-lining epithelial cells, and hyperactive mTOR signaling. Consistent ciliary abnormalities were observed in both the embryonic and adult zebrafish mutants compared with the wild-type fish, including shorter and fewer single cilia in the distal pronephros and all segments of the mesonephros and greater numbers of multiciliated cells (MCCs). Lack of single cilium preceded cystogenesis, suggestive of a primary defect. Expansion of MCCs occurred after pronephric cyst formation and was inversely correlated with the severity of cystogenesis in young adult zebrafish, suggesting an adaptive action. Interestingly, mTOR inhibition ameliorated renal cysts in both the embryonic and adult zebrafish models; however, it only rescued ciliary abnormalities in the adult mutants. In summary, we have established a tmem67 mutant as the first adult zebrafish PKD model, revealed a novel aspect of cilium regulation, and identified sustained mTOR inhibition as a candidate therapeutic strategy for tmem67-based PKD.
Significance Statement While zebrafish embryos are well recognized for their value in studying ciliogenesis and polycystic kidney disease (PKD), adult zebrafish have not commonly been used. Here, we report the establishment of the first adult zebrafish model for PKD, which exhibits characteristics of mammalian PKD and shows kidney ciliary abnormalities consistent with those observed in an embryonic model. We also provide evidence for mTOR inhibition as a therapeutic strategy for this particular type of cystogenesis. Compared to the embryonic model, the adult fish model exhibits a spectrum of progressive pathogeneses and enables ciliary abnormalities to be discerned as either primary or secondary to cystogenesis. We believe that this novel adult fish model will facilitate mechanistic studies and therapeutic development for PKD.
Introduction
TMEM67 is a major causative gene for Meckel syndrome (MKS; the gene was formerly named MKS3), accounting for approximately 15% of MKS cases; it is also a causative gene for Joubert syndrome (JBS).1–4 MKS is an autosomal recessive and perinatal lethal disorder that is characterized by a spectrum of abnormalities, including renal cysts in more than 95% of patients, central nervous system defects such as encephalocele, liver fibrosis, and sometimes polydactyly.5–7 Spontaneous animal models have been used to study MKS3, including the wpk rat carrying a naturally occurring single point mutation in the Tmem67 gene, the bpck mouse with the causative Tmem67 gene encompassed in a large 245-kb deletion, and even a sheep model.1, 8–10 The murine models capture some characteristics of MKS3 patients, such as polycystic kidneys and hydrocephalus, but not encephalocele, biliary abnormalities, polydactyly, while the sheep has the characteristic hepatorenal disease.8–12 A targeted knockout mouse (Tmem67tm1(Dgen/H)) has also been generated that exhibits pulmonary hypoplasia, cardiac malformation, kidney cysts, and some encephalocele and dies by postnatal day 1 (P1) due to pulmonary and/or cardiac defects.13–15 Of note, an effective therapy for MKS has not yet been reported.
TMEM67 encodes a transmembrane protein (meckelin) that is localized in the ciliary transition zone of the renal epithelium;1, 13, 16 thus, TMEM67-based polycystic kidney disease (PKD) is considered a ciliopathy. However, a range of ciliary phenotypes has been noted among different MKS3 models. For instance, bpck mice, wpk rats, and human MKS3 fetuses have elongated renal cilia; murine Tmem67 mutants exhibit shorter and fewer cilia than wild-type mice; and ovine TMEM67 mutants display both very long and very short cilia in their cystic kidneys.9–11, 13 Moreover, compared with those from wild-type mice, MEFs derived from Tmem67 knockout mice present longer, normal, or no cilia; similar findings have been obtained with Tmem67 shRNA-expressing cells.11, 13, 14, 17 Therefore, additional studies are needed to define ciliary defects in MKS3 models and to clarify the relationship between ciliary defects and cyst development.
