Systems Analysis Implicates WAVE2 Complex in the Pathogenesis of Developmental Left-Sided Obstructive Heart Defects.

Genetic variants are the primary driver of congenital heart disease (CHD) pathogenesis. However, our ability to identify causative variants is limited. To identify causal CHD genes that are associated with specific molecular functions, the study used prior knowledge to filter de novo variants from 2,881 probands with sporadic severe CHD. This approach enabled the authors to identify an association between left ventricular outflow tract obstruction lesions and genes associated with the WAVE2 complex and regulation of small GTPase-mediated signal transduction. Using CRISPR zebrafish knockdowns, the study confirmed that WAVE2 complex proteins brk1, nckap1, and wasf2 and the regulators of small GTPase signaling cul3a and racgap1 are critical to cardiac development.

The Paf1 complex and P-TEFb have reciprocal and antagonist roles in maintaining multipotent neural crest progenitors.

Multipotent progenitor populations are necessary for generating diverse tissue types during embryogenesis. We show the RNA polymerase-associated factor 1 complex (Paf1C) is required to maintain multipotent progenitors of the neural crest (NC) lineage in zebrafish. Mutations affecting each Paf1C component result in near-identical NC phenotypes; alyron mutant embryos carrying a null mutation in paf1 were analyzed in detail. In the absence of zygotic paf1 function, definitive premigratory NC progenitors arise but fail to maintain expression of the sox10 specification gene. The mutant NC progenitors migrate aberrantly and fail to differentiate appropriately. Blood and germ cell progenitor development is affected similarly. Development of mutant NC could be rescued by additional loss of positive transcription elongation factor b (P-TEFb) activity, a key factor in promoting transcription elongation. Consistent with the interpretation that inhibiting/delaying expression of some genes is essential for maintaining progenitors, mutant embryos lacking the CDK9 kinase component of P-TEFb exhibit a surfeit of NC progenitors and their derivatives. We propose Paf1C and P-TEFb act antagonistically to regulate the timing of the expression of genes needed for NC development.

De novo and recessive forms of congenital heart disease have distinct genetic and phenotypic landscapes.

The genetic architecture of sporadic congenital heart disease (CHD) is characterized by enrichment in damaging de novo variants in chromatin-modifying genes. To test the hypothesis that gene pathways contributing to de novo forms of CHD are distinct from those for recessive forms, we analyze 2391 whole-exome trios from the Pediatric Cardiac Genomics Consortium. We deploy a permutation-based gene-burden analysis to identify damaging recessive and compound heterozygous genotypes and disease genes, controlling for confounding effects, such as background mutation rate and ancestry. Cilia-related genes are significantly enriched for damaging rare recessive genotypes, but comparatively depleted for de novo variants. The opposite trend is observed for chromatin-modifying genes. Other cardiac developmental gene classes have less stratification by mode of inheritance than cilia and chromatin-modifying gene classes. Our analyses reveal dominant and recessive CHD are associated with distinct gene functions, with cilia-related genes providing a reservoir of rare segregating variation leading to CHD.

Maternal Gdf3 is an obligatory cofactor in Nodal signaling for embryonic axis formation in zebrafish.

Zebrafish Gdf3 (Dvr1) is a member of the TGFß superfamily of cell signaling ligands that includes Xenopus Vg1 and mammalian Gdf1/3. Surprisingly, engineered homozygous mutants in zebrafish have no apparent phenotype. Elimination of Gdf3 in oocytes of maternal-zygotic mutants results in embryonic lethality that can be fully rescued with gdf3 RNA, demonstrating that Gdf3 is required only early in development, beyond which mutants are viable and fertile. Gdf3 mutants are refractory to Nodal ligands and Nodal repressor Lefty1. Signaling driven by TGFß ligand Activin and constitutively active receptors Alk4 and Alk2 remain intact in gdf3 mutants, indicating that Gdf3 functions at the same pathway step as Nodal. Targeting gdf3 and ndr2 RNA to specific lineages indicates that exogenous gdf3 is able to fully rescue mutants only when co-expressed with endogenous Nodal. Together, these findings demonstrate that Gdf3 is an essential cofactor of Nodal signaling during establishment of the embryonic axis.

