The intracellular domain of Sema6A is essential for development of the zebrafish retina.

Semaphorin6A (Sema6A) is a repulsive guidance molecule that plays many roles in central nervous system, heart and bone development, as well as immune system responses and cell signaling in cancer. Loss of Sema6A or its receptor PlexinA2 in zebrafish leads to smaller eyes and improper retinal patterning. Here, we investigate a potential role for the Sema6A intracellular domain in zebrafish eye development and dissect which phenotypes rely on forward signaling and which rely on reverse signaling. We performed rescue experiments on zebrafish Sema6A morphants with either full-length Sema6A (Sema6A-FL) or Sema6A lacking its intracellular domain (Sema6A-ΔC). We identified that the intracellular domain is not required for eye size and retinal patterning, however it is required for retinal integrity, the number and end feet strength of Müller glia and protecting against retinal cell death. This novel function for the intracellular domain suggests a role for Sema6A reverse signaling in zebrafish eye development.

Crk adaptor proteins are necessary for the development of the zebrafish retina.

BACKGROUND: The development of the central nervous system (CNS) requires critical cell signaling molecules to coordinate cell proliferation and migration in order to structure the adult tissue. Chicken tumor virus #10 Regulator of Kinase (CRK) and CRK-like (CRKL) are adaptor proteins with pre-metazoan ancestry and are known to be required for patterning laminated structures downstream of Reelin (RELN), such as the cerebral cortex, cerebellum, and hippocampus. CRK and CRKL also play crucial roles in a variety of other growth factor and extracellular matrix signaling cascades. The neuronal retina is another highly laminated structure within the CNS that is dependent on migration for proper development, but the cell signaling mechanisms behind neuronal positioning in the retina are only partly understood. RESULTS: We find that crk and crkl have largely overlapping expression within the developing zebrafish nervous system. We find that their disruption results in smaller eye size and loss of retinal lamination. CONCLUSIONS: Our data indicate that Crk adaptors are critical for proper development of the zebrafish neural retina in a crk/crkl dose-dependent manner.

FYN and ABL Regulate the Interaction Networks of the DCBLD Receptor Family.

The Discoidin, CUB, and LCCL domain-containing protein (DCBLD) family consists of two type-I transmembrane scaffolding receptors, DCBLD1 and DCBLD2, which play important roles in development and cancer. The nonreceptor tyrosine kinases FYN and ABL are known to drive phosphorylation of tyrosine residues in YXXP motifs within the intracellular domains of DCBLD family members, which leads to the recruitment of the Src homology 2 (SH2) domain of the adaptors CT10 regulator of kinase (CRK) and CRK-like (CRKL). We previously characterized the FYN- and ABL-driven phosphorylation of DCBLD family YXXP motifs. However, we have identified additional FYN- and ABL-dependent phosphorylation sites on DCBLD1 and DCBLD2. This suggests that beyond CRK and CRKL, additional DCBLD interactors may be regulated by FYN and ABL activity. Here, we report a quantitative proteomics approach in which we map the FYN- and ABL-regulated interactomes of DCBLD family members. We found FYN and ABL regulated the binding of several signaling molecules to DCBLD1 and DCBLD2, including members of the 14-3-3 family of adaptors. Biochemical investigation of the DCBLD2/14-3-3 interaction revealed ABL-induced binding of 14-3-3 family members directly to DCBLD2.

Shootin-1 is required for nervous system development in zebrafish.

BACKGROUND: Semaphorin6A (Sema6A) and its PlexinA2 (PlxnA2) receptor canonically function as repulsive axon guidance cues. To understand downstream signaling mechanisms, we performed a microarray screen and identified the "clutch molecule" shootin-1 (shtn-1) as a transcriptionally repressed target. Shtn-1 is a key proponent of cell migration and neuronal polarization and must be regulated during nervous system development. The mechanisms of Shtn-1 regulation and the phenotypic consequences of loss of repression are poorly understood. RESULTS: We demonstrate shtn-1 overexpression results in impaired migration of the optic vesicles, lack of retinal pigmented epithelium, and pathfinding errors of retinotectal projections. We also observed patterning defects in the peripheral nervous system. Importantly, these phenotypes were rescued by overexpressing PlxnA2. CONCLUSIONS: We demonstrate a functional role for repression of shtn-1 by PlxnA2 in development of the eyes and peripheral nervous system in zebrafish. These results demonstrate that careful regulation of shtn-1 is critical for development of the nervous system.

