Next-Generation Sequencing Identifies the Danforth's Short Tail Mouse Mutation as a Retrotransposon Insertion Affecting <em>Ptf1a</em> Expression

<div><p>The semidominant Danforth's short tail (<i>Sd</i>) mutation arose spontaneously in the 1920s. The homozygous <i>Sd</i> phenotype includes severe malformations of the axial skeleton with an absent tail, kidney agenesis, anal atresia, and persistent cloaca. The <i>Sd</i> mutant phenotype mirrors features seen in human caudal malformation syndromes including urorectal septum malformation, caudal regression, VACTERL association, and persistent cloaca. The <i>Sd</i> mutation was previously mapped to a 0.9 cM region on mouse chromosome 2qA3. We performed Sanger sequencing of exons and intron/exon boundaries mapping to the <i>Sd</i> critical region and did not identify any mutations. We then performed DNA enrichment/capture followed by next-generation sequencing (NGS) of the critical genomic region. Standard bioinformatic analysis of paired-end sequence data did not reveal any causative mutations. Interrogation of reads that had been discarded because only a single end mapped correctly to the <i>Sd</i> locus identified an early transposon (ETn) retroviral insertion at the <i>Sd</i> locus, located 12.5 kb upstream of the <i>Ptf1a</i> gene. We show that <i>Ptf1a</i> expression is significantly upregulated in <i>Sd</i> mutant embryos at E9.5. The identification of the <i>Sd</i> mutation will lead to improved understanding of the developmental pathways that are misregulated in human caudal malformation syndromes.</p> </div

The <i>Sd</i> phenotype.

<p>A) Heterozygous (<i>Sd</i>/+) mouse. B) Wildtype (+/+) mouse. C) Homozygous (<i>Sd/Sd</i>) P0 neonate. Note the lack of urogenital openings and tail. D) Wildtype (+/+) P0 neonate. E) Homozygous (<i>Sd/Sd</i>) mutant neonate showing bilateral kidney agenesis. Kidney location indicated by asterisks below the normally formed adrenal glands. F) Homozygous (<i>Sd/Sd</i>) mutant intestine. The colon ends blindly (indicated by arrow), never connecting to the rectum which is never formed (see panel C). The asterisk indicates normal formation of the caecum.</p

Confirmation and mapping of the <i>Sd</i> mutation.

<p>A) Southern analysis and PCR showing the presence of a large DNA insertion at the <i>Sd</i> locus. B) Multiplex PCR from inbred mouse lines showing the mutation is only present in <i>Sd</i> mice, and is not a polymorphism. In this three primer PCR reaction the <i>Sd</i> amplimer is 406 bp, while the WT amplimer is 510 bp. C) Mapping of the <i>Sd</i> mutation which we identified as an ETn (early transposon) in relation to nearby gene/ESTs, figure not to scale.</p

Physical map of the <i>Sd</i> critical region as viewed on the UCSC genome browser (

<p><a href="http://www.genome.ucsc.edu" target="_blank">http://www.genome.ucsc.edu</a><b>).</b> The <i>Sd</i> region on chromosome 2 is defined by the flanking genetic markers D2Mit362 proximally and D2Mit364 distally (markers are highlighted in red) and represented by bases 18,686,882–20,915,358 on the July 2007 genome build (mm9). The <i>Sd</i> critical region contains 11 known genes. The mutation location is denoted by the red X.</p

Genes and ESTs mapping to the <i>Sd</i> critical region.

<p>Genes and ESTs mapping to the <i>Sd</i> critical region.</p

Gene expression at E9.5 in <i>Sd</i> mutant embryos.

<p>Quantitative real time PCR plot of gene expression in RNA isolated from <i>Sd/Sd</i> (black bars), <i>Sd/</i>+ (hatched bars), and +/+ (grey bars) embryos of RefSeq genes mapping to the <i>Sd</i> interval. Y-axis shows fold change normalized to 1 for wildtype (+/+) expression. * denotes significance in expression levels where p<0.05 (For <i>Ptf1a</i>; <i>Sd</i>/+ versus +/+ p = 0.029, <i>Sd/Sd</i> versus +/+ p = 0.044).</p

Novel Bioinformatics Method for Identification of Genome-Wide Non-Canonical Spliced Regions Using RNA-Seq Data