Embryonic zebrafish have long been used to study PKD owing to their transparency and the efficiency of genetic manipulation.18–21 Embryonic zebrafish have also proven to be excellent models for in vivo analyses of ciliogenesis and cilium maintenance.22 The cilia in the zebrafish pronephric kidney include single motile cilia arising from the majority of epithelial cells and cilia arranged in multicilia bundles existing in the proximal straight tubule (PST) and distal early (DE) segment.23–26 Single-ciliated cells (SCCs) and multiciliated cells (MCCs) form an intercalated ‘salt and pepper” pattern in the PST-DE region that is controlled by Notch signaling.25, 26
Because the pathogenesis of PKD cannot be fully recapitulated during 8 days of embryogenesis, we turned our attention to adult zebrafish. The adult zebrafish mesonephric kidney undergoes similar branching morphogenesis and segment organization (proximal tubule (PT), distal tubule (DT), and collecting duct (CD)) as the mammalian kidney, although it contains much fewer nephrons (~200 vs 1 million in humans) and lacks the loop of Henle. Because the loop of Henle functions to preserve water in mammals, it is unnecessary for freshwater zebrafish to have this segment.27–30 Whether adult zebrafish can be used to model PKD and to develop therapies has not been explored.
Here, we report the generation of a zebrafish tmem67 mutant and the characterization of its renal and ciliary phenotypes. We noted pronephric cysts in the embryos and mesonephric cysts in the adult fish; thus, we have established the first adult zebrafish model of PKD. We defined ciliary abnormalities during embryogenesis and adulthood and correlated these defects with cyst development. Finally, we revealed that mTOR inhibition is a candidate therapeutic strategy for Tmem67-associated renal cyst formation.
Methods
Zebrafish strains
Zebrafish (WIK) were maintained under standard laboratory conditions, and experiments were carried out in accordance with the policies of the Mayo Clinic Institutional Animal Care and Use Committee. A hypomorphic mtor strain was identified from our insertional mutagenesis screen.31
Zebrafish tmem67 mutants were generated using the Golden Gate TALEN assembly protocol and library.21, 32–34 TALEN mRNAs were injected into embryos at the one-cell stage. F1 adults carrying germline mutations were identified by PCR (forward primer: 5′-TGTATAGGACTGGCATGTGAG-3′; reverse primer: 5′-AGGGATTGCCATTCCCATC-3′) and XmnI restriction enzyme digestion. Frameshift mutations were identified by sequencing, and the zebrafish were further outcrossed to reduce potential off-target effects. All experimental fish used in this study were F3 or F4 animals.
In situ hybridization
Whole-mount in situ hybridization was performed as previously described using riboprobes that were generated from T7 promoter sequence-tagged PCR products.35
Histological analysis
Zebrafish embryos were analyzed by hematoxylin and eosin (HE) staining as we have described previously.21 Adult zebrafish kidneys were also collected as described in another study.36 Briefly, each fish was euthanized with 0.2% Tricaine. All of its internal organs were removed except the kidney, which was attached to the dorsal wall of the abdominal cavity. The fish body with the kidney was then fixed in 4% paraformaldehyde (PFA) overnight at 4°C. On the next day, the kidney was carefully detached from the abdominal wall and subjected to paraffin embedding and HE staining. If a tubule was dilated to an area exceeding 0.03% of the total kidney area, it was considered a cyst. The areas of renal cysts as percentages of the total tissue area were calculated using ImageJ software.
Immunofluorescence labeling
Cilia in zebrafish embryos were visualized by whole-mount immunofluorescence staining using antibodies against acetylated α-tubulin (Sigma-Aldrich), as previously described.37, 38 Antibodies against α6F (Developmental Studies Hybridoma Bank) were included for staining of the pronephros.