Mechanical Characteristics of Ultrafast Zebrafish Larval Swimming Muscles.

Zebrafish (Danio rerio) swim within days of fertilization, powered by muscles of the axial myotomes. Forces generated by these muscles can be measured rapidly in whole, intact larval tails by adapting protocols developed for ex vivo muscle mechanics. But it is not known how well these measurements reflect the function of the underlying muscle fibers and sarcomeres. Here, we consider the anatomy of the 5-day-old, wild-type larval tail, and implement technical modifications to measuring muscle physiology in intact tails. Specifically, we quantify fundamental relationships between force, length, and shortening velocity, and capture the extreme contractile speeds required to swim with tail-beat frequencies of 80-100 Hz. Therefore, we analyze 1000 frames/s videos to track the movement of structures, visible in the transparent tail, which correlate with sarcomere length. We also characterize the passive viscoelastic properties of the preparation to isolate forces contributed by nonmuscle structures within the tail. Myotomal muscles generate more than 95% of their maximal isometric stress (76 ± 3 mN/mm2) over the range of muscle lengths used in vivo. They have rapid twitch kinetics (full width at half-maximal stress: 11 ± 1 ms) and a high twitch/tetanus ratio (0.91 ± 0.05), indicating adaptations for fast excitation-contraction coupling. Although contractile stress is relatively low, myotomal muscles develop high net power (134 ± 20 W/kg at 80 Hz) in cyclical work loop experiments designed to simulate the in vivo dynamics of muscle fibers during swimming. When shortening at a constant speed of 7 ± 1 muscle lengths/s, muscles develop 86 ± 2% of isometric stress, whereas peak instantaneous power during 100 Hz work loops occurs at 18 ± 2 muscle lengths/s. These approaches can improve the usefulness of zebrafish as a model system for muscle research by providing a rapid and sensitive functional readout for experimental interventions.

The DCBLD receptor family: emerging signaling roles in development, homeostasis and disease.

The discoidin, CUB, and LCCL domain-containing (DCBLD) receptor family are composed of the type-I transmembrane proteins DCBLD1 and DCBLD2 (also ESDN and CLCP1). These proteins are highly conserved across vertebrates and possess similar domain structure to that of neuropilins, which act as critical co-receptors in developmental processes. Although DCBLD1 remains largely uncharacterized, the functional and mechanistic roles of DCBLD2 are emerging. This review provides a comprehensive discussion of this presumed receptor family, ranging from structural and signaling aspects to their associations with cancer, physiology, and development.

An in silico proteomics screen to predict and prioritize protein-protein interactions dependent on post-translationally modified motifs.

Motivation: The development of proteomic methods for the characterization of domain/motif interactions has greatly expanded our understanding of signal transduction. However, proteomics-based binding screens have limitations including that the queried tissue or cell type may not harbor all potential interacting partners or post-translational modifications (PTMs) required for the interaction. Therefore, we sought a generalizable, complementary in silico approach to identify potentially novel motif and PTM-dependent binding partners of high priority. Results: We used as an initial example the interaction between the Src homology 2 (SH2) domains of the adaptor proteins CT10 regulator of kinase (CRK) and CRK-like (CRKL) and phosphorylated-YXXP motifs. Employing well-curated, publicly-available resources, we scored and prioritized potential CRK/CRKL-SH2 interactors possessing signature characteristics of known interacting partners. Our approach gave high priority scores to 102 of the >9000 YXXP motif-containing proteins. Within this 102 were 21 of the 25 curated CRK/CRKL-SH2-binding partners showing a more than 80-fold enrichment. Several predicted interactors were validated biochemically. To demonstrate generalized applicability, we used our workflow to predict protein-protein interactions dependent upon motif-specific arginine methylation. Our data demonstrate the applicability of our approach to, conceivably, any modular binding domain that recognizes a specific post-translationally modified motif. Supplementary information: Supplementary data are available at Bioinformatics online.

Dynamic multi-site phosphorylation by Fyn and Abl drives the interaction between CRKL and the novel scaffolding receptors DCBLD1 and DCBLD2.