<div><p>Setting</p><p>During endoplasmic reticulum (ER) stress, the endoribonuclease (RNase) <i>Ire1</i>α initiates removal of a 26 nt region from the mRNA encoding the transcription factor <i>Xbp1</i> via an unconventional mechanism (atypically within the cytosol). This causes an open reading frame-shift that leads to altered transcriptional regulation of numerous downstream genes in response to ER stress as part of the unfolded protein response (UPR). Strikingly, other examples of targeted, unconventional splicing of short mRNA regions have yet to be reported.</p><p>Objective</p><p>Our goal was to develop an approach to identify non-canonical, possibly very short, splicing regions using RNA-Seq data and apply it to ER stress-induced <i>Ire1</i>α heterozygous and knockout mouse embryonic fibroblast (MEF) cell lines to identify additional <i>Ire1</i>α targets.</p><p>Results</p><p>We developed a bioinformatics approach called the Read-Split-Walk (RSW) pipeline, and evaluated it using two <i>Ire1</i>α heterozygous and two <i>Ire1</i>α-null samples. The 26 nt non-canonical splice site in <i>Xbp1</i> was detected as the top hit by our RSW pipeline in heterozygous samples but not in the negative control <i>Ire1</i>α knockout samples. We compared the <i>Xbp1</i> results from our approach with results using the alignment program BWA, Bowtie2, STAR, Exonerate and the Unix “<i>grep</i>” command. We then applied our RSW pipeline to RNA-Seq data from the SKBR3 human breast cancer cell line. RSW reported a large number of non-canonical spliced regions for 108 genes in chromosome 17, which were identified by an independent study.</p><p>Conclusions</p><p>We conclude that our RSW pipeline is a practical approach for identifying non-canonical splice junction sites on a genome-wide level. We demonstrate that our pipeline can detect novel splice sites in RNA-Seq data generated under similar conditions for multiple species, in our case mouse and human.</p></div

A <i>Poc1a</i> BAC transgene rescues <i>chagun</i>, and a null allele phenocopies <i>chagun</i>.

<p>A. Female and male mice of the indicated genotypes were weighed at 6 wks of age. N = 5-19/group. B. Adult male animals of the indicated genotypes were analyzed for testis morphology (0.8X objective) and histology. Testis sections were stained with hematoxylin and eosin. Scale bar = 100 μm. C. Bones were collected from <i>Poc1a</i>^+/+ and <i>Poc1a</i>^-/- animals at P21 and testes from P40 animals. Samples were fixed, sectioned and stained with hematoxylin and eosin. Scale bar = 100 μm.</p

Insertion of a processed cDNA into exon 8 of <i>Poc1a</i> underlies the <i>chagun</i> phenotype.

<p>A. Normal and <i>chagun</i> mutant genomic DNA samples (N = 4 mice per genotype) were amplified with primers flanking <i>Poc1a</i> exon 8. The products were separated by gel electrophoresis and stained with ethidium bromide. A 1,100 bp product was produced from the <i>chagun</i> genomic DNA samples and the predicted product 613 bp in wild-type genomic DNA. B. <i>Poc1a</i> transcripts were analyzed by RT-PCR using RNA from tibia (N = 3 per genotype) and primers specific to exons 5 and 10. A 526 bp product was detected in wild types, but a ~450 bp product was detected in <i>chagun</i> mutants. C. <i>Poc1a</i> transcripts in tibia were analyzed using primers in exons 7 and 8. The expected size product was detected in wild types but no amplification products were detected in <i>chagun</i> mutants. D. Protein extracts from P3 tibiae of wild type (+/+), heterozygote (+/-) and <i>chagun</i> mutants (-/-) were run on a 12% SDS-PAGE gel, transferred to a membrane, and probed with primary antibodies specific for POC1A and tubulin. Both images are exposures taken from the same blot. E. Schematic diagram of the processed <i>Cenpw</i> cDNA insertion into <i>Poc1a</i>, the effects on mRNA splicing, and the predicted protein. A portion of the <i>Poc1a</i> coding sequence is duplicated (hatched regions) at the 3’ end of the <i>Cenpw</i> insertion (black) within exon 8. The insertion results in skipping exon 8, and the predicted protein lacks a portion of the last WD-40 repeat domain. F. Molecular modeling of WD40 domains and amino acid interactions. i) Molecular model of the WD domains of mouse POC1A. The N- and C-termini of POC1A, which do not form part of the WD domains, have not been modeled. The <i>chagun</i> mutation is indicated in white within WD7. ii) A predicted hotspot of amino acid interactions, Asn233, forms a hydrogen bond with Ala274, which resides within WD7 and the <i>chagun</i> deletion. LCD: Low complexity domain, CCD: Coiled-coil domain, no template (nt), Genomic DNA (g).</p

The <i>Poc1a</i>^cha/cha long bone growth plates become disorganized.

<p>A. Standard hematoxylin and eosin histological staining was carried out on paraffin sections from P0 and P15 tibial growth plates. Scale bar = 50 μm. B. Immunohistochemistry using a primary antibody against the Golgi marker GM130 or acetylated tubulin counterstained with DAPI in wild type and mutant P15 tibia sections. Scale bar = 25 μm. C. Immunohistochemistry for primary antibodies against the proliferation marker Ki67 (i) or cell death marker TUNEL (ii), with quantification of the number of TUNEL-positive cells per field of view in wild type and <i>chagun</i> tibia (iii). Scale bar = 100 μm.</p