Immunofluorescence analysis of adult zebrafish kidneys was performed on cryo-sections as described previously.21, 28, 29 The kidneys were dissected, fixed in 4% PFA/0.1% DMSO overnight at 4°C and then permeabilized with 5% sucrose for 30 minute followed by 30% sucrose overnight. The following day, the kidneys were embedded in tissue freezing medium (Electron Microscopy Sciences) and cryo-cut at 10 μm (Leica CM3050S). Immunofluorescence analysis of adult zebrafish kidneys was also conducted on paraffin sections as described previously.39 Renal tubular segments were labeled with alkaline phosphatase (AP, Invitrogen), rhodamine Dolichos biflorus agglutinin (DBA), and Lotus tetragonolobus lectin (LTL) (Vector Laboratories). Nuclei were labeled with SYTOX, DAPI (Vector Laboratories), or propidium iodide (PI) (Sigma-Aldrich). Anti-PCNA and anti-acetylated α-tubulin antibodies (Sigma-Aldrich) were used for immunostaining. Images were acquired using a Zeiss Axioplan II microscope equipped with ApoTome and AxioVision software (Carl Zeiss Microscopy).
Ultrastructural analysis
Adult zebrafish kidneys were fixed in Trump fixative (4% PFA, 1% glutaraldehyde). The remaining procedures for transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were performed according to standard methods at the Electron Microscopy Core Facility of the Mayo Clinic in Rochester, Minnesota.
Western blotting
Adult zebrafish kidneys were homogenized in RIPA lysis buffer (Sigma-Aldrich) as described previously.21 The following antibodies were used for western blotting: anti-S6 ribosomal protein (Cell Signaling Technology), anti-phospho-S6 ribosomal protein (Ser240/244) (Cell Signaling Technology), and anti-actin (Sigma-Aldrich).
Rapamycin treatment of adult zebrafish
Rapamycin (LC Laboratories) was administered at the specified dose to adult zebrafish via oral gavage as described previously.40 Treatment was conducted daily and lasted for one month.
Statistical analysis
The data are presented as the mean ± s.d. Comparisons between two groups were performed with two-tailed Student’s t tests, and a P value <0.05 was considered to indicate significance.
Results
tmem67e3/e3 embryos exhibit pronephric cysts with partial penetrance
tmem67, the only zebrafish homologue of mammalian TMEM67, is located on chromosome 16 (Figure 1A).12 Its transcript was detected in the kidney, neural tube, otic vesicle, brain, and retina during embryogenesis (Figure 1B) and in all major organs of adult fish (Supplemental Figure 1). To investigate the role of TMEM67 in renal cyst formation and ciliogenesis, we designed TALEN pairs targeting exon 3 of tmem67, because pathogenic mutations are mostly found in the exons encoding the N-terminal domains of meckelin.3, 41, 42 We analyzed two tmem67 mutant alleles that resulted in a premature stop codon, including the M1 allele containing a 1-bp insertion and the M2 allele containing a 5-bp deletion (Figure 1A,C). Because both alleles exhibited the same phenotypes, we have presented data only from the M1 allele and have renamed this allele tmem67e3.
In tmem67e3/e3 embryos, tmem67 transcript levels were reduced by nearly 40%, likely due to nonsense-mediated mRNA decay (Figure 1D). We could not quantify meckelin levels due to the lack of antibodies recognizing the zebrafish protein. Approximately 40% of the tmem67e3/e3 embryos exhibited ventral body curvature, and 20% developed pronephric cysts; furthermore, cystic embryos were more likely to be curved than non-cystic embryos (Figure 1E-G). Unlike rodent knockout animals and zebrafish morphants, the tmem67e3/e3 mutants did not exhibit apparent defects in convergence extension or neural tube development and rarely developed hydrocephalus (Supplemental Figure 2 and data not shown).8, 9, 12, 14 We assessed the possibility that the mutations were hypomorphic but we did not note any skipping of the targeted exon via alternative splicing events (Supplemental Figure 3).