Discoidin, CUB, and LCCL domain containing 2 (DCBLD2) is a neuropilin-like transmembrane scaffolding receptor with known and anticipated roles in vascular remodeling and neuronal positioning. DCBLD2 is also up-regulated in several cancers and can drive glioblastomas downstream of activated epidermal growth factor receptor. While a few studies have shown either a positive or negative role for DCBLD2 in regulating growth factor receptor signaling, little is known about the conserved signaling features of DCBLD family members that drive their molecular activities. We previously identified DCBLD2 tyrosine phosphorylation sites in intracellular YxxP motifs that are required for the phosphorylation-dependent binding of the signaling adaptors CRK and CRKL (CT10 regulator of kinase and CRK-like). These intracellular YxxP motifs are highly conserved across vertebrates and between DCBLD family members. Here, we demonstrate that, as for DCBLD2, DCBLD1 YxxP motifs are required for CRKL-SH2 (Src homology 2) binding. We report that Src family kinases (SFKs) and Abl differentially promote the interaction between the CRKL-SH2 domain and DCBLD1 and DCBLD2, and while SFKs and Abl each promote DCBLD1 and DCBLD2 binding to the CRKL-SH2 domain, the effect of Abl is more pronounced for DCBLD1. Using high-performance liquid chromatography coupled with tandem mass spectrometry, we quantified phosphorylation at several YxxP sites in DCBLD1 and DCBLD2, mapping site-specific preferences for SFKs and Abl. Together, these data provide a platform to decipher the signaling mechanisms by which these novel receptors drive their biological activities.

Identification of target genes downstream of semaphorin6A/PlexinA2 signaling in zebrafish.

BACKGROUND: Semaphorin (Sema)/Plexin (Plxn) signaling is important for many aspects of neuronal development, however, the transcriptional regulation imposed by this signaling pathway is unknown. Previously, we identified an essential role for Sema6A/PlxnA2 signaling in regulating proliferation and cohesion of retinal precursor cells (RPCs) during early eye development. This study used RNA isolated from control, Sema6A-deficient and PlxnA2-deficient zebrafish embryos in a microarray analysis to identify genes that were differentially expressed when this signaling pathway was disrupted. RESULTS: We uncovered a set of 58 transcripts, and all but 1 were up-regulated in both sema6A and plxnA2 morphants. We validated gene expression changes in subset of candidates that are suggested to be involved in proliferation, migration or neuronal positioning. We further functionally evaluated one gene, rasl11b, as contributing to disrupted proliferation in sema6A and plxna2 morphants. Our results suggest rasl11b negatively regulates proliferation of RPCs in the developing zebrafish eye. CONCLUSIONS: Microarray analysis has generated a resource of target genes downstream of Sema6A/PlxnA2 signaling, which can be further investigated to elucidate the downstream effects of this well-studied neuronal and vascular guidance signaling pathway. Developmental Dynamics 246:539-549, 2017. © 2017 Wiley Periodicals, Inc.

Sema6a and Plxna2 mediate spatially regulated repulsion within the developing eye to promote eye vesicle cohesion.

Organs are generated from collections of cells that coalesce and remain together as they undergo a series of choreographed movements to give the organ its final shape. We know little about the cellular and molecular mechanisms that regulate tissue cohesion during morphogenesis. Extensive cell movements underlie eye development, starting with the eye field separating to form bilateral vesicles that go on to evaginate from the forebrain. What keeps eye cells together as they undergo morphogenesis and extensive proliferation is unknown. Here, we show that plexina2 (Plxna2), a member of a receptor family best known for its roles in axon and cell guidance, is required alongside the repellent semaphorin 6a (Sema6a) to keep cells integrated within the zebrafish eye vesicle epithelium. sema6a is expressed throughout the eye vesicle, whereas plxna2 is restricted to the ventral vesicle. Knockdown of Plxna2 or Sema6a results in a loss of vesicle integrity, with time-lapse microscopy showing that eye progenitors either fail to enter the evaginating vesicles or delaminate from the eye epithelium. Explant experiments, and rescue of eye vesicle integrity with simultaneous knockdown of sema6a and plxna2, point to an eye-autonomous requirement for Sema6a/Plxna2. We propose a novel, tissue-autonomous mechanism of organ cohesion, with neutralization of repulsion suggested as a means to promote interactions between cells within a tissue domain.

