NCK-associated protein 1 like (nckap1l) minor splice variant regulates intrahepatic biliary network morphogenesis

Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in the nckap1l gene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediated nckap1l deletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver of nckap1l mutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show that nckap1l genetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that although nckap1l was previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.     

Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b

The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis.     

Fast homozygosity mapping and identification of a zebrafish ENU-induced mutation by whole-genome sequencing

Forward genetics using zebrafish is a powerful tool for studying vertebrate development through large-scale mutagenesis. Nonetheless, the identification of the molecular lesion is still laborious and involves time-consuming genetic mapping. Here, we show that high-throughput sequencing of the whole zebrafish genome can directly locate the interval carrying the causative mutation and at the same time pinpoint the molecular lesion. The feasibility of this approach was validated by sequencing the m1045 mutant line that displays a severe hypoplasia of the exocrine pancreas. We generated 13 Gb of sequence, equivalent to an eightfold genomic coverage, from a pool of 50 mutant embryos obtained from a map-cross between the AB mutant carrier and the WIK polymorphic strain. The chromosomal region carrying the causal mutation was localized based on its unique property to display high levels of homozygosity among sequence reads as it derives exclusively from the initial AB mutated allele. We developed an algorithm identifying such a region by calculating a homozygosity score along all chromosomes. This highlighted an 8-Mb window on chromosome 5 with a score close to 1 in the m1045 mutants. The sequence analysis of all genes within this interval revealed a nonsense mutation in the snapc4 gene. Knockdown experiments confirmed the assertion that snapc4 is the gene whose mutation leads to exocrine pancreas hypoplasia. In conclusion, this study constitutes a proof-of-concept that whole-genome sequencing is a fast and effective alternative to the classical positional cloning strategies in zebrafish.     

A genetic screen in zebrafish identifies the mutants vps18, nf2 and foie gras as models of liver disease

Hepatomegaly is a sign of many liver disorders. To identify zebrafish mutants to serve as models for hepatic pathologies, we screened for hepatomegaly at day 5 of embryogenesis in 297 zebrafish lines bearing mutations in genes that are essential for embryonic development. Seven mutants were identified, and three have phenotypes resembling different liver diseases. Mutation of the class C vacuolar protein sorting gene vps18 results in hepatomegaly associated with large, vesicle-filled hepatocytes, which we attribute to the failure of endosomal-lysosomal trafficking. Additionally, these mutants develop defects in the bile canaliculi and have marked biliary paucity, suggesting that vps18 also functions to traffic vesicles to the hepatocyte apical membrane and may play a role in the development of the intrahepatic biliary tree. Similar findings have been reported for individuals with arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, which is due to mutation of another class C vps gene. A second mutant, resulting from disruption of the tumor suppressor gene nf2, develops extrahepatic choledochal cysts in the common bile duct, suggesting that this gene regulates division of biliary cells during development and that nf2 may play a role in the hyperplastic tendencies observed in biliary cells in individuals with choledochal cysts. The third mutant is in the novel gene foie gras, which develops large, lipid-filled hepatocytes, resembling those in individuals with fatty liver disease. These mutants illustrate the utility of zebrafish as a model for studying liver development and disease, and provide valuable tools for investigating the molecular pathogenesis of congenital biliary disorders and fatty liver disease.     

Zebrafish fat-free is required for intestinal lipid absorption and Golgi apparatus structure

The zebrafish fat-free (ffr) mutation was identified in a physiological screen for genes that regulate lipid metabolism. ffr mutant larvae are morphologically indistinguishable from wild-type sibling larvae, but their absorption of fluorescent lipids is severely impaired. Through positional cloning, we have identified a causative mutation in a highly conserved and ubiquitously expressed gene within the ffr locus. The Ffr protein contains a Dor-1 like domain typical of oligomeric Golgi complex (COG) gene, cog8. Golgi complex ultrastructure is disrupted in the ffr digestive tract. Consistent with a possible role in COG-mediated Golgi function, wild-type Ffr-GFP and COG8-mRFP fusion proteins partially colocalize in zebrafish blastomeres. Enterocyte retention of an endosomal lipid marker in ffr larvae support the idea that altered vesicle trafficking contributes to the ffr mutant defect. These data indicate that ffr is required for both Golgi structure and vesicular trafficking, and ultimately lipid transport.     