tmem67e3/e3 embryos progressively lose single cilia in the pronephros
Next, we assessed renal cilia. We noted significantly shorter cilia in both the proximal and distal pronephron segments in tmem67e3/e3 embryos compared to wild-type at 26 hours post fertilization (hpf) (Figure 2A,F), when developing pronephros do not yet have filtration functions and when multicilia clusters are not yet present.18, 25, 26 Because pronephric cysts are visually detectable at 2 days post fertilization (dpf),18 manifesting as dilations in the glomerulus-neck region, we grouped the mutants into those with cysts (designated as +EC) and without cysts (designated as −EC). Compared with wild-type organisms, both +EC and −EC mutants had significantly shorter and fewer single cilia at the segments caudal to the multiciliated region (Figure 2B,E,G,H) and had less but only marginally shorter single cilia at the segments rostral to the multiciliated region (Figure 2B,C,G). The overall reductions in both cilium length and number were comparable between +EC and −EC embryos (Figure 2G-H). In contrast, multicilia bundles and MCCs, as indicated by odf3b expression, remained normal at this stage (Figure 2B,D,I-K). At 4 dpf, distal single cilia were absent from all mutants, but the number of multicilia bundles continued to be normal; however, the numbers of cells with MCC fates were increased in the +EC embryos, suggesting that the number of cilia bundles may increase at a later time (Supplemental Figure 4). In contrast to the pronephros, other tissues, such as Kupffer’s vesicle and the neural tube, had unaffected cilia (Supplemental Figure 5).
The +EC embryos showed failure of anterior migration and convolution of tubular epithelial cells (Supplemental Figure 6A). Consistently, these embryos had a condensed MCC domain and shortened PST and DE segments (Figure 2K, Supplemental Figures 4D and 6B,C). Because anterior migration and convolution require fluid flow in the tubule,24, 43–45 these data suggest impaired renal fluid movement in the +EC embryos, which could contribute to pronephric cyst formation.
Adult tmem67e3/e3 fish develop progressive mesonephric cysts
Most tmem67e3/e3 embryos, including cystic ones, survived for at least 15 months, with approximately 40% of fish exhibiting wavy bodies (Figure 3A). Independent of body shape, the mutant kidneys were significantly enlarged (Figure 3B,C), and the renal tubules were progressively dilated in male fish and, to a much less extent, female fish (Figure 3D,E and data not shown). Because the renal phenotype was milder than that in rodent models, we examined the expression of other MKS genes. While tmem67 transcript levels were reduced by approximately 50%, mks1, cep290, and cc2d2a were activated in the mutant kidney, suggesting genetic compensation that has been associated with TALEN-mediated mutagenesis (Supplemental Figure 7).46–48 Biliary abnormalities were not observed in the tmem67 fish up to 12 months.
In rodent Tmem67 models, renal cysts present as a mixture of PT dilation and CD enlargement in newborns and are mainly restricted to the CD at the later stage.8, 9 A similar progression was noted in the tmem67e3/e3 zebrafish. The proportion of PT cysts, marked by AP, decreased from 85% of all cysts to 33% from 6 to 12 months; DT cysts, labeled by DBA, slightly increased; and AP/DBA(−) cysts, presumptively a mixture of CDs and tubules with dedifferentiated epithelial cells, significantly increased (Figure 4A,B). We further costained samples with DBA and LTL, which labels PTs as well as the major CDs in the zebrafish kidney,28, 29 and found that the numbers of LTL cysts only moderately reduced and the numbers of unstained cysts moderately increased with time, indicating more CD cysts at a later stage (Figure 4C,D).
One of the characteristics of mammalian PKD is increased proliferation of cyst-lining epithelial cells.49, 50 Consistently, we detected more PCNA-positive cells in the tmem67e3/e3 mutants than in those of wild-type fish (Figure 5A,B). Hyperproliferation was observed after cyst formation and was correlated with disease severity (Figures 5B and 7B).
