Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.     

Exocrine pancreas development in zebrafish

Although many of the genes that regulate development of the endocrine pancreas have been identified, comparatively little is known about how the exocrine pancreas forms. Previous studies have shown that exocrine pancreas development may be modeled in zebrafish. However, the timing and mechanism of acinar and ductal differentiation and morphogenesis have not been described. Here, we characterize zebrafish exocrine pancreas development in wild type and mutant larvae using histological, immunohistochemical and ultrastructural analyses. These data allow us to identify two stages of zebrafish exocrine development. During the first stage, the exocrine anlage forms from rostral endodermal cells. During the second stage, proto-differentiated progenitor cells undergo terminal differentiation followed by acinar gland and duct morphogenesis. Immunohistochemical analyses support a model in which the intrapancreatic ductal system develops from progenitors that join to form a contiguous network rather than by branching morphogenesis of the pancreatic epithelium, as described for mammals. Contemporaneous appearance of acinar glands and ducts in developing larvae and their disruption in pancreatic mutants suggest that common molecular pathways may regulate gland and duct morphogenesis and differentiation of their constituent cells. By contrast, analyses of mind bomb mutants and jagged morpholino-injected larvae suggest that Notch signaling principally regulates ductal differentiation of bipotential exocrine progenitors.     

Inactivation of dispatched 1 by the chameleon mutation disrupts Hedgehog signalling in the zebrafish embryo

Searches of zebrafish EST and whole genome shotgun sequence databases for sequences encoding the sterol-sensing domain (SSD) protein motif identified two sets of DNA sequences with significant homology to the Drosophila dispatched gene required for release of secreted Hedgehog protein. Using morpholino antisense oligonucleotides, we found that inhibition of one of these genes, designated Disp1, results in a phenotype similar to that of the "you-type" mutants, previously implicated in signalling by Hedgehog proteins in the zebrafish embryo. Injection of disp1 mRNA into embryos homozygous for one such mutation, chameleon (con) results in rescue of the mutant phenotype. Radiation hybrid mapping localised disp1 to the same region of LG20 to which the con mutation was mapped by meiotic recombination analysis. Sequence analysis of disp1 cDNA derived from homozygous con mutant embryos revealed that both mutant alleles are associated with premature termination codons in the disp1 coding sequence. By analysing the expression of markers of specific cell types in the neural tube, pancreas and myotome of con mutant and Disp1 morphant embryos, we conclude that Disp1 activity is essential for the secretion of lipid-modified Hh proteins from midline structures.     

Targeted next-generation sequencing of a 12.5 Mb homozygous region reveals ANO10 mutations in patients with autosomal-recessive cerebellar ataxia

Autosomal-recessive cerebellar ataxias comprise a clinically and genetically heterogeneous group of neurodegenerative disorders. In contrast to their dominant counterparts, unraveling the molecular background of these ataxias has proven to be more complicated and the currently known mutations provide incomplete coverage for genotyping of patients. By combining SNP array-based linkage analysis and targeted resequencing of relevant sequences in the linkage interval with the use of next-generation sequencing technology, we identified a mutation in a gene and have shown its association with autosomal-recessive cerebellar ataxia. In a Dutch consanguineous family with three affected siblings a homozygous 12.5 Mb region on chromosome 3 was targeted by array-based sequence capture. Prioritization of all detected sequence variants led to four candidate genes, one of which contained a variant with a high base pair conservation score (phyloP score: 5.26). This variant was a leucine-to-arginine substitution in the DUF 590 domain of a 16K transmembrane protein, a putative calcium-activated chloride channel encoded by anoctamin 10 (ANO10). The analysis of ANO10 by Sanger sequencing revealed three additional mutations: a homozygous mutation (c.1150_1151del [p.Leu384fs]) in a Serbian family and a compound-heterozygous splice-site mutation (c.1476+1G>T) and a frameshift mutation (c.1604del [p.Leu535X]) in a French family. This illustrates the power of using initial homozygosity mapping with next-generation sequencing technology to identify genes involved in autosomal-recessive diseases. Moreover, identifying a putative calcium-dependent chloride channel involved in cerebellar ataxia adds another pathway to the list of pathophysiological mechanisms that may cause cerebellar ataxia.     

