Inversin (NPHP2) and Vangl2 are required for normal zebrafish cloaca formation.

Nephronophthisis (NPH), an autosomal recessive ciliopathy, results from mutations in more than 20 different genes (NPHPs). These gene products form protein complexes that regulate trafficking within the cilium, a microtubular structure that plays a crucial role in developmental processes. Several NPHPs, including NPHP2/Inversin, have been linked to extraciliary functions. In addition to defining a specific segment of primary cilia (Inversin compartment), NPHP2 participates in planar cell polarity (PCP) signaling along with Dishevelled and Vangl family members. We used the mutant zebrafish line invssa36157, containing a stop codon at amino acid 314, to characterize tissue-specific functions of zebrafish Nphp2. The invssa36157 line exhibits mild ciliopathy phenotypes and increased glomerular and cloaca cyst formation. These mutants showed enhanced susceptibility to the simultaneous depletion of the nphp1/nphp2/nphp8 module, known to be involved in the cytoskeletal organization of epithelial cells. Notably, simultaneous depletion of zebrafish nphp1 and vangl2 led to a pronounced increase in cloaca malformations in the invssa36157 mutant embryos. Time-lapse imaging showed that the pronephric cells correctly migrated towards the ectodermal cells in these embryos, but failed to form the cloaca opening. Despite these abnormal developments, cellular fate does not seem to be affected in nphp1 and vangl2 MO-depleted invssa36157 mutants, as shown by in situ hybridizations for markers of pronephros and ectodermal cell development. However, significantly reduced apoptotic activity was observed in this double knockdown model, signifying the role of apoptosis in cloacal morphogenesis. Our findings underscore the critical interplay of nphp1, nphp2/Inversin, and vangl2 in orchestrating normal cloaca formation in zebrafish, shedding light on the complex molecular mechanisms underlying ciliopathy-associated phenotypes.

MARVEL domain containing CMTM4 affects CXCR4 trafficking.

The MARVEL proteins CMTM4 and CMTM6 control PD-L1, thereby influencing tumor immunity. We found that defective zebrafish cmtm4 slowed the development of the posterior lateral line (pLL) by altering the Cxcr4b gradient across the pLL primordium (pLLP). Analysis in mammalian cells uncovered that CMTM4 interacted with CXCR4, altering its glycosylation pattern, but did not affect internalization or degradation of CXCR4 in the absence of its ligand CXCL12. Synchronized release of CXCR4 from the endoplasmic reticulum revealed that CMTM4 slowed CXCR4 trafficking from the endoplasmic reticulum to the plasma membrane without affecting overall cell surface expression. Altered CXCR4 trafficking reduced ligand-induced CXCR4 degradation and affected AKT but not ERK1/2 activation. CMTM4 expression, in contrast to that of CXCR4, correlated with the survival of patients with renal cell cancer in the TCGA cohort. Furthermore, we observed that cmtm4 depletion promotes the separation of cells from the pLLP cell cluster in zebrafish embryos. Collectively, our findings indicate that CMTM4 exerts general roles in the biosynthetic pathway of cell surface molecules and seems to affect CXCR4-dependent cell migration.

Clock genes rescue nphp mutations in zebrafish.

The zebrafish pronephros model, using morpholino oligonucleotides (MO) to deplete target genes, has been extensively used to characterize human ciliopathy phenotypes. Recently, discrepancies between MO and genetically defined mutants have questioned this approach. We analyzed zebrafish with mutations in the nphp1-4-8 module to determine the validity of MO-based results. While MO-mediated depletion resulted in glomerular cyst and cloaca malformation, these ciliopathy-typical manifestations were observed at a much lower frequency in zebrafish embryos with defined nphp mutations. All nphp1-4-8 mutant zebrafish were viable and displayed decreased manifestations in the next (F2) generation, lacking maternal RNA contribution. While genetic compensation was further supported by the observation that nphp4-deficient mutants became partially refractory to MO-based nphp4 depletion, zebrafish embryos, lacking one nphp gene, became more sensitive to MO-based depletion of additional nphp genes. Transcriptome analysis of nphp8 mutant embryos revealed an upregulation of the circadian clock genes cry1a and cry5. MO-mediated depletion of cry1a and cry5 caused ciliopathy phenotypes in wild-type embryos, while cry1a and cry5 depletion in maternal zygotic nphp8 mutant embryos increased the frequency of glomerular cysts compared to controls. Importantly, cry1a and cry5 rescued the nephropathy-related phenotypes in nphp1, nphp4 or nphp8-depleted zebrafish embryos. Our results reveal that nphp mutant zebrafish resemble the MO-based phenotypes, albeit at a much lower frequency. Rapid adaption through upregulation of circadian clock genes seems to ameliorate the loss of nphp genes, contributing to phenotypic differences.

Single-cell mRNA profiling reveals changes in solute carrier expression and suggests a metabolic switch during zebrafish pronephros development.