The mesonephric kidneys of tmem67e3/e3 fish have shorter and fewer single cilia but more MCCs than wild-type fish
Because adult zebrafish kidney cilia have not been characterized, we conducted EM analysis. In agreement with the light microscopic observations,27, 29 we found that some tubules were lined with thick brush borders, which are indicative of PT segments (Figure 6A,A’), while others had scanty microvilli and could not be clearly distinguished as DTs or CDs (Figure 6B,B’). Cilia bundles were observed in PTs, as noted in other teleost fish, but also in DTs/CDs (Figure 6A,A’,B,B’,G).51 Approximately 20-30 cilia measuring 100-150 μm in length were typically clustered together with the distinctive “9+2” motile cilia structure (Figure 6C,G). Single cilia were scarcely detected by SEM (Figure 6H), probably because longitudinal sections were rarely obtained from the thin kidney tissues. In the tmem67e3/e3 kidneys, the epithelial cells in dilated PTs often had loose brush borders (Figure 6D,D’). Clusters of 100-150-μm-long cilia also resided in the PTs and DTs/CDs and retained the 9+2 organization (Figure 6D-F,I). Notably, the mutants appeared to have shorter single cilia than the wild-type fish, although only a few were identified (Figure 6J), and contained more cilia bundles in the PT segment (Figure 6K).
In parallel, we examined single cilia by immunostaining. We noted that single cilia were shorter in DTs than in PTs and AP/DBA(−) tubules, and the latter two had comparable cilium lengths (Figure 6L,N). In tmem67e3/e3 fish, the single cilia were stunted, and the numbers of SCCs were reduced in all segments assessed (Figure 6M-O). Consistent with EM analysis, cilia bundles, as specified by strong α-tubulin staining, were present in all tubular segments and were significantly increased in number in the mutants (Figure 6L,M,P). Together, our data reveal the occurrence of consistent ciliary defects in tmem67e3/e3 embryos and adult fish.
Ciliary defects are more prominent than cyst development in the mesonephric kidneys of adult tmem67e3/e3 fish
To assess whether pronephric tubule dilation affects mesonephric cyst formation, we raised +EC embryos separately from −EC embryos. The cystic index in +EC fish progressively increased from 4 to 12 months; however, surprisingly, it was lower at 4 months in +EC fish than in −EC fish (Figure 7A). Consistently, epithelial cell hyperproliferation was detected only in −EC kidneys (Figure 7B). Thus, we conclude that cyst development during embryogenesis does not predispose tmem67 fish to mesonephric cyst formation.
Prompted by the significant difference in cyst formation between +EC and −EC fish at 4 months of age, we compared their renal cilia. We found more cilia bundles in the LTL tubules in +EC animals than in −EC animals, although the numbers of MCCs were also substantially increased in the −EC group (Figure 7C,D). On the other hand, the single-cilium length and number were similarly reduced in +EC and −EC fish (Figure 7C,E,F). Taken together, our data suggest that the loss of single cilia is a primary defect of cystogenesis, while MCC expansion might be a later compensatory event.
mTOR inhibition ameliorates cyst formation, increased proliferation of cyst-lining cells, and ciliary abnormality in tmem67e3/e3 fish
Because the mTOR pathway is often activated and mTOR inhibition is therapeutic in PKD models,52–54 we examined mTOR activity in tmem67e3/e3 fish. Indeed, the S6 ribosomal protein was hyperphosphorylated in both +EC embryos and adult mutants (Figure 8A, Supplemental Figure 8A). We also found mTOR activation in bpck mice, suggesting a conserved mechanism (Supplemental Figure 9).
