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Visual Abstract, Highlights • Combining CHD phenotype–driven gene set enrichment and CRISPR knockdown screening in zebrafish is an effective approach to identifying novel CHD genes. • Mutations affecting genes coding for the WAVE2 protein complex and small GTPase-mediated signaling are associated with LVOTO lesions. • WAVE2 complex genes brk1, nckap1, and wasf2 and regulators of small GTPase signaling cul3a and racgap1 are critical to zebrafish heart development., Summary 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 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.

During zebrafish cardiac development, 3-OST-7 constrains BMP signaling to the atrioventricular junction and precludes it from contractile myocardium, allowing tropomyosin-dependent sarcomere assembly and contraction., The 3-O-sulfotransferase (3-OST) family catalyzes rare modifications of glycosaminoglycan chains on heparan sulfate proteoglycans, yet their biological functions are largely unknown. Knockdown of 3-OST-7 in zebrafish uncouples cardiac ventricular contraction from normal calcium cycling and electrophysiology by reducing tropomyosin4 (tpm4) expression. Normal 3-OST-7 activity prevents the expansion of BMP signaling into ventricular myocytes, and ectopic activation of BMP mimics the ventricular noncontraction phenotype seen in 3-OST-7 depleted embryos. In 3-OST-7 morphants, ventricular contraction can be rescued by overexpression of tropomyosin tpm4 but not by troponin tnnt2, indicating that tpm4 serves as a lynchpin for ventricular sarcomere organization downstream of 3-OST-7. Contraction can be rescued by expression of 3-OST-7 in endocardium, or by genetic loss of bmp4. Strikingly, BMP misregulation seen in 3-OST-7 morphants also occurs in multiple cardiac noncontraction models, including potassium voltage-gated channel gene, kcnh2, affected in Romano-Ward syndrome and long-QT syndrome, and cardiac troponin T gene, tnnt2, affected in human cardiomyopathies. Together these results reveal 3-OST-7 as a key component of a novel pathway that constrains BMP signaling from ventricular myocytes, coordinates sarcomere assembly, and promotes cardiac contractile function., Author Summary A highly complex environment at the cell surface and in the space between cells is thought to modulate cell behavior. Heparan sulfate proteoglycans are cell surface and extracellular matrix molecules that are covalently linked to long chains of repeating sugar units called glycosaminoglycan chains. These chains can be subjected to rare modifications and they are believed to influence specific cell signaling events in a lineage specific fashion in what is called the “glycocode.” Here we explore the functions of one member of a family of enzymes, 3-O-sulfotransferases (3-OSTs) that catalyzes a rare modification (3-O-sulfation) of glycosaminoglycans in zebrafish. We show that knockdown of 3-OST-7 results in a very specific phenotype, including loss of cardiac ventricle contraction. Knockdown of other 3-OST family members did not result in the same phenotype, suggesting that distinct 3-OST family members have distinct functions in vertebrates and lending in vivo evidence for the glycocode hypothesis. Mechanistically, we found that cardiac contraction can be rescued by reducing the amount of endogenous BMP4, and can be blocked by increasing BMP signaling, suggesting that the glycocode generated by 3-OST-7 is necessary to constrain BMP signaling in the heart for normal cardiac contraction. Furthermore, we show that tropomyosin4 (tpm4) is downstream of 3-OST-7 function, indicating that Tpm4 is key in this pathway to building the sarcomere, the functional contraction unit of the cardiomyocyte.

Real-time imaging coupled with a permeabilized cell system presents a very versatile platform to visualize the dynamic and intricate nature of nuclear envelope breakdown, one of the major morphological changes of mitosis. Here, we describe such a strategy in which the plasma membrane of cells expressing fluorescently tagged nucleoporin POM121 and Histone H2B is permeabilized with digitonin. These cells are then incubated with mitotic Xenopus egg extract to create conditions that recapitulate the major events of mitotic nuclear remodeling seen in live-cell imaging, providing the opportunity to probe mechanisms and pathways that coordinate nuclear disassembly.

By most measures, the University of Utah Centralized Zebrafish Animal Resource is a successful zebrafish core facility: we house ∼4000-5000 tanks for over 16 research groups; provide services and equipment for ∼150 users; are currently undergoing an expansion by 3000 tanks; and have been praised by institutional and national regulatory agencies for the cleanliness and efficiency of our facility. In recent years, we have implemented new programs to improve the overall health of our colony and believe we have seen a reduction in apparently sick fish. However, there are still deficiencies in our monitoring and pathogen control programs. Our histopathology sample sizes have been insufficient to estimate prevalence, but our sentinel tank program reveals the presence of Pseudoloma neurophilia and myxozoan, presumably Myxidium streisinger, in our facility. As we develop protocols to further reduce the burden of disease, we are focused on defining our baseline, establishing goals, and implementing methods to monitor our progress. The data generated by this approach will allow us to evaluate and implement the most cost-effective protocols to improve fish health.