Developing organisms need to adapt to environmental variations as well as to rapid changes in substrate availability and energy demands imposed by fast-growing tissues and organs. Little is known about the adjustments that kidneys undergo in response to these challenges. We performed single-cell RNA sequencing of zebrafish pronephric duct cells to understand how the developing kidney responds to changes in filtered substrates and intrinsic energy requirements. We found high levels of glucose transporters early in development and increased expression of monocarboxylate transporters at later times. This indicates that the zebrafish embryonic kidney displays a high glucose transporting capacity during early development, which is replaced by the ability to absorb monocarboxylates and amino acids at later stages. This change in transport capacity was accompanied by the upregulation of mitochondrial carriers, indicating a switch to increased oxidative phosphorylation to meet the increasing energy demand of a developing kidney.NEW & NOTEWORTHY The zebrafish embryonic kidney has high levels of glucose transporters during early development, which are replaced by monocarboxylate and amino acid transporters later on. Inhibition of Na+-glucose cotransporter-dependent glucose transport by sotagliflozin also increased slc2a1a expression, supporting the idea that the glucose transport capacity is dynamically adjusted during zebrafish pronephros development. Concurrent upregulation of mitochondrial SCL25 transporters at later stages supports the idea that the pronephros adjusts to changing substrate supplies and/or energy demands during embryonic development.

Nephronophthisis gene products display RNA-binding properties and are recruited to stress granules.

Mutations of cilia-associated molecules cause multiple developmental defects that are collectively termed ciliopathies. However, several ciliary proteins, involved in gating access to the cilium, also assume localizations at other cellular sites including the nucleus, where they participate in DNA damage responses to maintain tissue integrity. Molecular insight into how these molecules execute such diverse functions remains limited. A mass spectrometry screen for ANKS6-interacting proteins suggested an involvement of ANKS6 in RNA processing and/or binding. Comparing the RNA-binding properties of the known RNA-binding protein BICC1 with the three ankyrin-repeat proteins ANKS3, ANKS6 (NPHP16) and INVERSIN (NPHP2) confirmed that certain nephronophthisis (NPH) family members can interact with RNA molecules. We also observed that BICC1 and INVERSIN associate with stress granules in response to translational inhibition. Furthermore, BICC1 recruits ANKS3 and ANKS6 into TIA-1-positive stress granules after exposure to hippuristanol. Our findings uncover a novel function of NPH family members, and provide further evidence that NPH family members together with BICC1 are involved in stress responses to maintain tissue and organ integrity.

CXCL12 and MYC control energy metabolism to support adaptive responses after kidney injury.

Kidney injury is a common complication of severe disease. Here, we report that injuries of the zebrafish embryonal kidney are rapidly repaired by a migratory response in 2-, but not in 1-day-old embryos. Gene expression profiles between these two developmental stages identify cxcl12a and myca as candidates involved in the repair process. Zebrafish embryos with cxcl12a, cxcr4b, or myca deficiency display repair abnormalities, confirming their role in response to injury. In mice with a kidney-specific knockout, Cxcl12 and Myc gene deletions suppress mitochondrial metabolism and glycolysis, and delay the recovery after ischemia/reperfusion injury. Probing these observations in zebrafish reveal that inhibition of glycolysis slows fast migrating cells and delays the repair after injury, but does not affect the slow cell movements during kidney development. Our findings demonstrate that Cxcl12 and Myc facilitate glycolysis to promote fast migratory responses during development and repair, and potentially also during tumor invasion and metastasis.

Cilia-localized LKB1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney.

Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy-related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell-autonomous manner and results in peritubular accumulation of CCR2+ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.

EpCAM controls morphogenetic programs during zebrafish pronephros development.

Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is dynamically expressed in human and murine renal epithelia during development. The levels of EpCAM in the renal epithelium are upregulated both during regeneration after ischemia/reperfusion injury and in renal-derived carcinomas. The role of EpCAM in early kidney development, however, has remained unclear. The zebrafish pronephros shows a similar segmentation pattern to the mammalian metanephric nephron, and has recently emerged as a tractable model to study the regulatory programs governing early nephrogenesis. Since EpCAM shows persistent expression in the pronephros throughout early development, we developed a method to study the global changes in gene expression in specific pronephric segments of wild type and EpCAM-deficient zebrafish embryos. In epcam mutants, we found 379 differentially expressed genes. Gene ontology analysis revealed that EpCAM controls various developmental programs, including uretric bud development, morphogenesis of branching epithelium, regulation of cell differentiation and cilium morphogenesis.

FAT1 mutations cause a glomerulotubular nephropathy.

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease (CKD). Here we show that recessive mutations in FAT1 cause a distinct renal disease entity in four families with a combination of SRNS, tubular ectasia, haematuria and facultative neurological involvement. Loss of FAT1 results in decreased cell adhesion and migration in fibroblasts and podocytes and the decreased migration is partially reversed by a RAC1/CDC42 activator. Podocyte-specific deletion of Fat1 in mice induces abnormal glomerular filtration barrier development, leading to podocyte foot process effacement. Knockdown of Fat1 in renal tubular cells reduces migration, decreases active RAC1 and CDC42, and induces defects in lumen formation. Knockdown of fat1 in zebrafish causes pronephric cysts, which is partially rescued by RAC1/CDC42 activators, confirming a role of the two small GTPases in the pathogenesis. These findings provide new insights into the pathogenesis of SRNS and tubulopathy, linking FAT1 and RAC1/CDC42 to podocyte and tubular cell function.