The vesicular integral protein-like gene is essential for development of a mechanosensory system in zebrafish

The zebrafish hi472 mutation is caused by a retroviral insertion into the vesicular integral protein-like gene, or zVIPL, a poorly studied lectin implicated in endoplasmic reticulum (ER)-Golgi trafficking. A mutation in the shorter isoform of zVIPL (zVIPL-s) results in a reduction of mechanosensitivity and consequent loss of escape behavior. Here we show that motoneurons and hindbrain reticulospinal neurons, which normally integrate mechanosensory inputs, failed to fire in response to tactile stimuli in hi472 larvae, suggesting a perturbation in sensory function. The hi472 mutant larvae in fact suffered from a severe loss of functional neuromasts of the lateral line mechanosensory system, a reduction of zVIPL labeling in support cells, and a reduction or even a complete loss of hair cells in neuromasts. The Delta-Notch signaling pathway is implicated in cellular differentiation of neuromasts, and we observed an increase in Notch expression in neuromasts of hi472 mutant larvae. Treatment of hi472 mutant larvae with DAPT, an inhibitor of Notch signaling, or overexpression of the Notch ligand deltaB in hi472 mutant blastocysts produced partial rescue of the morphological defects and of the startle response behavior. We conclude that zVIPL-s is a necessary component of Delta-Notch signaling during neuromast development in the lateral line mechanosensory system.     

A novel keratin18 promoter that drives reporter gene expression in the intrahepatic and extrahepatic biliary system allows isolation of cell-type specific transcripts from zebrafish liver

Heritable and acquired biliary disorders are an important cause of acute and chronic human liver disease. Biliary development and physiology have been studied extensively in rodent models and more recently, zebrafish have been used to uncover pathogenic mechanisms and potential therapies for these conditions. Here we report development of novel transgenic lines labeling the intrahepatic and extrahepatic biliary system of zebrafish larvae that can be used for lineage tracing and isolation of biliary-specific RNAs from mixed populations of liver cells. We show that GFP expression driven by a 4.4 kilobase promoter fragment from the zebrafish keratin18 (krt18) gene allows visualization of all components of the developing biliary system as early as 3 days post-fertilization. In addition, expression of a ribosomal fusion protein (EGFP-Rpl10a) in krt18:TRAP transgenic fish allows for enrichment of translated biliary cell mRNAs via translating ribosome affinity purification (TRAP). Future studies utilizing these reagents will enhance our understanding of the morphologic and molecular processes involved in biliary development and disease.     

Congenital end-plate acetylcholinesterase deficiency caused by a nonsense mutation and an A-->G splice-donor-site mutation at position +3 of the collagenlike-tail-subunit gene (COLQ): how does G at position +3 result in aberrant splicing?

Congenital end-plate acetylcholinesterase (AChE) deficiency (CEAD), the cause of a disabling myasthenic syndrome, arises from defects in the COLQ gene, which encodes the AChE triple-helical collagenlike-tail subunit that anchors catalytic subunits of AChE to the synaptic basal lamina. Here we describe a patient with CEAD with a nonsense mutation (R315X) and a splice-donor-site mutation at position +3 of intron 16 (IVS16+3A-->G) of COLQ. Because both A and G are consensus nucleotides at the +3 position of splice-donor sites, we constructed a minigene that spans exons 15-17 and harbors IVS16+3A-->G for expression in COS cells. We found that the mutation causes skipping of exon 16. The mutant splice-donor site of intron 16 harbors five discordant nucleotides (at -3, -2, +3, +4, and +6) that do not base-pair with U1 small-nuclear RNA (snRNA), the molecule responsible for splice-donor-site recognition. Versions of the minigene harboring, at either +4 or +6, nucleotides complementary to U1 snRNA restore normal splicing. Analysis of 1,801 native splice-donor sites reveals that presence of a G nucleotide at +3 is associated with preferential usage, at positions +4 to +6, of nucleotides concordant to U1 snRNA. Analysis of 11 disease-associated IVS+3A-->G mutations indicates that, on average, two of three nucleotides at positions +4 to +6 fail to base-pair, and that the nucleotide at +4 never base-pairs, with U1 snRNA. We conclude that, with G at +3, normal splicing generally depends on the concordance that residues at +4 to +6 have with U1 snRNA, but other cis-acting elements may also be important in assuring the fidelity of splicing.     

