The type IIS restriction enzyme MmeI can cut across a double-strand break.

BACKGROUND: Type-IIS restriction enzymes cut outside their recognition sites, allowing them to remove their binding sites upon digestion. This feature has resulted in their wide application in molecular biology techniques, including seamless cloning methods, enzymatic CRISPR library generation, and others. We studied the ability of the Type-IIS restriction enzyme MmeI, which recognizes an asymmetric sequence TCCRAC and cuts 20 bp downstream, to cut across a double-strand break (DSB). METHODS AND RESULTS: We used synthetic double-stranded oligos with MmeI recognition sites close to 5' end and different overhang lengths to measure digestion after different periods of time and at different temperatures. We found that the MmeI binding and cutting sites can be situated on opposite sides of a DSB if the edges of the DNA molecules are held together by transient base-pairing interactions between compatible overhangs. CONCLUSION: We found that MmeI can cut across a DSB, and the efficiency of the cutting depends on both overhang length and temperature.

A simple and rapid method for enzymatic synthesis of CRISPR-Cas9 sgRNA libraries.

CRISPR-Cas9 sgRNA libraries have transformed functional genetic screening and have enabled several innovative methods that rely on simultaneously targeting numerous genetic loci. Such libraries could be used in a vast number of biological systems and in the development of new technologies, but library generation is hindered by the cost, time, and sequence data required for sgRNA library synthesis. Here, we describe a rapid enzymatic method for generating robust, variant-matched libraries from any source of cDNA in under 3 h. This method, which we have named SLALOM, utilizes a custom sgRNA scaffold sequence and a novel method for detaching oligonucleotides from solid supports by a strand displacing polymerase. With this method, we constructed libraries targeting the E. coli genome and the transcriptome of developing zebrafish hearts, demonstrating its ability to expand the reach of CRISPR technology and facilitate methods requiring custom libraries.

Increased Dissociation of Adamantanamines in Influenza A M2 S31N with Partial Block by Rimantadine.

The ubiquitous mutation from serine (WT) to asparagine at residue 31 (S31N) in the influenza A M2 channel renders it insensitive to amantadine (AMT) and rimantadine (RMT) block, but it is unknown whether the inhibition results from weak binding or incomplete block. Two-electrode voltage clamp (TEVC) of transfected Xenopus oocytes revealed that the M2 S31N channel is essentially fully blocked by AMT at 10 mM, demonstrating that, albeit weak, AMT binding in a channel results in complete block of its proton current. In contrast, RMT achieves only a modest degree of block in the M2 S31N channel at 1 mM, with very little increase in block at 10 mM, indicating that the RMT binding site in the channel saturates with only modest block. From exponential curve fits to families of proton current wash-in and wash-out traces, the association rate constant (k1) is somewhat decreased for both AMT and RMT in the S31N, but the dissociation rate constant (k2) is dramatically increased compared with WT. The potentials of mean force (PMF) from adaptive biasing force (ABF) molecular dynamics simulations predict that rate constants should be exquisitely sensitive to the charge state of the His37 selectivity filter of M2. With one exception out of eight cases, predictions from the simulations with one and three charged side chains bracket the experimental rate constants, as expected for the acidic bath used in the TEVC assay. From simulations, the weak binding can be accounted for by changes in the potentials of mean force, but the partial block by RMT remains unexplained.

Heart morphogenesis gene regulatory networks revealed by temporal expression analysis.

During embryogenesis the heart forms as a linear tube that then undergoes multiple simultaneous morphogenetic events to obtain its mature shape. To understand the gene regulatory networks (GRNs) driving this phase of heart development, during which many congenital heart disease malformations likely arise, we conducted an RNA-seq timecourse in zebrafish from 30 hpf to 72 hpf and identified 5861 genes with altered expression. We clustered the genes by temporal expression pattern, identified transcription factor binding motifs enriched in each cluster, and generated a model GRN for the major gene batteries in heart morphogenesis. This approach predicted hundreds of regulatory interactions and found batteries enriched in specific cell and tissue types, indicating that the approach can be used to narrow the search for novel genetic markers and regulatory interactions. Subsequent analyses confirmed the GRN using two mutants, Tbx5 and nkx2-5, and identified sets of duplicated zebrafish genes that do not show temporal subfunctionalization. This dataset provides an essential resource for future studies on the genetic/epigenetic pathways implicated in congenital heart defects and the mechanisms of cardiac transcriptional regulation.