The C175R mutation alters nuclear localization and transcriptional activity of the nephronophthisis NPHP7 gene product.

Nephronophthisis (NPH) is a rare autosomal ciliopathy, but the leading cause for hereditary end-stage renal disease in children. Most NPH family members form large protein networks, which appear to participate in structural elements of the cilium and/or function to restrict access of molecules to the ciliary compartment. The zinc-finger protein GLIS2/NPHP7 represents an exception as it has been implicated in transcriptional regulation; only two families with GLIS2/NPHP7 mutations and typical NPH manifestations have been identified so far. We describe here that the recently identified GLIS2/NPHP7(C175R) point mutation abolished the nuclear localization of GLIS2/NPHP7. Forced nuclear import did not rescue the transcriptional defects of GLIS2/NPHP7(C175R), indicating additional defects as DNA-binding protein. We further observed that wild type, but not GLIS2/NPHP7(C175R), prevented the cyst formation caused by depletion of nphp7 in zebrafish embryos. Taken together, our findings indicate that the C175R mutation affects both localization and function of GLIS2/NPHP7, supporting a role of this mutation in NPH, but questioning the direct involvement of GLIS2/NPHP7 in ciliary functions.

The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells.

Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.

Anks3 alters the sub-cellular localization of the Nek7 kinase.

Nephronophthisis (NPH) is an autosomal recessive cystic kidney disease, and a frequent cause of end-stage renal failure in children. To date, 17 NPH-associated gene products (NPHPs) have been identified. Most NPHPs participate in large multi-protein complexes that localize to the cilium and/or basal body; however, the precise composition of these complexes and their biological function remain largely unknown. We recently observed that the ankyrin repeat protein Anks3 interacts with the NPH family member Anks6. Both Anks3 and Anks6 form complexes with multiple other NPHPs, suggesting that both proteins function in similar or overlapping signaling pathways. Here, we show that Anks3, but not Anks6 interacted with the NIMA-related kinase Nek7, and was heavily modified in the presence of Nek7, resulting in an approximately 20 kD increase in molecular weight. Although mass spectrometry revealed increased serine and threonine phosphorylation of Anks3 primarily within the N-terminal ankyrin repeats also required for Nek7 interaction, the molecular weight increase occurred even in the presence of a kinase-dead Nek7 mutant, indicating that this modification was not caused by Nek7-dependent Anks3 phosphorylation. Furthermore, the Anks3 modification was specific for Nek7, and did not occur in the presence of Nek8. Importantly, Anks3 retained Nek7 in the cytoplasm, suggesting that, Nek7 triggers the modification of Anks3, which in turn prevents the nuclear localization of Nek7.

Anks3 interacts with nephronophthisis proteins and is required for normal renal development.

Nephronophthisis (NPH) is a heterogenetic autosomal recessive disorder associated with kidney cysts and multiple extrarenal manifestations. The disease-associated gene products (NPHPs) typically contain domains involved in protein-protein interactions, and appear to exert their tissue-specific functions in large protein complexes. Most NPHPs localize to the cilium and/or basal body; however, their precise molecular functions remain largely unknown. We have recently identified the SAM-domain containing protein Anks3 as a potential ANKS6/NPHP16-interacting protein, and report now that Anks3 interacts with several NPHPs as well as with Bicc1 and the oxygen-sensitive asparaginyl hydroxylase HIF1AN. Knockdown of anks3 in zebrafish embryos was associated with NPH-typical manifestations, including ciliary abnormalities, cyst formation, and laterality defects. In multi-ciliated epidermal cells, GFP-tagged Anks3 localizes to the cilium, but forms large aggregates in the absence of NPHP1, indicating that the negatively charged NPHP1 curtails the polymerization of Anks3. Collectively, these findings suggest that Anks3 is a cilia-associated molecule that partners with the ANKS6- and via NPHP1 to the NPHP1-4-8 module. Thus, developmental defects associated with Anks3 depletion in zebrafish suggest that ANKS3 mutations may cause NPH or NPH-like disease in humans.

Bällchen is required for self-renewal of germline stem cells in Drosophila melanogaster.

Self-renewing stem cells are pools of undifferentiated cells, which are maintained in cellular niche environments by distinct tissue-specific signalling pathways. In Drosophila melanogaster, female germline stem cells (GSCs) are maintained in a somatic niche of the gonads by BMP signalling. Here we report a novel function of the Drosophila kinase Bällchen (BALL), showing that its cell autonomous role is to maintain the self-renewing capacity of female GSCs independent of BMP signalling. ball mutant GSCs are eliminated from the niche and subsequently differentiate into mature eggs, indicating that BALL is largely dispensable for differentiation. Similar to female GSCs, BALL is required to maintain self-renewal of male GSCs, suggesting a tissue independent requirement of BALL for self-renewal of germline stem cells.