Nucleoporin 133 deficiency leads to glomerular damage in zebrafish

Although structural nuclear pore proteins (nucleoporins) are seemingly required in every cell type to assemble a functional nuclear transport machinery, mutations or deregulation of a subset of them have been associated with specific human hereditary diseases. In particular, previous genetic studies of patients with nephrotic syndrome identified mutations in Nup107 that impaired the expression or the localization of its direct partner at nuclear pores, Nup133. In the present study, we characterized the zebrafish nup133 orthologous gene and its expression pattern during larval development. Morpholino-mediated gene knockdown revealed that Nup133 depletion in zebrafish larvae leads to the formation of kidney cysts, a phenotype that can be rescued by co-injection of wild type mRNA. Analysis of different markers for tubular and glomerular development shows that the overall kidney development is not affected by nup133 knockdown. On the other hand, we demonstrate that nup133 is essential for the organization and functional integrity of the pronephric glomerular filtration barrier, as its downregulation results in proteinuria and moderate foot process effacement, mimicking some of the abnormalities typically featured by patients with nephrotic syndrome. These data indicate that nup133 is a new gene required for proper glomerular structure and function in zebrafish.


Introduction
Efficient and regulated bidirectional transport between the cytoplasm and the nucleus is an essential process in all eukaryotic cells. This function is achieved by nuclear pore complexes (NPCs), huge assemblies anchored within the nuclear envelope and composed of about 30 different proteins, termed nucleoporins (Nups) (reviewed in 1 ). Despite the universal role of NPCs in all nucleated cells, some Nups are linked to human hereditary diseases affecting specific cell types or organs (reviewed in 2,3,4 ).
In particular, genetic studies have implicated a restricted number of structural nucleoporins in specific kidney diseases termed nephrotic syndromes (NS). NS arise from defects or damages that impair the selectivity of the glomerular filtration barrier and lead to massive proteinuria and hypoalbuminemia, which in turn cause edema and dyslipidemia. The glomerular filtration barrier (GFB) surrounds the glomerular capillaries and comprises three layers: (i) a fenestrated endothelium, (ii) a basement membrane, and (iii) the podocytes. The latter are highly specialized epithelial cells characterized by long and thin cytoplasmic projections, termed foot processes (FPs), that interdigitate and are connected by specialized cell-cell junctions, the slit diaphragms (reviewed in 5 ). While most patients with childhoodonset NS respond well to steroid treatments, 10-20% of the affected children do not achieve remission upon corticosteroid therapy. Steroid-resistant NS (SRNS) is associated with a high risk of progression to end-stage renal disease (ESRD) 6 . It frequently manifests histologically as focal segmental glomerulosclerosis (FSGS), characterized by scattered scarring of some glomeruli and is often associated with retractions ("effacement") of podocytes foot processes (reviewed in 7 ).
Although nonhereditary forms of SRNS seem to be prevalent, studies over the last years have identified over 50 dominant or recessive single-gene mutations in a significant percentage (30%) of patients with early-onset SRNS and FSGS (reviewed or discussed in 6,[8][9][10][11] ). While some of these genes have podocyte-specific or -restricted functions, these studies also unveiled the implication of multiple cellular processes in the establishment or maintenance of the glomerular filtration barrier 7,12,13 . In particular, these genetic studies have identified mutations in Nup93 and Nup205, two constituents of the inner ring of the NPC 14 and in Nup107, a constituent of the Y-complex (Nup107/160-complex) that builds up the cytoplasmic and nuclear rings of the NPC 15,16,17 . Mutations within Nup107 were also identified in patients with a rare co-occurrence of microcephaly with nephrotic syndrome, similar to Galloway-Mowat syndrome (GAMOS) 18 . While patients with GAMOS-like presentation had a strong reduction in Nup107 protein level accompanied by decreased levels of Nup133, its direct partners within the Y-complex 18 , another SRNS-linked mutation affecting Nup107 was shown to impair its interaction with Nup133 16 . These data thus pointed towards a possible implication of Nup133 in NS.
In mice, a previous characterization of a Nup133 null (mermaid, or merm) mutant revealed that mouse embryos lacking a functional Nup133 allele developed through midgestation but die at e9.5-e10.5 19 . While this indicates that Nup133 is not an obligate NPC component, merm mutants displayed abnormalities in a number of tissues, indicating that cell differentiation towards several epiblast-derived lineages likely requires Nup133 19 . However, the possible contribution of Nup133 to kidney development or function has never been assessed. To address this question, we used morpholino-mediated nup133 inactivation in zebrafish (Danio rerio, Dr), a well-established vertebrate model to study kidney development and model renal diseases [20][21][22][23] . We report here that knockdown of zebrafish nup133, while not impairing early stages of kidney development, leads to glomerular abnormality that mimic nephrotic syndrome.