Resistance mechanisms and cross-resistance for a pyridine-pyrimidine amide inhibitor of microtubule polymerization.

Despite their clinical importance, drug resistance remains problematic for microtubule targeting drugs. D4-9-31, a novel microtubule destabilizing agent, has pharmacology that suggests it can overcome common resistance mechanisms and has been shown to remain efficacious in cell and animal models with acquired taxane resistance. To better understand resistance mechanisms and the breadth of cross-resistance with D4-9-31, this study examines the A2780 ovarian cancer cell line as it develops acquired resistance with continuous exposure to D4-9-31. Analyzing cellular responses to D4-9-31 reveals that D4-9-31 resistance is associated with increased mitochondrial respiration, but no cross-resistance to other microtubule targeting agents is observed. Sequencing of transcripts of parental cells and resistant counterparts reveals mutations and altered expression of microtubule-associated genes, but not in genes commonly associated with resistance to microtubule targeting drugs. Additionally, our findings suggest distinct mechanisms drive short- and long-term drug resistance.

MMAPPR: mutation mapping analysis pipeline for pooled RNA-seq.

Forward genetic screens in model organisms are vital for identifying novel genes essential for developmental or disease processes. One drawback of these screens is the labor-intensive and sometimes inconclusive process of mapping the causative mutation. To leverage high-throughput techniques to improve this mapping process, we have developed a Mutation Mapping Analysis Pipeline for Pooled RNA-seq (MMAPPR) that works without parental strain information or requiring a preexisting SNP map of the organism, and adapts to differential recombination frequencies across the genome. MMAPPR accommodates the considerable amount of noise in RNA-seq data sets, calculates allelic frequency by Euclidean distance followed by Loess regression analysis, identifies the region where the mutation lies, and generates a list of putative coding region mutations in the linked genomic segment. MMAPPR can exploit RNA-seq data sets from isolated tissues or whole organisms that are used for gene expression and transcriptome analysis in novel mutants. We tested MMAPPR on two known mutant lines in zebrafish, nkx2.5 and tbx1, and used it to map two novel ENU-induced cardiovascular mutants, with mutations found in the ctr9 and cds2 genes. MMAPPR can be directly applied to other model organisms, such as Drosophila and Caenorhabditis elegans, that are amenable to both forward genetic screens and pooled RNA-seq experiments. Thus, MMAPPR is a rapid, cost-efficient, and highly automated pipeline, available to perform mutant mapping in any organism with a well-assembled genome.

Hectd1 is required for development of the junctional zone of the placenta.

The placenta plays a critical role in the growth and survival of the fetus. Here we demonstrate that the Homologous to the E6-AP Carboxyl Terminus (HECT) domain E3 ubiquitin ligase, Hectd1, is essential for development of the mouse placenta. Hectd1 is widely expressed during placentation with enrichment in trophoblast giant cells (TGCs) and other trophoblast-derived cell subtypes in the junctional and labyrinth zones of the placenta. Disruption of Hectd1 results in mid-gestation lethality and intrauterine growth restriction (IUGR). Variable defects in the gross structure of the mutant placenta are found including alterations in diameter, thickness and lamination. The number and nuclear size of TGCs is reduced. Examination of subtype specific markers reveals altered TGC development with decreased expression of Placental lactogen-1 and -2 (Pl1 and Pl2) and increased expression of Proliferin (Plf). Reduced numbers of spongiotrophoblasts and glycogen trophoblasts were also found at the junctional zone of the Hectd1 mutant placenta. Finally, there was an increase in immature uterine natural killer (uNK) cells in the maternal decidua of the Hectd1 mutant placenta. Proliferation and apoptosis are differentially altered in the layers of the placenta with an increase in both apoptosis and proliferation in the maternal decidua, a decrease in proliferation and increase in apoptosis in the labyrinth layer and both unchanged in the junctional zone. Together these data demonstrate that Hectd1 is required for development of multiple cell types within the junctional zone of the placenta.

Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products.

BACKGROUND: Genome editing techniques, including ZFN, TALEN, and CRISPR, have created a need to rapidly screen many F1 individuals to identify carriers of indels and determine the sequences of the mutations. Current techniques require multiple clones of the targeted region to be sequenced for each individual, which is inefficient when many individuals must be analyzed. Direct Sanger sequencing of a polymerase chain reaction (PCR) amplified region surrounding the target site is efficient, but Sanger sequencing genomes heterozygous for an indel results in a string of "double peaks" due to the mismatched region. RESULTS: To facilitate indel identification, we developed an online tool called Poly Peak Parser (available at http://yost.genetics.utah.edu/software.php) that is able to separate chromatogram data containing ambiguous base calls into wild-type and mutant allele sequences. This tool allows the nature of the indel to be determined from a single sequencing run per individual performed directly on a PCR product spanning the targeted site, without cloning. CONCLUSIONS: The method and algorithm described here facilitate rapid identification and sequence characterization of heterozygous mutant carriers generated by genome editing. Although designed for screening F1 individuals, this tool can also be used to identify heterozygous indels in many contexts.

Heterochronic shift in gene expression leads to ontogenetic morphological divergence between two closely related polyploid species.

Heterochrony-alteration to the rate or timing of development-is an important mechanism of trait differentiation associated with speciation. Heterochrony may explain the morphological divergence between two polyploid species, June sucker (Chasmistes liorus) and Utah sucker (Catostomus ardens). The larvae of both species have terminal mouths; however, as adults, June sucker and Utah sucker develop subterminal and ventral mouths, respectively. We document a difference in the timing of shape development and a corresponding change in the timing of gene expression, suggesting the distinctive mouth morphology in June suckers may result from paedomorphosis. Specifically, adult June suckers exhibit an intermediate mouth morphology between the larval (terminal) and ancestral (ventral) states. Endemic and sympatric Chasmistes/Catostomus pairs in two other lakes also are morphologically divergent, but genetically similar. These species pairs could have resulted from the differential expression of genes and corresponding divergence in trait development. Paedomorphosis may lead to adaptive diversification in Catostomids.

Valproic Acid Causes Redox-Regulated Post-Translational Protein Modifications That Are Dependent upon P19 Cellular Differentiation States.

Valproic acid (VPA) is a common anti-epileptic drug and known neurodevelopmental toxicant. Although the exact mechanism of VPA toxicity remains unknown, recent findings show that VPA disrupts redox signaling in undifferentiated cells but has little effect on fully differentiated neurons. Redox imbalances often alter oxidative post-translational protein modifications and could affect embryogenesis if developmentally critical proteins are targeted. We hypothesize that VPA causes redox-sensitive post-translational protein modifications that are dependent upon cellular differentiation states. Undifferentiated P19 cells and P19-derived neurons were treated with VPA alone or pretreated with D3T, an inducer of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant pathway, prior to VPA exposure. Undifferentiated cells treated with VPA alone exhibited an oxidized glutathione redox couple and increased overall protein oxidation, whereas differentiated neurons were protected from protein oxidation via increased S-glutathionylation. Pretreatment with D3T prevented the effects of VPA exposure in undifferentiated cells. Taken together, our findings support redox-sensitive post-translational protein alterations in undifferentiated cells as a mechanism of VPA-induced developmental toxicity and propose NRF2 activation as a means to preserve proper neurogenesis.

RNA Sequencing Demonstrates Ex Vivo Neocortical Transcriptomic Changes Induced by Epileptiform Activity in Male and Female Mice.