Linkage analysis and gene expression profile of pancreatic acinar atrophy in the German Shepherd Dog

Pancreatic acinar atrophy (PAA) is a degenerative disease of the exocrine pancreas and is the most common cause of exocrine pancreatic insufficiency in the German Shepherd Dog. Analyses of inheritance have shown that a single gene segregating in an autosomal recessive fashion is causative for PAA. To date the gene and causative mutation have not been determined. To identify a region of interest and/or candidate genes, we conducted linkage and gene expression studies. Analysis of 384 microsatellite markers resulted in a maximum two-point LOD score of 2.5 for FH2107 on CFA03. We used an oligonucleotide array to generate gene expression profiles for normal and affected pancreata. It revealed 244 genes with greater than two-fold difference in expression levels. Five genes of interest were further assessed by TaqMan quantitative real-time RT-PCR that confirmed trends observed using the microarray. One gene, gp25L, located on CFA03, was found to be downregulated by more than 500-fold in affected pancreata and was further investigated as a candidate gene. Sequence data did not reveal a mutation in the coding sequence that segregates with PAA.     

Microtubule Actin Crosslinking Factor 1 Regulates the Balbiani Body and Animal-Vegetal Polarity of the Zebrafish Oocyte

Although of fundamental importance in developmental biology, the genetic basis for the symmetry breaking events that polarize the vertebrate oocyte and egg are largely unknown. In vertebrates, the first morphological asymmetry in the oocyte is the Balbiani body, a highly conserved, transient structure found in vertebrates and invertebrates including Drosophila, Xenopus, human, and mouse. We report the identification of the zebrafish magellan (mgn) mutant, which exhibits a novel enlarged Balbiani body phenotype and a disruption of oocyte polarity. To determine the molecular identity of the mgn gene, we positionally cloned the gene, employing a novel DNA capture method to target region-specific genomic DNA of 600 kb for massively parallel sequencing. Using this technique, we were able to enrich for the genomic region linked to our mutation within one week and then identify the mutation in mgn using massively parallel sequencing. This is one of the first successful uses of genomic DNA enrichment combined with massively parallel sequencing to determine the molecular identity of a gene associated with a mutant phenotype. We anticipate that the combination of these technologies will have wide applicability for the efficient identification of mutant genes in all organisms. We identified the mutation in mgn as a deletion in the coding sequence of the zebrafish microtubule actin crosslinking factor 1 (macf1) gene. macf1 is a member of the highly conserved spectraplakin family of cytoskeletal linker proteins, which play diverse roles in polarized cells such as neurons, muscle cells, and epithelial cells. In mgn mutants, the oocyte nucleus is mislocalized; and the Balbiani body, localized mRNAs, and organelles are absent from the periphery of the oocyte, consistent with a function for macf1 in nuclear anchoring and cortical localization. These data provide the first evidence for a role for spectraplakins in polarization of the vertebrate oocyte and egg.     

Identification of EMS-Induced Mutations in Drosophila melanogaster by Whole-Genome Sequencing

Next-generation methods for rapid whole-genome sequencing enable the identification of single-base-pair mutations in Drosophila by comparing a chromosome bearing a new mutation to the unmutagenized sequence. To validate this approach, we sought to identify the molecular lesion responsible for a recessive EMS-induced mutation affecting egg shell morphology by using Illumina next-generation sequencing. After obtaining sufficient sequence from larvae that were homozygous for either wild-type or mutant chromosomes, we obtained high-quality reads for base pairs composing ~70% of the third chromosome of both DNA samples. We verified 103 single-base-pair changes between the two chromosomes. Nine changes were nonsynonymous mutations and two were nonsense mutations. One nonsense mutation was in a gene, encore, whose mutations produce an egg shell phenotype also observed in progeny of homozygous mutant mothers. Complementation analysis revealed that the chromosome carried a new functional allele of encore, demonstrating that one round of next-generation sequencing can identify the causative lesion for a phenotype of interest. This new method of whole-genome sequencing represents great promise for mutant mapping in flies, potentially replacing conventional methods.     

C. elegans mutant identification with a one-step whole-genome-sequencing and SNP mapping strategy

Whole-genome sequencing (WGS) is becoming a fast and cost-effective method to pinpoint molecular lesions in mutagenized genetic model systems, such as Caenorhabditis elegans. As mutagenized strains contain a significant mutational load, it is often still necessary to map mutations to a chromosomal interval to elucidate which of the WGS-identified sequence variants is the phenotype-causing one. We describe here our experience in setting up and testing a simple strategy that incorporates a rapid SNP-based mapping step into the WGS procedure. In this strategy, a mutant retrieved from a genetic screen is crossed with a polymorphic C. elegans strain, individual F2 progeny from this cross is selected for the mutant phenotype, the progeny of these F2 animals are pooled and then whole-genome-sequenced. The density of polymorphic SNP markers is decreased in the region of the phenotype-causing sequence variant and therefore enables its identification in the WGS data. As a proof of principle, we use this strategy to identify the molecular lesion in a mutant strain that produces an excess of dopaminergic neurons. We find that the molecular lesion resides in the Pax-6/Eyeless ortholog vab-3. The strategy described here will further reduce the time between mutant isolation and identification of the molecular lesion.     