Results
Zebrafish nup133 ortholog is broadly expressed at early stages and becomes restricted to specific tissues at later stages  (Fig. 1b). At 3 days post fertilization (dpf), in addition to a diffuse staining notably in the brain, we found evidence of nup133 mRNA enrichment in the liver, in the intestine and in neuromasts of the lateral line organ (Fig. 1c,d). At 5 dpf, the overall expression of nup133 is weaker, but the mRNA is still visible in the brain, and enriched in the liver, the intestine and the swim bladder (Fig. 1e,f). Cross sections at 4 and 5 dpf confirmed the enrichment of nup133 mRNA in the liver and highlighted the presence of nup133 mRNA also in the pronephric proximal tubules and the glomerulus (Fig. 1g,h). Because structural nucleoporins were reported to be long-lived proteins, with a very low turnover in at least some non-dividing cells 24,25 , we next sought to determine whether higher nup133 mRNA expression in the larvae was associated with highly proliferating tissues. To evaluate cell proliferation, we treated 5 dpf zebrafish larvae with the thymidine analog 5-ethynyl-2′deoxyuridine (EdU) for 1 h and assayed for EdU-positive cells in the different tissues of the larvae. As shown in Figure 1i, EdU positive cells are more frequent in the developing fins, the intestine and the liver, that with exception to the liver, do not correspond to tissues with major enrichment of nup133 mRNA. This indicates that nup133 expression in zebrafish is not directly correlated with cell proliferation.

nup133 knockdown causes glomerular cysts in zebrafish
In order to characterize the in vivo function of nup133 in zebrafish larvae, we generated . This demonstrates that the glomerular phenotype observed in nup133 morphants is due to a specific effect of nup133 knockdown.

nup133 morphants properly express molecular markers of kidney development
We next wanted to determine whether the glomerular expansion in nup133 morphants is caused by a developmental defect of the glomerulus and/or of the pronephros. For this purpose, we used whole-mount ISH to screen established markers of pronephros and glomerulus development. We first assayed expression of the intermediate mesoderm marker pax2a (paired box gene 2a) at 24 hpf. pax2a is expressed during early somitogenesis in the developing pronephric tubules and is important in establishing the boundary between podocytes and the neck segment of the nephron 27,28 . In nup133 morphants, expression of pax2a at 24 hpf was similar to that of wild type embryos, suggesting that the tubular development is not compromised in the morphants (Fig. 3a). We further examined the pronephric tubules and ducts in nup133 knockdown embryos by checking the expression of the kidney specific marker cadherin 17 (cdh17) at 24 hpf and 72 hpf. Again, no obvious difference in expression of cdh17 was observed between control and nup133 morphant larvae at 24 or 72 hpf (Fig. 3b). Based on these markers, we conclude that tubular development is not impaired upon nup133 knockdown.
We next used an mRNA probe for Wilms tumor suppressor 1a (wt1a) that is predominantly expressed in podocytes throughout pronephric development 29 . At 24 hpf, wt1a is still expressed in two distinct domains, the glomerular primordia, that fuse to form a single glomerulus by 40 hpf 30,31 . In nup133 morphants, expression of wt1a was comparable at 30 and 48 hpf to that of wild type embryos (Fig. 3c). wt1a expression sometimes revealed some glomerular enlargement already at 48hpf, a phenotype that became more evident at 58 hpf, after the onset of the glomerular filtration (Fig. 3c, bottom panels). Nevertheless, wt1a transcripts were still expressed in nup133 morphants podocytes. These results suggest that knockdown of nup133 does not affect the gross development of zebrafish glomerulus. were imaged. In the control larvae (n= 6, arising from two distinct experiments), we did not observe fluorescent signal from the injected BSA in the apical endosomes of the proximal tubules, indicating that BSA was not able to pass through the intact filtration barrier (Fig. 4, left panels). In most (5/6 imaged larvae) nup133 morphants, however, fluorescently labeled endosomes were detectable in the proximal tubules (Fig. 4, right panels). This suggests that the filtration barrier of the glomerulus is impaired upon nup133 knockdown, allowing the passage of macromolecules with a size exceeding the size selectivity of an intact GFB, a condition defined in patients as proteinuria.

nup133 knockdown results in podocyte FPs effacement
In order to determine whether the glomerular filtration impairment observed in nup133 morphants was a consequence of a defect in the glomerular filtration barrier (GFB), we conducted electron microscopy studies to compare the ultrastructure of the glomerulus in nup133 morphants and wild type larvae at 5 dpf, a time point when the larval glomerulus development is considered nearly complete 30,35 . In wild type larvae, the glomerulus showed the typical organization with podocytes harboring well-developed interdigitated foot processes on the outer side, and fenestrated endothelial cells on the inner side of the GBM (Fig. 5b, left panels). In nup133 knockdown larvae, however, a moderate foot process effacement was observed in many areas of the glomerulus, with no detectable alteration of the fenestrated endothelial cells (Fig. 5b, right panels). These data thus indicate that zebrafish nup133 contributes to the structural integrity of the GFB.