Seizures are generally associated with epilepsy but may also be a symptom of many other neurological conditions. A hallmark of a seizure is the intensity of the local neuronal activation, which can drive large-scale gene transcription changes. Such changes in the transcriptional profile likely alter neuronal function, thereby contributing to the pathological process. Therefore, there is a strong clinical imperative to characterize how gene expression is changed by seizure activity. To this end, we developed a simplified ex vivo technique for studying seizure-induced transcriptional changes. We compared the RNA sequencing profile in mouse neocortical tissue with up to 3 h of epileptiform activity induced by 4-aminopyridine (4AP) relative to control brain slices not exposed to the drug. We identified over 100 genes with significantly altered expression after 4AP treatment, including multiple genes involved in MAPK, TNF, and neuroinflammatory signaling pathways, all of which have been linked to epilepsy previously. Notably, the patterns in male and female brain slices were almost identical. Various immediate early genes were among those showing the largest upregulation. The set of down-regulated genes included ones that might be expected either to increase or to decrease neuronal excitability. In summary, we found the seizure-induced transcriptional profile complex, but the changes aligned well with an analysis of published epilepsy-associated genes. We discuss how simple models may provide new angles for investigating seizure-induced transcriptional changes.

Single-Cell RNA Sequencing Characterizes the Molecular Heterogeneity of the Larval Zebrafish Optic Tectum.

The optic tectum (OT) is a multilaminated midbrain structure that acts as the primary retinorecipient in the zebrafish brain. Homologous to the mammalian superior colliculus, the OT is responsible for the reception and integration of stimuli, followed by elicitation of salient behavioral responses. While the OT has been the focus of functional experiments for decades, less is known concerning specific cell types, microcircuitry, and their individual functions within the OT. Recent efforts have contributed substantially to the knowledge of tectal cell types; however, a comprehensive cell catalog is incomplete. Here we contribute to this growing effort by applying single-cell RNA Sequencing (scRNA-seq) to characterize the transcriptomic profiles of tectal cells labeled by the transgenic enhancer trap line y304Et(cfos:Gal4;UAS:Kaede). We sequenced 13,320 cells, a 4X cellular coverage, and identified 25 putative OT cell populations. Within those cells, we identified several mature and developing neuronal populations, as well as non-neuronal cell types including oligodendrocytes and microglia. Although most mature neurons demonstrate GABAergic activity, several glutamatergic populations are present, as well as one glycinergic population. We also conducted Gene Ontology analysis to identify enriched biological processes, and computed RNA velocity to infer current and future transcriptional cell states. Finally, we conducted in situ hybridization to validate our bioinformatic analyses and spatially map select clusters. In conclusion, the larval zebrafish OT is a complex structure containing at least 25 transcriptionally distinct cell populations. To our knowledge, this is the first time scRNA-seq has been applied to explore the OT alone and in depth.

Shutdown corner, a large deletion mutant isolated from a haploid mutagenesis screen in zebrafish.

Morphogenesis, the formation of three-dimensional organ structures, requires precise coupling of genetic regulation and complex cell behaviors. The genetic networks governing many morphogenetic systems, including that of the embryonic eye, are poorly understood. In zebrafish, several forward genetic screens have sought to identify factors regulating eye development. These screens often look for eye defects at stages after the optic cup is formed and when retinal neurogenesis is under way. This approach can make it difficult to identify mutants specific for morphogenesis, as opposed to neurogenesis. To this end, we carried out a forward genetic, small-scale haploid mutagenesis screen in zebrafish (Danio rerio) to identify factors that govern optic cup morphogenesis. We screened ∼100 genomes and isolated shutdown corner (sco), a mutant that exhibits multiple tissue defects and harbors a ∼10-Mb deletion that encompasses 89 annotated genes. Using a combination of live imaging and antibody staining, we found cell proliferation, cell death, and tissue patterning defects in the sco optic cup. We also observed other phenotypes, including paralysis, neuromuscular defects, and ocular vasculature defects. To date, the largest deletion mutants reported in zebrafish are engineered using CRISPR-Cas9 and are less than 300 kb. Because of the number of genes within the deletion interval, shutdown corner [Df(Chr05:sco)z207] could be a useful resource to the zebrafish community, as it may be helpful for gene mapping, understanding genetic interactions, or studying many genes lost in the mutant.