To test whether mTOR inhibition exerts beneficial effects, we treated zebrafish with rapamycin. We found that rapamycin alleviated pronephric cysts in +EC embryos and diminished mesonephric cysts in the adult mutants, without causing apparent defects on wild-type fish (Figure 8B,C, Supplemental Figure 8B). Moreover, genetic inhibition of mTOR signaling with a hypomorphic mtor+/− mutant also significantly inhibited cyst growth in the adult fish (Figure 8D).31 At the cellular level, we noticed that hyperproliferation of renal epithelial cells was reversed, the numbers of MCCs were normalized, and the single-cilium lengths and percentages of SCCs were restored in tmem67e3/e3;mtor+/− double mutants (Figure 8E-H). However, given that short-term rapamycin treatment was unable to alleviate ciliary defects in the embryos (Supplemental Figure 8C,D), the therapeutic benefit of sustained mTOR inhibition likely does not occur via direct regulation of ciliogenesis.
Discussion
The adult zebrafish tmem67 model exhibits key features of mammalian PKD
In this study, we have demonstrated, for the first time, that renal cystogenesis and cilium synthesis can be studied in adult zebrafish, thus establishing a new vertebrate model of PKD that can be used for therapeutic development. Conserved pathogenic processes were noted in adult tmem67e3/e3 fish mesonephric kidneys, including progressive cystogenesis, an age-dependent switch in tubular cyst origin, and increased cell proliferation. Unlike zebrafish embryonic models, the adult model provides a platform for the quantification of cyst size and number. Notably, by following fish with or without pronephric cysts to adulthood, we found that embryonic cysts do not predispose but rather delay mesonephric cystogenesis in young adults. While we cannot rule out the possibility of distinct mechanisms underlying pronephric and mesonephric cyst formation, our observations underscore the additional research opportunities offered by our adult fish model, which can be used to study longitudinal progresses consisting of both compensatory and decompensatory processes.
Compared to MKS3 patients and related rodent models, the adult zebrafish tmem67 mutants exhibited milder cystic phenotypes. This difference is unlikely an issue related to freshwater species per se, since naturally occurring pc/glis3 mutants of medaka, another freshwater teleost, develop massive fluid-filled cysts within 6 months.51, 55 We hypothesize that this difference can be explained by at least two mechanisms. First, zebrafish have a strong regenerative capacity, not only repairing nephrons (as mammals do) but also forming new ones de novo.56–60 Because impairments in renal tubule repair after injury have been suggested to trigger cystogenesis,61, 62 an elevated regeneration potential would certainly help to slow disease progression. Second, TALEN/CRISPR-mediated mutagenesis has recently been shown to sometimes induce genetic compensation by family members of the targeted gene or other genes in the same biological pathway.46, 47 Indeed, transcriptional adaptation of other MKS genes was noted in the tmem67 kidneys. Thus, identification of critical regeneration/compensation genes or pathways in this fish model might suggest new therapeutic interventions for MKS3 treatment in mammals.
Role of ciliary abnormality in the zebrafish tmem67 models
In contrast to the partial penetrance of pronephric cysts, all mutant embryos uniformly exhibited defective single cilia in the distal pronephros, even before cyst formation. The loss of single cilia phenotype also extended to other renal tubular segments of adult fish, including +EC fish at the precyst stage of 4 months. The fact that a lack of cilia preceded cyst development strongly suggests that abnormal ciliogenesis is a primary defect in the tmem67 model.
On the other hand, multicilia clusters seemed unaffected before 4 dpf. This observation could explain the absence of pronephric cysts in most tmem67 embryos, because fluid can be propelled out of the pronephros by beating multicilia.23, 25 The +EC population likely has additional as-yet-unidentified defects in fluid output. In adult zebrafish kidneys, MCCs were present in all tubular segments, suggesting their roles in normal renal physiology, which are different from those in mammals. MCCs are rarely detected in adult mammals; however, they have been observed in human fetuses, patients with hypercalcemia or nephrotic syndrome, and rodent models of ARPKD.11, 14, 63–68 With regard to TMEM67 in particular, more MCCs have been noted in human MKS3 fetuses than in normal fetuses;11 in addition, multiple centrosomes and more than one cilium have been detected in wpk rat and Tmem67-deficient cells in vitro, though further investigation is required to determine whether these cells are MCC-like.11, 14 Nonetheless, the reappearance of MCCs in diseased kidneys indicates their importance and has been postulated as an adaptive event to relieve local fluid accumulation.69 Our data indicating that +EC fish have more MCCs from 4 dpf to 4 months and fewer renal cysts at 4 months than −EC fish provide the first experimental evidence to support this hypothesis.