Functional role of myosin-binding protein H in thick filaments of developing vertebrate fast-twitch skeletal muscle.

Myosin-binding protein H (MyBP-H) is a component of the vertebrate skeletal muscle sarcomere with sequence and domain homology to myosin-binding protein C (MyBP-C). Whereas skeletal muscle isoforms of MyBP-C (fMyBP-C, sMyBP-C) modulate muscle contractility via interactions with actin thin filaments and myosin motors within the muscle sarcomere "C-zone," MyBP-H has no known function. This is in part due to MyBP-H having limited expression in adult fast-twitch muscle and no known involvement in muscle disease. Quantitative proteomics reported here reveal MyBP-H is highly expressed in prenatal rat fast-twitch muscles and larval zebrafish, suggesting a conserved role in muscle development, and promoting studies to define its function. We take advantage of the genetic control of the zebrafish model and a combination of structural, functional, and biophysical techniques to interrogate the role of MyBP-H. Transgenic, FLAG-tagged MyBP-H or fMyBP-C both localize to the C-zones in larval myofibers, whereas genetic depletion of endogenous MyBP-H or fMyBP-C leads to increased accumulation of the other, suggesting competition for C-zone binding sites. Does MyBP-H modulate contractility from the C-zone? Globular domains critical to MyBP-C's modulatory functions are absent from MyBP-H, suggesting MyBP-H may be functionally silent. However, our results suggest an active role. Small angle x-ray diffraction of intact larval tails revealed MyBP-H contributes to the compression of the myofilament lattice accompanying stretch or contraction, while in vitro motility experiments indicate MyBP-H shares MyBP-C's capacity as a molecular "brake". These results provide new insights and raise questions about the role of the C-zone during muscle development.

CRK and NCK adaptors may functionally overlap in zebrafish neurodevelopment, as indicated by common binding partners and overlapping expression patterns.

CRK adaptor proteins are important for signal transduction mechanisms driving cell proliferation and positioning during vertebrate central nervous system development. Zebrafish lacking both CRK family members exhibit small, disorganized retinas with 50% penetrance. The goal of this study was to determine whether another adaptor protein might functionally compensate for the loss of CRK adaptors. Expression patterns in developing zebrafish, and bioinformatic analyses of the motifs recognized by their SH2 and SH3 domains, suggest NCK adaptors are well-positioned to compensate for loss of CRK adaptors. In support of this hypothesis, proteomic analyses found CRK and NCK adaptors share overlapping interacting partners including known regulators of cell adhesion and migration, suggesting their functional intersection in neurodevelopment.

Voltage-gated calcium channel CACNB2 (ß2.1) protein is required in the heart for control of cell proliferation and heart tube integrity.

BACKGROUND: L-type calcium channels (LTCC) regulate calcium entry into cardiomyocytes. CACNB2 (ß2) LTCC auxiliary subunits traffic the pore-forming CACNA subunit to the membrane and modulate channel kinetics. ß2 is a membrane associated guanylate kinase (MAGUK) protein. A major role of MAGUK proteins is to scaffold cellular junctions and multiprotein complexes. RESULTS: To investigate developmental functions for ß2.1, we depleted it in zebrafish using morpholinos. ß2.1-depleted embryos developed compromised cardiac function by 48 hr postfertilization, which was ultimately lethal. ß2.1 contractility defects were mimicked by pharmacological depression of LTCC, and rescued by LTCC stimulation, suggesting ß2.1 phenotypes are at least in part LTCC-dependent. Morphological studies indicated that ß2.1 contributes to heart size by regulating the rate of ventricle cell proliferation, and by modulating the transition of outer curvature cells to an elongated cell shape during chamber ballooning. In addition, ß2.1-depleted cardiomyocytes failed to accumulate N-cadherin at the membrane, and dissociated easily from neighboring myocytes under stress. CONCLUSIONS: Hence, we propose that ß2.1 may also function in the heart as a MAGUK scaffolding unit to maintain N-cadherin-based adherens junctions and heart tube integrity.

Neuropathy-associated histidyl-tRNA synthetase variants attenuate protein synthesis in vitro and disrupt axon outgrowth in developing zebrafish.