Novel computational analysis of protein binding array data identifies direct targets of Nkx2.2 in the pancreas.

BACKGROUND: The creation of a complete genome-wide map of transcription factor binding sites is essential for understanding gene regulatory networks in vivo. However, current prediction methods generally rely on statistical models that imperfectly model transcription factor binding. Generation of new prediction methods that are based on protein binding data, but do not rely on these models may improve prediction sensitivity and specificity. RESULTS: We propose a method for predicting transcription factor binding sites in the genome by directly mapping data generated from protein binding microarrays (PBM) to the genome and calculating a moving average of several overlapping octamers. Using this unique algorithm, we predicted binding sites for the essential pancreatic islet transcription factor Nkx2.2 in the mouse genome and confirmed >90% of the tested sites by EMSA and ChIP. Scores generated from this method more accurately predicted relative binding affinity than PWM based methods. We have also identified an alternative core sequence recognized by the Nkx2.2 homeodomain. Furthermore, we have shown that this method correctly identified binding sites in the promoters of two critical pancreatic islet ß-cell genes, NeuroD1 and insulin2, that were not predicted by traditional methods. Finally, we show evidence that the algorithm can also be applied to predict binding sites for the nuclear receptor Hnf4α. CONCLUSIONS: PBM-mapping is an accurate method for predicting Nkx2.2 binding sites and may be widely applicable for the creation of genome-wide maps of transcription factor binding sites.

Lack of skeletal muscle liver kinase B1 alters gene expression, mitochondrial content, inflammation and oxidative stress without affecting high-fat diet-induced obesity or insulin resistance.

Ad libitum high-fat diet (HFD) induces obesity and skeletal muscle metabolic dysfunction. Liver kinase B1 (LKB1) regulates skeletal muscle metabolism by controlling the AMP-activated protein kinase family, but its importance in regulating muscle gene expression and glucose tolerance in obese mice has not been established. The purpose of this study was to determine how the lack of LKB1 in skeletal muscle (KO) affects gene expression and glucose tolerance in HFD-fed, obese mice. KO and littermate control wild-type (WT) mice were fed a standard diet or HFD for 14 weeks. RNA sequencing, and subsequent analysis were performed to assess mitochondrial content and respiration, inflammatory status, glucose and insulin tolerance, and muscle anabolic signaling. KO did not affect body weight gain on HFD, but heavily impacted mitochondria-, oxidative stress-, and inflammation-related gene expression. Accordingly, mitochondrial protein content and respiration were suppressed while inflammatory signaling and markers of oxidative stress were elevated in obese KO muscles. KO did not affect glucose or insulin tolerance. However, fasting serum insulin and skeletal muscle insulin signaling were higher in the KO mice. Furthermore, decreased muscle fiber size in skmLKB1-KO mice was associated with increased general protein ubiquitination and increased expression of several ubiquitin ligases, but not muscle ring finger 1 or atrogin-1. Taken together, these data suggest that the lack of LKB1 in skeletal muscle does not exacerbate obesity or insulin resistance in mice on a HFD, despite impaired mitochondrial content and function and elevated inflammatory signaling and oxidative stress.

Identifying Toxicant-Interacting Genes Using Forward Genetic Screening in Zebrafish.

Forward genetic screening is an extremely powerful method for identifying novel genes driving a broad range of phenotypes. This protocol describes the complete process for conducting a forward genetic screen in zebrafish, including mutagenesis with N-ethyl-N-nitrosourea (ENU), mating, phenotypic screening, and genetic mapping.

SOX10 regulates multiple genes to direct eumelanin versus pheomelanin production in domestic rock pigeon.