The zebrafish tmem67 model recapitulates tissue-specific single cilium loss and MCC induction but not the ciliary elongation phenotype present in some mammalian models.9, 11, 13, 15 Cilium length elongation has also been proposed as an adaptive response.70, 71 Whether such an adaptive event predominantly presents as cilium lengthening in mammals and MCC expansion in zebrafish due to species-specific expression of ciliogenic factors and/or different susceptibility to cell fate transition warrants further study.
The adult zebrafish MKS3 model facilitates the identification of candidate therapeutic strategies such as mTOR inhibition
Consistent with findings in PKD patients and various animal models, we noted hyperactivation of the mTOR pathway and beneficial effects of mTOR inhibition in both tmem67 embryos and adult fish. Because mTOR is also activated in bpck mice, mTOR inhibition is anticipated to also be beneficial in rodent MKS3 models. Our results thus support the hypothesis that abnormal mTOR signaling is a common pathological event for PKDs of different etiologies and that mTOR inhibition is a broadly applicable therapeutic strategy.
mTOR signaling has been shown to regulate cilium biogenesis, but the conclusions of the related studies have not always been consistent, and the relationship between mTOR and cilia during cystogenesis remains elusive.72–78 We found that long-term mTOR inhibition normalized ciliary abnormalities in adult fish, while short-term inhibition in embryos did not, suggesting an indirect role of mTOR in ciliogenesis. Therefore, we favor the hypothesis that sustained mTOR inhibition exerts therapeutic benefits via a cilia-independent mechanism. Future studies using the adult zebrafish model are anticipated to uncover this mechanism, which could potentially be shared among PKDs of different etiologies, thus facilitating our understanding of the interplay among mTOR signaling, ciliogenesis, and cystogenesis.
Author Contributions
X.L. and X.X. designed the study; P.Z., Q.Q. and X.L. carried out experiments; P.Z. and X.L. analyzed the data and made the figures. X.L., X.X. and P.C.H. drafted and revised the paper; all authors approved the final version of the manuscript.
Acknowledgements and Financial Disclosures
We thank Bingquan Huang and Scott I. Gamb of the Mayo Clinic Electron Microscopy Core Facility for expert TEM and SEM assistance, Amanda C. Leightner for kindly providing us with mouse kidney samples. This work was supported by the Mayo Translational Polycystic Kidney Disease Center (MTPC) Pilot and Feasibility grant (NIDDK DK90728) (to X.L.) and the Mayo Foundation for Medical Education and Research (to X.X.).
Disclosures: None
Table of Contents for the Supplemental material
Supplemental Figure 1. tmem67 is expressed in multiple tissues of the adult zebrafish
Supplemental Figure 2. Small percentage of tmem67e3/e3 embryos develops hydrocephalous
Supplemental Figure 3. tmem67e3/e3 mutants do not show exon skipping events
Supplemental Figure 4. Ciliary and MCC defects in tmem67e3/e3 embryos at 4 dpf
Supplemental Figure 5. Cilium lengths are not significantly altered in the Kupffer’s vesicle and neural tubes
Supplemental Figure 6. Anterior migration of the pronephric epithelial cells and convolution of the proximal tubules are defective in tmem67e3/e3 embryos
Supplemental Figure 7. The expression of other MKS genes in tmem67e3/e3 kidneys
Supplemental Figure 8. The effect of rapamycin on pronephric cyst formation and cilium biosynthesis in tmem67e3/e3 embryos
Supplemental Figure 9. mTOR is activated in bpck mice