Charcot-Marie-Tooth disease (CMT) encompasses a set of genetically and clinically heterogeneous neuropathies characterized by length-dependent dysfunction of the peripheral nervous system. Mutations in over 80 diverse genes are associated with CMT, and aminoacyl-tRNA synthetases (ARS) constitute a large gene family implicated in the disease. Despite considerable efforts to elucidate the mechanistic link between ARS mutations and the CMT phenotype, the molecular basis of the pathology is unknown. In this work, we investigated the impact of three CMT-associated substitutions (V155G, Y330C, and R137Q) in the cytoplasmic histidyl-tRNA synthetase (HARS1) on neurite outgrowth and peripheral nervous system development. The model systems for this work included a nerve growth factor-stimulated neurite outgrowth model in rat pheochromocytoma cells (PC12), and a zebrafish line with GFP/red fluorescent protein reporters of sensory and motor neuron development. The expression of CMT-HARS1 mutations led to attenuation of protein synthesis and increased phosphorylation of eIF2α in PC12 cells and was accompanied by impaired neurite and axon outgrowth in both models. Notably, these effects were phenocopied by histidinol, a HARS1 inhibitor, and cycloheximide, a protein synthesis inhibitor. The mutant proteins also formed heterodimers with wild-type HARS1, raising the possibility that CMT-HARS1 mutations cause disease through a dominant-negative mechanism. Overall, these findings support the hypothesis that CMT-HARS1 alleles exert their toxic effect in a neuronal context, and lead to dysregulated protein synthesis. These studies demonstrate the value of zebrafish as a model for studying mutant alleles associated with CMT, and for characterizing the processes that lead to peripheral nervous system dysfunction.

Developmental expression patterns of protein kinase A catalytic subunits in zebrafish.

Protein kinase A (PKA), also known as cAMP dependent protein kinase, is an essential component of many signaling pathways, many of which regulate key developmental processes. Inactive PKA is a tetrameric holoenzyme, comprised of two catalytic (PRKAC), and two regulatory subunits. Upon cAMP binding, the catalytic subunits are released and thereby activated. There are multiple isoforms of PKA catalytic subunits, but their individual roles are not well understood. In order to begin studying their roles in zebrafish development, it is first necessary to identify the spatial and temporal expression profiles for each prkac subunit. Here we evaluate the expression profiles for the four zebrafish prkacs: prkacαa, αb, ßa, and ßb, at key developmental time points: 24, 48 and 72 h post fertilization. We show that zebrafish prkacs are expressed throughout the developing nervous system, each showing unique expression patterns. This body of work will inform future functional studies into the roles of PKA during development.

PKC induces release of a functional ectodomain of the guidance cue semaphorin6A.

Semaphorins (Semas) are a family of secreted and transmembrane proteins that play critical roles in development. Interestingly, several vertebrate transmembrane Sema classes are capable of producing functional soluble ectodomains. However, little is known of soluble Sema6 ectodomains in the nervous system. Herein, we show that the soluble Sema6A ectodomain, sSema6A, exhibits natural and protein kinase C (PKC)-induced release. We show that PKC mediates Sema6A phosphorylation at specific sites and while this phosphorylation is not the primary mechanism regulating sSema6A production, we found that the intracellular domain confers resistance to ectodomain release. Finally, sSema6A is functional as it promotes the cohesion of zebrafish early eye field explants. This suggests that in addition to its canonical contact-mediated functions, Sema6A may have regulated, long-range, forward-signaling capacity.

Genomic organization, expression, and phylogenetic analysis of Ca2+ channel beta4 genes in 13 vertebrate species.