CRISPR/Cas9-Directed Gene Editing for the Generation of Loss-of-Function Mutants in High-Throughput Zebrafish F0 Screens.

The ability to perform reverse genetics in the zebrafish model organism has been greatly advanced with the advent of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated) system. The high level of efficiency in generating mutations when using the CRISPR/Cas9 system combined with the rapid generation time of the zebrafish model organism has made the possibility of performing F0 screens in this organism a reality. This unit describes a detailed protocol for performing an F0 screen using the CRISPR/Cas9 system in zebrafish starting with the design and production of custom CRISPR/Cas9 reagents for injection. Next, two approaches for determining the efficiency of mutation induction by the custom CRISPR/Cas9 reagents that are easily performed using standard molecular biology protocols are detailed. Finally, screening for F0 induced phenotypes using the zebrafish flh gene as an example is discussed. © 2017 by John Wiley & Sons, Inc.

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.

Differential roles for 3-OSTs in the regulation of cilia length and motility.

As cells integrate molecular signals from their environment, cell surface receptors require modified proteoglycans for the robust activation of signaling pathways. Heparan sulfate proteoglycans (HSPGs) have long unbranched chains of repetitive disaccharide units that can be sulfated at specific positions by heparan sulfate O-sulfotransferase (OST) families. Here, we show that two members of the 3-OST family are required in distinct signaling pathways to control left-right (LR) patterning through control of Kupffer's vesicle (KV) cilia length and motility. 3-OST-5 functions in the fibroblast growth factor pathway to control cilia length via the ciliogenic transcription factors FoxJ1a and Rfx2. By contrast, a second 3-OST family member, 3-OST-6, does not regulate cilia length, but regulates cilia motility via kinesin motor molecule (Kif3b) expression and cilia arm dynein assembly. Thus, two 3-OST family members cell-autonomously control LR patterning through distinct pathways that regulate KV fluid flow. We propose that individual 3-OST isozymes create distinct modified domains or 'glycocodes' on cell surface proteoglycans, which in turn regulate the response to diverse cell signaling pathways.

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.

RFX2 is essential in the ciliated organ of asymmetry and an RFX2 transgene identifies a population of ciliated cells sufficient for fluid flow.

Motile cilia create asymmetric fluid flow in the evolutionarily conserved ciliated organ of asymmetry (COA) and play a fundamental role in establishing the left-right (LR) axis in vertebrate embryos. The transcriptional control of the large group of genes that encode proteins that contribute to ciliary structure and function remains poorly understood. In this study we find that the winged helix transcription factor Rfx2 is expressed in motile cilia in mouse and zebrafish embryos. Morpholino knockdown of Rfx2 function in the whole embryo or specifically in cells of the zebrafish COA (Kupffer's Vesicle, KV) leads to reduced KV cilia length and perturbations in LR asymmetry. LR patterning defects include randomization of the early asymmetric Nodal signaling pathway genes southpaw, lefty1 and lefty2 and subsequent reversals in the organ primordia of the heart and gut. Rfx2 is also required for ciliogenesis in zebrafish pronephric duct. We further show that by restoring Left-Right dynein (LRD) expression and motility specifically in a subset of ciliated cells of the mouse COA (posterior notochord, PNC), we can restore fluid flow, asymmetric expression of Pitx2 and partially rescue situs defects.

FGF signalling during embryo development regulates cilia length in diverse epithelia.

Cilia are cell surface organelles found on most epithelia in vertebrates. Specialized groups of cilia have critical roles in embryonic development, including left-right axis formation. Recently, cilia have been implicated as recipients of cell-cell signalling. However, little is known about cell-cell signalling pathways that control the length of cilia. Here we provide several lines of evidence showing that fibroblast growth factor (FGF) signalling regulates cilia length and function in diverse epithelia during zebrafish and Xenopus development. Morpholino knockdown of FGF receptor 1 (Fgfr1) in zebrafish cell-autonomously reduces cilia length in Kupffer's vesicle and perturbs directional fluid flow required for left-right patterning of the embryo. Expression of a dominant-negative FGF receptor (DN-Fgfr1), treatment with SU5402 (a pharmacological inhibitor of FGF signalling) or genetic and morpholino reduction of redundant FGF ligands Fgf8 and Fgf24 reproduces this cilia length phenotype. Knockdown of Fgfr1 also results in shorter tethering cilia in the otic vesicle and shorter motile cilia in the pronephric ducts. In Xenopus, expression of a dn-fgfr1 results in shorter monocilia in the gastrocoel roof plate that control left-right patterning and in shorter multicilia in external mucociliary epithelium. Together, these results indicate a fundamental and highly conserved role for FGF signalling in the regulation of cilia length in multiple tissues. Abrogation of Fgfr1 signalling downregulates expression of two ciliogenic transcription factors, foxj1 and rfx2, and of the intraflagellar transport gene ift88 (also known as polaris), indicating that FGF signalling mediates cilia length through an Fgf8/Fgf24-Fgfr1-intraflagellar transport pathway. We propose that a subset of developmental defects and diseases ascribed to FGF signalling are due in part to loss of cilia function.