Discussion
In this study we used zebrafish as model organism to evaluate the contribution of Nup133 to vertebrate kidney function. Using MO-based depletion of Nup133 followed by rescue experiments with nup133 mRNA, we have identified nup133 as a new regulator of glomerular structure and functional integrity.
While we report here a kidney-specific alteration caused by Nup133 depletion in zebrafish, more severe developmental phenotypes were previously observed upon Nup133 inactivation in mouse 19 . This likely reflects the complete absence of Nup133 protein in the mouse mutant as compared to its incomplete MO-mediated depletion in zebrafish. Of note, the extent of depletion of structural nucleoporins may also lead to distinct phenotypes as revealed by previous studies of zebrafish nup107. Indeed, while MO-induced depletion of zebrafish Nup107 was reported to cause glomerular defects including podocytes foot processes effacement in one study 16  Nup133 knockdown may also alter nuclear mechanotransduction, for instance by interfering with the 'linker of nucleoskeleton and cytoskeleton' (LINC) complex that establish a stable and cross-linked network between the cytoskeleton and the lamina underneath the NE (reviewed in 41 ). Indeed, while Nup133 is required for the proper assembly of the NPC basket 42 , intimate links between the NPC basket, the LINC subunit Sun1 and Lamin-C have been reported 43,44 . As previously discussed 16  Since appearance of kidney cysts in zebrafish can also be caused by inactivation of several ciliopathy genes (reviewed in 51,52 ), the hypothesis that Nup133 depletion may contribute to kidney cysts formation in zebrafish by affecting cilia, might deserve attention in the future.
So far, the only Y-complex nucleoporin gene found to be mutated in SRNS is Nup107 [15][16][17][18] . Our data now indicate that Nup133, its direct partner within the Y-complex, is also required for proper glomerular function in zebrafish. In addition, a recent study revealed that normal expression level of another Y-complex component, Nup160, is critical for the proliferation and viability of podocytes in vitro 53 . These data indicate that in addition to Nup107, at least these two other Y-Nups, or possibly all the nine Y-complex subunits (Nup107, Nup133, Nup160, Nup96, Nup85/75, Nup43, Nup37, Sec1, and Sec13) are candidate genes that would be worth testing in SRNS patients for which the mutated gene is yet unidentified. Our study, together with an increasing number of studies correlating single-gene mutations in Nups with cell type-specific defects, thus contributes to strengthen the rise of the "nucleoporopathies" concept 1 an exciting development in the nuclear pore complex field.

Fish maintenance and breeding
Zebrafish (Danio rerio) were kept at 26°C under a 14-h/10-h light/dark cycle and bred as previously described 54 . Larval stages were raised at 28°C in E3 embryo medium (

Whole-mount in situ hybridization
Sequences were identified and annotated using combined information from expressed sequence tags and genome databases (GeneBank, http://www.ncbi.nlm.nih.gov; Ensembl, http://www.ensembl.org/index.html). The primers used for probe preparation are listed in supplementary Table S1. nup133, pax2a, wt1a and cdh17 cDNAs were all isolated by RT-PCR from total RNA from 24-48 hpf embryos and cloned into TOPO pCRII vector (TA Cloning Kit Dual Promoter, Invitrogen) as previously described 55 . The resulting plasmids were linearized for SP6 and T7 in vitro transcription and purified with phenol-chloroform.
Digoxigenin (DIG)-labeled antisense (or sense, as negative control for nup133 expression) RNA probes were generated using DIG-RNA-labeling kit (Roche Diagnostic). Zebrafish embryos were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C overnight and whole mount in situ hybridization was performed as previously described 56 .
Briefly, on day 1 the larvae were treated with proteinase K and then fixed with 4% paraformaldehyde (PFA) before prehybridization at 64°C. Hybridization of RNA probes was done at 64°C overnight. On day 2, after several stringency washes at 64°C, probes were blocked in 1× Roche blocking solution in Tris/NaCl/Tween. Anti-DIG AP antibody was applied overnight at 4°C. On day 3, after several washing steps, signal was detected by incubation in staining buffer. Stained embryos were postfixed with PFA and imaged in glycerol with an Olympus BX61 light microscope or with a stereomicroscope (Olympus MVX10). Images were processed and assembled using Adobe Photoshop and Adobe Illustrator CS6.