The domesticated rock pigeon (Columba livia) has been bred for hundreds of years to display an immense variety of ornamental attributes such as feather color and color patterns. Color is influenced by multiple loci that impact the type and amount of melanin deposited on the feathers. Pigeons homozygous for the "recessive red" mutation, which causes downregulation of Sox10, display brilliant red feathers instead of blue/black feathers. Sox10 encodes a transcription factor important for melanocyte differentiation and function, but the genes that mediate its promotion of black versus red pigment are unknown. Here, we present a transcriptomic comparison of regenerating feathers from wild-type and recessive red pigeons to identify candidate SOX10 targets. Our results identify both known and novel targets, including many genes not previously implicated in pigmentation. These data highlight the value of using novel, emerging model organisms to gain insight into the genetic basis of pigment variation.

Imbalanced sphingolipid signaling is maintained as a core proponent of a cancerous phenotype in spite of metabolic pressure and epigenetic drift.

Tumor heterogeneity may arise through genetic drift and environmentally driven clonal selection for metabolic fitness. This would promote subpopulations derived from single cancer cells that exhibit distinct phenotypes while conserving vital pro-survival pathways. We aimed to identify significant drivers of cell fitness in pancreatic adenocarcinoma (PDAC) creating subclones in different nutrient formulations to encourage differential metabolic reprogramming. The genetic and phenotypic expression profiles of each subclone were analyzed relative to a healthy control cell line (hTert-HPNE). The subclones exhibited distinct variations in protein expression and lipid metabolism. Relative to hTert-HPNE, PSN-1 subclones uniformly maintained modified sphingolipid signaling and specifically retained elevated sphingosine-1-phosphate (S1P) relative to C16 ceramide (C16 Cer) ratios. Each clone utilized a different perturbation to this pathway, but maintained this modified signaling to preserve cancerous phenotypes, such as rapid proliferation and defense against mitochondria-mediated apoptosis. Although the subclones were unique in their sensitivity, inhibition of S1P synthesis significantly reduced the ratio of S1P/C16 Cer, slowed cell proliferation, and enhanced sensitivity to apoptotic signals. This reliance on S1P signaling identifies this pathway as a promising drug-sensitizing target that may be used to eliminate cancerous cells consistently across uniquely reprogrammed PDAC clones.

ZeMo: An Open Source Water Quality Monitoring System for Aquaria.

Zebrafish and other aquatic organisms depend on careful monitoring and adjustment of water quality for health and survival. This ideally includes continuous monitoring of several water parameters, including temperature, pH, conductivity, and dissolved oxygen. However, manual readings can be laborious, and commercially available monitors are cost-prohibitive for many installations, especially in small laboratories and classrooms. To address these issues, we have created ZeMo, a high-end open-source water monitoring system that includes a touchscreen, web interface, and email alerts-making continuous water quality monitoring attainable for a wide range of aquarium installations.

FOXN3 Regulates Hepatic Glucose Utilization.

A SNP (rs8004664) in the first intron of the FOXN3 gene is associated with human fasting blood glucose. We find that carriers of the risk allele have higher hepatic expression of the transcriptional repressor FOXN3. Rat Foxn3 protein and zebrafish foxn3 transcripts are downregulated during fasting, a process recapitulated in human HepG2 hepatoma cells. Transgenic overexpression of zebrafish foxn3 or human FOXN3 increases zebrafish hepatic gluconeogenic gene expression, whole-larval free glucose, and adult fasting blood glucose and also decreases expression of glycolytic genes. Hepatic FOXN3 overexpression suppresses expression of mycb, whose ortholog MYC is known to directly stimulate expression of glucose-utilization enzymes. Carriers of the rs8004664 risk allele have decreased MYC transcript abundance. Human FOXN3 binds DNA sequences in the human MYC and zebrafish mycb loci. We conclude that the rs8004664 risk allele drives excessive expression of FOXN3 during fasting and that FOXN3 regulates fasting blood glucose.