Isolation and characterization of phi X174 mutants carrying lethal missense mutations in gene G.

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.     

Caenorhabditis elegans mutant allele identification by whole-genome sequencing

Identification of the molecular lesion in Caenorhabditis elegans mutants isolated through forward genetic screens usually involves time-consuming genetic mapping. We used Illumina deep sequencing technology to sequence a complete, mutant C. elegans genome and thus pinpointed a single-nucleotide mutation in the genome that affects a neuronal cell fate decision. This constitutes a proof-of-principle for using whole-genome sequencing to analyze C. elegans mutants.     

Nil per os encodes a conserved RNA recognition motif protein required for morphogenesis and cytodifferentiation of digestive organs in zebrafish

Digestive organ development occurs through a sequence of morphologically distinct stages, from overtly featureless endoderm, through organ primordia to, ultimately, adult form. The developmental controls that govern progression from one stage to the next are not well understood. To identify genes required for the formation of vertebrate digestive organs we performed a genetic screen in zebrafish. We isolated the nil per os (npo) mutation, which arrests morphogenesis and cytodifferentiation of the gut and exocrine pancreas in a primordial state. We identified the npo gene by positional cloning. It encodes a conserved protein, with multiple RNA recognition motifs, that is related to the yeast protein Mrd1p. During development npo is expressed in a dynamic fashion, functioning cell autonomously to promote organ cytodifferentiation. Antisense-mediated knockdown of npo results in organ hypoplasia, and overexpression of npo causes an overgrowth of gastrointestinal organs. Thus, npo is a gene essential for a key step in the gut morphogenetic sequence.     

Effect of genetic variation of CEBPA gene on body measurement and carcass traits of Qinchuan cattle

In our study, genetic variation in coding region of cattle CCAAT enhancer binding protein alpha(namely CEBPA)gene was detected by PCR-RFLP and DNA sequencing methods in 215 individuals from Qinchuan cattle breed. Two haplotypes (A and B) and three observed genotypes (AA, AB, and BB) were detected. The result of DNA sequence showed one mutation by comparisons with NC_007316. The mutation at nt963 (T>G) were located in coding region of the CEBPA gene. Associations between the CEBPA gene genetic variation and the carcass traits were revealed in Qinchuan cattle. Least squares analysis revealed a significant statistical effect of the CEBPA gene different genotypes on slaughter weight and carcass weight in Qinchuan cattle. Individuals with BB genotype showed higher slaughter weight and carcass weight than individuals with AA and AB genotypes. Therefore, these results suggest that the CEBPA gene is a strong candidate gene that affects carcass traits in Qinchuan cattle.     

Homozygosity for multiple contiguous single-nucleotide polymorphisms as an indicator of large heterozygous deletions: identification of a novel heterozygous 8-kb intragenic deletion (IVS7-19 to IVS15-17) in a patient with glycogen storage disease type II

Current methods for detection of mutations by polymerase chain reaction (PCR) and sequence analysis frequently are not able to detect heterozygous large deletions. We report the successful use of a novel approach to identify such deletions, based on detection of apparent homozygosity of contiguous single-nucleotide polymorphisms (SNPs). The sequence analysis of genomic DNA PCR products containing all coding exons and flanking introns identified only a single heterozygous mutation (IVS18+2t-->a) in a patient with classic infantile-onset autosomal recessive glycogen storage disease type II (GSDII). Apparent homozygosity for multiple contiguous SNPs detected by this sequencing suggested presence of a large deletion as the second mutation; primers flanking the region of homozygous SNPs permitted identification and characterization by PCR of a large genomic deletion (8.26 kb) extending from IVS7 to IVS15. The data clearly demonstrate the utility of SNPs as markers for large deletions in autosomal recessive diseases when only a single mutation is found, thus complementing currently standard DNA PCR sequence methods for identifying the molecular basis of disease.     

Escherichia coli DNA repair genes radA and sms are the same gene.

Escherichia coli strains carrying radA100 or sms mutations were identical in their sensitivities to either methyl methanesulfonate or UV radiation treatment and in their plasmid complementation patterns for UV radiation survival. DNA sequencing analysis of the radA mutant and radA+ strains and comparison of their sequences with the published sms gene sequence showed the radA mutant to differ only by a G-to-A transition mutation, which is predicted to change a cysteine in a zinc-finger motif to tyrosine. The sms gene is concluded to be identical to the previously described radA gene.