Baf60c is a nuclear Notch signaling component required for the establishment of left-right asymmetry.

Notch-mediated induction of Nodal at the vertebrate node is a critical step in initiating left-right (LR) asymmetry. In mice and zebrafish we show that Baf60c, a subunit of the Swi/Snf-like BAF chromatin remodeling complex, is essential for establishment of LR asymmetry. Baf60c knockdown mouse embryos fail to activate Nodal at the node and also have abnormal node morphology with mixing of crown and pit cells. In cell culture, Baf60c is required for Notch-dependent transcriptional activation and functions to stabilize interactions between activated Notch and its DNA-binding partner, RBP-J. Brg1 is also required for these processes, suggesting that BAF complexes are key components of nuclear Notch signaling. We propose a critical role for Baf60c in Notch-dependent transcription and LR asymmetry.

The roles of cilia in developmental disorders and disease.

Cilia are highly conserved organelles that have diverse motility and sensory functions. Recent discoveries have revealed that cilia also have crucial roles in cell signaling pathways and in maintaining cellular homeostasis. As such, defects in cilia formation or function have profound effects on the development of body pattern and the physiology of multiple organ systems. By categorizing syndromes that are due to cilia dysfunction in humans and from studies in vertebrate model organisms, molecular pathways that intersect with cilia formation and function have come to light. Here, we summarize an emerging view that in order to understand some complex developmental pathways and disease etiologies, one must consider the molecular functions performed by cilia.

Polaris and Polycystin-2 in dorsal forerunner cells and Kupffer's vesicle are required for specification of the zebrafish left-right axis.

Recently, it has become clear that motile cilia play a central role in initiating a left-sided signaling cascade important in establishing the LR axis during mouse and zebrafish embryogenesis. Two genes proposed to be important in this cilia-mediated signaling cascade are polaris and polycystin-2 (pkd2). Polaris is involved in ciliary assembly, while Pkd2 is proposed to function as a Ca(2+)-permeable cation channel. We have cloned zebrafish homologues of polaris and pkd2. Both genes are expressed in dorsal forerunner cells (DFCs) from gastrulation to early somite stages when these cells form a ciliated Kupffer's vesicle (KV). Morpholino-mediated knockdown of Polaris or Pkd2 in zebrafish results in misexpression of left-side-specific genes, including southpaw, lefty1 and lefty2, and randomization of heart and gut looping. By targeting morpholinos to DFCs/KV, we show that polaris and pkd2 are required in DFCs/KV for normal LR development. Polaris morphants have defects in KV cilia, suggesting that the laterality phenotype is due to problems in cilia function per se. We further show that expression of polaris and pkd2 is dependent on the T-box transcription factors no tail and spadetail, respectively, suggesting that these genes have a previously unrecognized role in regulating ciliary structure and function. Our data suggest that the functions of polaris and pkd2 in LR patterning are conserved between zebrafish and mice and that Kupffer's vesicle functions as a ciliated organ of asymmetry.

Genomic characterization and expression analysis of the first nonmammalian renin genes from zebrafish and pufferfish.