Cryosectioning
Embryos at desired stages were fixed in 4% PFA at 4°C overnight, cryoprotected in 30% sucrose overnight and embedded in tissue freezing medium (Richard-Allan Scientific Neg-50 Frozen Section Medium Thermo Fisher Scientific). 14-16 µm transverse sections were cut on a Microm HM 550 cryostat and imaged with a Olympus BX61 wide-field microscope (Volketswil, Switzerland).

EdU incorporation and detection
5 dpf zebrafish larvae were incubated in E3 medium containing 10 mM EdU in 1 % DMSO for 1h in the dark, followed by 1h recovery time in E3 medium. Larvae were fixed in 4% PFA for 1h, rinsed in PBT and processed for cryosections. EdU staining was then performed as previously described 57 .

Morpholino and RNA injections
The nup133 gene was targeted with specific antisense splice-blocking Morpholinos (MOs) (GeneTools, Philamath, OR) designed to target the exon-intron boundary (splice donor, nup133_E3I3) of exon 3 and the intron-exon boundary (splice acceptor, nup133_I3E4) of exon 4, respectively (MO sequences are provided in Supplemental Table S1). The morpholinos were diluted in RNase-free water with 0.1% phenol red as an injection tracer and ~1 nl was injected into fertilized embryos at 1-2 cell stage. Uninjected sibling embryos and embryos injected with standard control (SC) morpholinos were used as controls. Evaluation of morphological changes was performed at 3 dpf. A range of concentration was first used to determine the optimal MO amount required to induce a specific phenotype without inducing toxicity. The two MOs were then always injected at 3 mM each. To examine splicing defects caused by nup133_E3I3 and nup133_I3E4 injection, poly(A) RNA were extracted from control or MO-injected embryos at 1 dpf and RT-PCR and characterized by RT-PCR using primers designed from flanking exon-coding sequence (Figure 2a and Supplementary Table   S1 online) The RT-PCR products were purified from gel, re-amplified with nested primers and altered splicing was confirmed by sequencing.
For the rescue experiments, a pBluescript KSM vector containing 3xHA-mCherry-Dr nup133 was used. The plasmid was generated using PCR amplification using proofreading DNA polymerases (Phusion HF, NEB) and NEBuilder HiFi DNA Assembly Cloning Kits. Briefly, the sequence encoding three copies of the influenza virus hemagglutinin (HA) epitope was amplified from pFA6a-3HA-kanMX6 58 , the mCherry were amplified from plasmid #1937 42 , full-length zebrafish nup133 was amplified from a Dharmacon cDNA vector (Clone ID: 2600558) and the three PCR products were inserted by recombination in a pBluescript KSM vector (Stratagene). PCR-amplified fragments and junctions were checked by sequencing.
Plasmid map is available upon request. For in vitro mRNA production the 3xHA-mCherry-Dr nup133 coding fragment was linearized by digestion with the restriction enzyme NotI and purified with phenol-chloroform extraction. Capped and tailed RNA was in vitro transcribed using the mMessage mMachine T3 kit (Life Technologies, Zug, Switzerland) according to the manufacturer's instruction. A polyA tail was added with polyA tailing kit (Invitrogen by thermos Fischer Scientific) followed by purification with MEGAclear Transcription Clean-Up Kit (Ambion). The mRNA (300nM) was injected into the embryos at one cell stage before MO injection.

Whole mount live-animal imaging
To evaluate morphological changes, larvae were anesthetized with 200 mg/ml 3aminobenzoic acid methyl ester (MESAB, Sigma-Aldrich), mounted in 1.5% low melting temperature agarose in E3 medium and imaged using a stereomicroscope (Olympus MVX10) equipped with a color camera (ColorViewIII, Soft imaging System, Olympus).

Transmission electron microscopy and histology
Zebrafish larvae at 5 dpf were fixed in 2.5% glutaraldehyde in 0. Software.

Glomerular filtration assay
Zebrafish larvae at 4 dpf were anesthetized with 200 mg/ml 3-aminobenzoic acid methyl ester (MESAB, Sigma-Aldrich). 1 nl of Albumin from bovine serum (BSA), Alexa Fluor 647 conjugate (Thermoischer scientific) was injected into the common cardinal vein according to 34 . The larvae were transferred to E3 medium for recovery. 20 mins after injections, the larvae were fixed in 4% PFA overnight and processed for cryosections and imaging.     At 58 hpf (lower panels), after the onset of glomerular filtration, the increased area labeled by the wt1a probe (arrow) reflects the glomerular expansion observed in 7 out of 13 nup133 morphants analyzed. Scale bars, 500 µm.