Interactions between Nup50 and soluble transport factors underlie the efficiency of certain nucleocytoplasmic transport pathways. The platform on which these interactions take place is important to building a complete understanding of nucleocytoplasmic trafficking. Nup153 is the nucleoporin that provides this scaffold for Nup50. Here, we have delineated requirements for the interaction between Nup153 and Nup50, revealing a dual interface. An interaction between Nup50 and a region in the unique N-terminal region of Nup153 is critical for the nuclear pore localization of Nup50. A second site of interaction is at the distal tail of Nup153 and is dependent on importin α. Both of these interactions involve the N-terminal domain of Nup50. The configuration of the Nup153-Nup50 partnership suggests that the Nup153 scaffold provides not just a means of pore targeting for Nup50 but also serves to provide a local environment that facilitates bringing Nup50 and importin α together, as well as other soluble factors involved in transport. Consistent with this, disruption of the Nup153-Nup50 interface decreases efficiency of nuclear import.

Neuronal and glial deposition of misfolded, proteolytically processed, polyubiquitinated and abnormally phosphorylated C-terminal fragments (CTFs) of the TAR DNA binding protein-43 (TDP-43) is a pathological hallmark of frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) and certain cases of amyotrophic lateral sclerosis. We demonstrate that TDP-43 can be proteolytically processed by caspases upon induction of apoptosis to a major 35 kDa and a minor 25 kDa CTF. These fragments are initially soluble, but over time they accumulate as insoluble and pathologically phosphorylated derivatives. However, proteolytic processing appears not to be absolutely required for the deposition of insoluble TDP-43 species, since a caspase resistant mutant of TDP-43 is also converted into insoluble species. Phosphorylation at S409/410 apparently occurs late during the conversion of soluble to insoluble TDP-43, suggesting that phosphorylation is not a prerequisite for aggregation. Loss of function of the progranulin (PGRN) gene causes FTLD-U with TDP-43 positive inclusions and has been suggested to lead to caspase activation and subsequent TDP-43 processing. However, siRNA-mediated knockdown of PGRN in cell culture as well as a PGRN gene knockout in mice failed to cause the formation of the disease characterizing CTFs of TDP-43. Our findings therefore suggest that caspase-mediated processing generates CTFs of similar biochemical properties as those occurring in nuclear and cytoplasmic deposits of FTLD-U patients independent of PGRN levels.

Loss of function mutations in progranulin cause tau-negative frontotemporal lobar degeneration with ubiquitin-positive inclusions. A major protein component of these inclusions is TDP-43, which becomes hyperphosphorylated, ubiquitinated, and cleaved to generate C-terminal fragments, which apparently translocate from nuclei to the cytoplasm. Most progranulin mutations are nonsense mutations resulting in nonsense-mediated mRNA decay and consequently reduced progranulin protein levels. However, some missense mutations are described that occur within the signal sequence and mature progranulin. We now demonstrate that a progranulin mutation located within the signal sequence (PGRN A9D) results in cytoplasmic missorting with extremely low expression. In contrast, two other progranulin mutations (PGRN P248L and R432C) are expressed as immature proteins but are inefficiently transported through and partially degraded within the secretory pathway, resulting in a significantly reduced secretion. Thus apparently all progranulin mutations cause reduced protein expression or secretion, although by different cellular mechanisms. To investigate a putative relationship between reduced expression of progranulin and TDP-43 relocalization and deposition, we down-regulated progranulin in human cell lines and in zebrafish. Upon reduction of progranulin, neither a major redistribution of TDP-43 nor proteolytic processing to disease-characterizing C-terminal fragments could be observed.

Somitogenesis is the key developmental process that lays down the framework for a metameric body in vertebrates. Somites are generated from the un-segmented presomitic mesoderm (PSM) by a pre-patterning process driven by a molecular oscillator termed the segmentation clock. The Delta–Notch intercellular signaling pathway and genes belonging to the hairy (h) and Enhancer of split (E(spl))-related (h/E(spl)) family of transcriptional repressors are conserved components of this oscillator. A subset of these genes, called cyclic genes, is characterized by oscillating mRNA expression that sweeps anteriorly like a wave through the embryonic PSM. Periodic transcriptional repression by H/E(spl) proteins is thought to provide a critical part of a negative feedback loop in the oscillatory process, but it is an open question how many cyclic h/E(spl) genes are involved in the somitogenesis clock in any species, and what distinct roles they might play. From a genome-wide search for h/E(spl) genes in the zebrafish, we previously estimated a total of five cyclic members. Here we report that one of these, the mHes5 homologue her15 actually exists as a very recently duplicated gene pair. We investigate the expression of this gene pair and analyse its regulation and activity in comparison to the paralogous her12 gene, and the other cyclic h/E(spl) genes in the zebrafish. The her15 gene pair and her12 display novel and distinct expression features, including a caudally restricted oscillatory domain and dynamic stripes of expression in the rostral PSM that occur at the future segmental borders. her15 expression stripes demarcate a unique two-segment interval in the rostral PSM. Mutant, morpholino, and inhibitor studies show that her12 and her15 expression in the PSM is regulated by Delta–Notch signaling in a complex manner, and is dependent on her7, but not her1 function. Morpholino-mediated her12 knockdown disrupts cyclic gene expression, indicating that it is a non-redundant core component of the segmentation clock. Over-expression of her12, her15 or her7 disrupts cyclic gene expression and somite border formation, and structure function analysis of Her7 indicates that DNA binding, but not Groucho-recruitment seems to be important in this process. Thus, the zebrafish has five functional cyclic h/E(spl) genes, which are expressed in a distinct spatial configuration. We propose that this creates a segmentation oscillator that varies in biochemical composition depending on position in the PSM.