Nuclear suppression of mitochondrial defects in cells without the ND6 subunit

Previously, we characterized a mouse cell line, 4A, carrying a mitochondrial DNA mutation in the subunit for respiratory complex I, NADH dehydrogenase, in the ND6 gene. This mutation abolished the complex I assembly and disrupted the respiratory function of complex I. We now report here that a galactose-resistant clone, 4AR, was isolated from the cells carrying the ND6 mutation. 4AR still contained the homoplasmic mutation, and apparently there was no ND6 protein synthesis, whereas the assembly of other complex I subunits into complex I was recovered. Furthermore, the respiratory activity and mitochondrial membrane potential were fully recovered. To investigate the genetic origin of this compensation, the mitochondrial DNA (mtDNA) from 4AR was transferred to a new nuclear background. The transmitochondrial lines failed to grow in galactose medium. We further transferred mtDNA with a nonsense mutation at the ND5 gene to the 4AR nuclear background, and a suppression for mitochondrial deficiency was observed. Our results suggest that change(s) in the expression of a certain nucleus-encoded factor(s) can compensate for the absence of the ND6 or ND5 subunit.     

A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems.

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.     

Mutations in vacuolar H+ -ATPase subunits lead to biliary developmental defects in zebrafish

We identified three zebrafish mutants with defects in biliary development. One of these mutants, pekin (pn), also demonstrated generalized hypopigmentation and other defects, including disruption of retinal cell layers, lack of zymogen granules in the pancreas, and dilated Golgi in intestinal epithelial cells. Bile duct cells in pn demonstrated an accumulation of electron dense bodies. We determined that the causative defect in pn was a splice site mutation in the atp6ap2 gene that leads to an inframe stop codon. atp6ap2 encodes a subunit of the vacuolar H(+)-ATPase (V-H(+)-ATPase), which modulates pH in intracellular compartments. The Atp6ap2 subunit has also been shown to function as an intracellular renin receptor that stimulates fibrogenesis. Here we show that mutants and morphants involving other V-H(+)-ATPase subunits also demonstrated developmental biliary defects, but did not demonstrate the inhibition of fibrogenic genes observed in pn. The defects in pn are reminiscent of those we and others have observed in class C VPS (vacuolar protein sorting) family mutants and morphants, and we report here that knockdown of atp6ap2 and vps33b had an additive negative effect on biliary development. Our findings suggest that pathways which are important in modulating intracompartmental pH lead to defects in digestive organ development, and support previous studies demonstrating the importance of intracellular sorting pathways in biliary development.     

Osteopetrosis Mutation R444L Causes Endoplasmic Reticulum Retention and Misprocessing of Vacuolar H+-ATPase a3 Subunit

Osteopetrosis is a genetic bone disease characterized by increased bone density and fragility. The R444L missense mutation in the human V-ATPase a3 subunit (TCIRG1) is one of several known mutations in a3 and other proteins that can cause this disease. The autosomal recessive R444L mutation results in a particularly malignant form of infantile osteopetrosis that is lethal in infancy, or early childhood. We have studied this mutation using the pMSCV retroviral vector system to integrate the cDNA construct for green fluorescent protein (GFP)-fused a3(R445L) mutant protein into the RAW 264.7 mouse osteoclast differentiation model. In comparison with wild-type a3, the mutant glycoprotein localized to the ER instead of lysosomes and its oligosaccharide moiety was misprocessed, suggesting inability of the core-glycosylated glycoprotein to traffic to the Golgi. Reduced steady-state expression of the mutant protein, in comparison with wild type, suggested that the former was being degraded, likely through the endoplasmic reticulum-associated degradation pathway. In differentiated osteoclasts, a3(R445L) was found to degrade at an increased rate over the course of osteoclastogenesis. Limited proteolysis studies suggested that the R445L mutation alters mouse a3 protein conformation. Together, these data suggest that Arg-445 plays a role in protein folding, or stability, and that infantile malignant osteopetrosis caused by the R444L mutation in the human V-ATPase a3 subunit is another member of the growing class of protein folding diseases. This may have implications for early-intervention treatment, using protein rescue strategies.     