The Ca(2+) channel beta-subunits, encoded by CACNB genes 1-4, are membrane-associated guanylate kinase (MAGUK) proteins. As auxiliary subunits of voltage-gated Ca(2+) channels, the beta-subunits facilitate membrane trafficking of the pore-forming alpha1 subunits and regulate voltage-dependent channel gating. In this report, we investigate whether two zebrafish beta4 genes, beta4.1 and beta4.2, have diverged in structure and function over time. Comparative expression analyses indicated that beta4.1 and beta4.2 were expressed in separable domains within the developing brain and other tissues. Alternative splicing in both genes was subject to differential temporal and spatial regulation, with some organs expressing different subsets of beta4.1 and beta4.2 transcript variants. We used several genomic tools to identify and compare predicted cDNAs for eight teleost and five tetrapod beta4 genes. Teleost species had either one or two beta4 paralogs, whereas each tetrapod species contained only one. Teleost beta4.1 and beta4.2 genes had regions of sequence divergence, but compared with tetrapod beta4s, they exhibited similar exon/intron structure, strong conservation of residues involved in alpha1 subunit binding, and similar 5' alternative splicing. Phylogenetic results are consistent with the duplicate teleost beta4 genes resulting from the teleost whole genome duplication. Following duplication, the beta4.1 genes have evolved faster than beta4.2 genes. We identified disproportionately large second and third introns in several beta4 genes, which we propose may provide regulatory elements contributing to their differential tissue expression. In sum, both mRNA expression data and phylogenetic analysis support the evolutionary divergence of beta4.1 and beta4.2 subunit function.

The calcium channel beta2 (CACNB2) subunit repertoire in teleosts.

BACKGROUND: Cardiomyocyte contraction is initiated by influx of extracellular calcium through voltage-gated calcium channels. These oligomeric channels utilize auxiliary beta subunits to chaperone the pore-forming alpha subunit to the plasma membrane, and to modulate channel electrophysiology 1. Several beta subunit family members are detected by RT-PCR in the embryonic heart. Null mutations in mouse beta2, but not in the other three beta family members, are embryonic lethal at E10.5 due to defects in cardiac contractility 2. However, a drawback of the mouse model is that embryonic heart rhythm is difficult to study in live embryos due to their intra-uterine development. Moreover, phenotypes may be obscured by secondary effects of hypoxia. As a first step towards developing a model for contributions of beta subunits to the onset of embryonic heart rhythm, we characterized the structure and expression of beta2 subunits in zebrafish and other teleosts. RESULTS: Cloning of two zebrafish beta2 subunit genes (beta2.1 and beta2.2) indicated they are membrane-associated guanylate kinase (MAGUK)-family genes. Zebrafish beta2 genes show high conservation with mammals within the SH3 and guanylate kinase domains that comprise the "core" of MAGUK proteins, but beta2.2 is much more divergent in sequence than beta2.1. Alternative splicing occurs at the N-terminus and within the internal HOOK domain. In both beta2 genes, alternative short ATG-containing first exons are separated by some of the largest introns in the genome, suggesting that individual transcript variants could be subject to independent cis-regulatory control. In the Tetraodon nigrovidis and Fugu rubripes genomes, we identified single beta2 subunit gene loci. Comparative analysis of the teleost and human beta2 loci indicates that the short 5' exon sequences are highly conserved. A subset of 5' exons appear to be unique to teleost genomes, while others are shared with mammals. Alternative splicing is temporally and spatially regulated in embryo and adult. Moreover, a different subset of spliced beta2 transcript variants is detected in the embryonic heart compared to the adult. CONCLUSION: These studies refine our understanding of beta2 subunit diversity arising from alternative splicing, and provide the groundwork for functional analysis of beta2 subunit diversity in the embryonic heart.

Neuronal expression patterns of the PlexinA family during zebrafish development.

Plexins (Plxns) and Semaphorins (Semas) are key signaling molecules that regulate many aspects of development. Plxns are a family of transmembrane protein receptors that are activated upon extracellular binding by Semas. Activated Plxns trigger intracellular signaling cascades, which regulate a range of developmental processes, including axon guidance, neuronal positioning and vasculogenesis. Semas are a large family of both transmembrane and secreted signaling molecules, and show subtype specific binding to different Plxn family members. Each Plxn can play different roles in development, and so tightly regulated temporal and spatial expression of receptor subtypes is critical to ensure appropriate signaling. Here we elucidate the expression profiles of the plxnA family, plxnA1a, A1b, A2, A3 and A4 at 18, 24, 36, 48, 60 and 72 h post fertilization in the developing zebrafish. We show that PlxnA family members are expressed in neuronal tissues during zebrafish development, but exhibit key differences in expression within these tissues. We also highlight that plxnA1 has two genes in zebrafish, A1a and A1b, which show divergences in expression patterns during early development.

Correction: A single Danio rerio hars gene encodes both cytoplasmic and mitochondrial histidyl-tRNA synthetases.

[This corrects the article DOI: 10.1371/journal.pone.0185317.].