Renin is a key enzyme in the renin-angiotensin system (RAS), a pathway which plays an important physiological role in blood pressure and electrolyte homeostasis. The origin of the RAS is believed to have accompanied early evolution of vertebrates. However, renin genes have so far only been unequivocally identified in mammals. Whether or not a bona fide renin gene exists in nonmammalian vertebrates has been an intriguing question of physiological and evolutionary interest. Using a genomic analytical approach, we identified renin genes in two nonmammalian vertebrates, zebrafish (Danio rerio) and pufferfish (Takifugu rubripes). Phylogenetic analysis demonstrates that the predicted fish renins cluster together with mammalian renins to form a distinct subclass of vertebrate aspartyl proteases. RT-PCR results confirm generation of the predicted zebrafish mRNA and its expression in association with the opisthonephric kidney of adult zebrafish. Comparative in situ hybridization analysis of wild-type and developmental mutants indicates that renin expression is first detected bilaterally in cells of the interrenal primordia at 24 h postfertilization, which subsequently migrate to lie adjacent to, but distinct from, the glomerulus of the developing pronephric kidney. Our report provides the first molecular evidence for the existence of renin genes in lower vertebrates. The observation that the earliest renin-expressing cells, arising during ontogeny of this teleost vertebrate, are of adrenocortical lineage raises an interesting hypothesis as regards the origin of renin-expressing cells in the metanephric kidney of higher vertebrates.

Genetics of human laterality disorders: insights from vertebrate model systems.

Many internal organs in the vertebrate body are asymmetrically oriented along the left-right (L-R) body axis. Organ asymmetry and some components of the molecular signaling pathways that direct L-R development are highly conserved among vertebrate species. Although individuals with full reversal of organ L-R asymmetry (situs inversus totalis) are healthy, significant morbidity and mortality is associated with perturbations in laterality that result in discordant orientation of organ systems and complex congenital heart defects. In humans and other vertebrates, genetic alterations of L-R signaling pathways can result in a wide spectrum of laterality defects. In this review we categorize laterality defects in humans, mice, and zebrafish into specific classes based on altered patterns of asymmetric gene expression, organ situs defects, and midline phenotypes. We suggest that this classification system provides a conceptual framework to help consolidate the disparate laterality phenotypes reported in humans and vertebrate model organisms, thereby refining our understanding of the genetics of L-R development. This approach helps suggest candidate genes and genetic pathways that might be perturbed in human laterality disorders and improves diagnostic criteria.

Evolutionary Conservation of the Larval Serotonergic Nervous System in a Direct Developing Sea Urchin: (sea urchin development/larval nervous systems/heterochrony/direct development/Heliocidaris erythrogramma).

Development of the larval serotonergic nervous system is examined by indirect immunofluorescence in two congeneric species of sea urchins that exhibit divergent embryonic and larval development. Heliocidar is tuberculata undergoes indirect planktotrophic development via a pluteus larva, whereas Heliocidaris erythrogramma develops directly, passing through a brief, highly derived lecithotrophic larval stage. We have cleared the opaque embryos of H. erythrogramma and discuss internal features of its development. The serotonergic nervous system of H. tuberculata arises in the apical plate at the end of gastrulation and develops into a bilaterally symmetric ganglion lying between the anterolateral arms in the preoral hood. Putatively homologous neurons appear at the apical end of the modified larva of H. erythrogramma well after the completion of gastrulation, coincident with development of the primary podia of the adult rudiment. The neurons form a bilaterally symmetric ganglion whose orientation relative to the vestibule is conserved with respect to that found in planktotrophic larvae. This allows us to define a left and right side for this larva which lacks external points of asymmetry such as a larval mouth. The alteration in the time of nervous system development in H. erythrogramma relative to that of H. tuberculata, and other indirect developers, implicates heterochronies in cellular differentiation as an important component of the evolution of direct development.

Development of Serotonergic Neurons in Embryos of the Sea Urchin, Strongylocentrotus purpuratus: (serotonergic/neural development/embryo/echinoid).

The development of the serotonergic component of the nervous system of larvae of S. purpuratus is traced using indirect immunofluorescence with a polyclonal antibody against the neurotransmitter serotonin. Initially one or two neuroblasts can be detected in the thickened epithelium of the animal plate of late gastrulae (56 hr). The number of immunoreactive cells increases to about eight during formation of the pluteus (85-90 hr). Immunoreactive axons appear simultaneously from all neuroblasts present in the 79 hr prism stage larva and form the apical ganglion. It is proposed that this component of the larval nervous system is derived from a small number of ectodermal cells associated with the apical tuft.