Functional Analysis of the Integrator Subunit 12 Identifies a Microdomain That Mediates Activation of the Drosophila Integrator Complex

The Drosophila integrator complex consists of 14 subunits that associate with the C terminus of Rpb1 and catalyze the endonucleolytic cleavage of nascent snRNAs near their 3′ ends. Although disruption of almost any integrator subunit causes snRNA misprocessing, very little is known about the role of the individual subunits or the network of structural and functional interactions that exist within the complex. Here we developed an RNAi rescue assay in Drosophila S2 cells to identify functional domains within integrator subunit 12 (IntS12) required for snRNA 3′ end formation. Surprisingly, the defining feature of the Ints12 protein, a highly conserved and centrally located plant homeodomain finger domain, is not required for reporter snRNA 3′ end cleavage. Rather, we find a small, 45-amino acid N-terminal microdomain to be both necessary and nearly sufficient for snRNA biogenesis in cells depleted of endogenous IntS12 protein. This IntS12 microdomain can function autonomously, restoring full integrator processing activity when introduced into a heterologous protein. Moreover, mutations within the microdomain not only disrupt IntS12 function but also abolish binding to other integrator subunits. Finally, the IntS12 microdomain is sufficient to interact and stabilize the putative scaffold integrator subunit, IntS1. Collectively, these results identify an unexpected interaction between the largest and smallest integrator subunits that is essential for the 3′ end formation of Drosophila snRNA.     

Yeast ubiquinol: cytochrome c oxidoreductase is still active after inactivation of the gene encoding the 17-kDa subunit VI

The single nuclear gene encoding the 17-kDa subunit VI of yeast ubiquinol: cytochrome c oxidoreductase has been inactivated by one-step gene disruption. Disruption was verified by Southern blot analysis of nuclear DNA and immunoblotting. Cells lacking the 17-kDa protein are still capable of growth on glycerol and they contain all other subunits of complex III at wild-type levels, implying that the 17-kDa subunit is not essential for either assembly of complex III, or its function. In vitro, electron transport activity of complex III of mutant cells is about 40% of the wild-type complex, but for the total respiratory chain no significant differences in activity was measured between mutant and wild type. The energy-transducing capacity of the complex is not reduced in the absence of the 17-kDa protein. In a relatively high proportion of the transformants, disruption of the 17-kDa gene was accompanied by the appearance of a second mutation causing a petite phenotype. In these cells which lack cytochrome b, the presence of the 17-kDa protein (after complementation) results in stabilization of cytochrome c1.     

Sec13 safeguards the integrity of the endoplasmic reticulum and organogenesis of the digestive system in zebrafish

The Sec13-Sec31 heterotetramer serves as the outer coat in the COPII complex, which mediates protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus. Although it has been studied in depth in yeast and cultured cells, the role of COPII in organogenesis in a multicellular organism has not. We report here that a zebrafish sec13(sq198) mutant, which exhibits a phenotype of hypoplastic digestive organs, has a mutation in the sec13 gene. The mutant gene encodes a carboxyl-terminus-truncated Sec13 that loses its affinity to Sec31a, which leads to disintegration of the ER structure in various differentiated cells in sec13(sq198), including chondrocytes, intestinal epithelial cells and hepatocytes. Disruption of the ER structure activates an unfolded protein response that eventually causes the cells to undergo cell-cycle arrest and cell apoptosis, which arrest the growth of developing digestive organs in the mutant. Our data provide the first direct genetic evidence that COPII function is essential for the organogenesis of the digestive system.