Activated PI3K delta syndrome 1 mutations cause neutrophilia in zebrafish larvae.

People with activated PI3 kinase delta syndrome 1 (APDS1) suffer from immune deficiency and severe bronchiectasis. APDS1 is caused by dominant activating mutations of the PIK3CD gene that encodes the PI3 kinase delta (PI3Kδ) catalytic subunit. Despite the importance of innate immunity defects in bronchiectasis, there has been limited investigation of neutrophils or macrophages in APDS1 patients or mouse models.

Loss of Deacetylation Enzymes Hdac6 and Sirt2 Promotes Acetylation of Cytoplasmic Tubulin, but Suppresses Axonemal Acetylation in Zebrafish Cilia.

Cilia are evolutionarily highly conserved organelles with important functions in many organs. The extracellular component of the cilium protruding from the plasma membrane comprises an axoneme composed of microtubule doublets, arranged in a 9 + 0 conformation in primary cilia or 9 + 2 in motile cilia. These microtubules facilitate transport of intraflagellar cargoes along the axoneme.

The role of endothelial cilia in postembryonic vascular development.

Background: Cilia are essential for morphogenesis and maintenance of many tissues. Loss-of-function of cilia in early Zebrafish development causes a range of vascular defects, including cerebral hemorrhage and reduced arterial vascular mural cell coverage. In contrast, loss of endothelial cilia in mice has little effect on vascular development.

Gfi1aa and Gfi1b set the pace for primitive erythroblast differentiation from hemangioblasts in the zebrafish embryo.

The transcriptional repressors Gfi1(a) and Gfi1b are epigenetic regulators with unique and overlapping roles in hematopoiesis. In different contexts, Gfi1 and Gfi1b restrict or promote cell proliferation, prevent apoptosis, influence cell fate decisions, and are essential for terminal differentiation. Here, we show in primitive red blood cells (prRBCs) that they can also set the pace for cellular differentiation.

klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes.

Introduction And Objectives: The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2) transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish.

The role of Sox6 in zebrafish muscle fiber type specification.

Background: The transcription factor Sox6 has been implicated in regulating muscle fiber type-specific gene expression in mammals. In zebrafish, loss of function of the transcription factor Prdm1a results in a slow to fast-twitch fiber type transformation presaged by ectopic expression of sox6 in slow-twitch progenitors. Morpholino-mediated Sox6 knockdown can suppress this transformation but causes ectopic expression of only one of three slow-twitch specific genes assayed.

A method for high-throughput PCR-based genotyping of larval zebrafish tail biopsies.

Here we describe a method for high-throughput genotyping of live larval zebrafish as early as 72 h post-fertilization (hpf). Importantly, this technique allows rapid and cost-effective PCR-based genotyping from very small fin biopsies, which regenerate as the embryo develops, thereby allowing researchers to select embryos with desired genotypes to be raised to adulthood.

Positive and negative regulation of Gli activity by Kif7 in the zebrafish embryo.

Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Here, using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, we show that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor.

A zebrafish model to study and therapeutically manipulate hypoxia signaling in tumorigenesis.

Hypoxic signaling is a central modulator of cellular physiology in cancer. Core members of oxygen-sensing pathway including the von Hippel-Lindau tumor suppressor protein (pVHL) and the hypoxia inducible factor (HIF) transcription factors have been intensively studied, but improved organismal models might speed advances for both pathobiologic understanding and therapeutic modulation. To study HIF signaling during tumorigenesis and development in zebrafish, we developed a unique in vivo reporter for hypoxia, expressing EGFP driven by prolyl hydroxylase 3 (phd3) promoter/regulatory elements.

Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates.

Using zinc-finger nuclease-mediated mutagenesis, we have generated mutant alleles of the zebrafish orthologue of the chicken talpid3 (ta3) gene, which encodes a centrosomal protein that is essential for ciliogenesis. Animals homozygous for these mutant alleles complete embryogenesis normally, but manifest a cystic kidney phenotype during the early larval stages and die within a month of hatching. Elimination of maternally derived Ta3 activity by germline replacement resulted in embryonic lethality of ta3 homozygotes.

Analysis of Pax7 expressing myogenic cells in zebrafish muscle development, injury, and models of disease.

The transcription factor Pax7 is a marker and regulator of muscle progenitors and satellite cells that contribute to the embryonic development and postembryonic growth of skeletal muscle in vertebrates, as well as to its repair and regeneration. Here, we identify Pax7(+ve) myogenic cells in the zebrafish and characterize their behavior in postembryonic stages. Mononucleate Pax7(+ve) cells can first be found associated with myofibers at 72 hours post fertilization (hpf).

Prdm1- and Sox6-mediated transcriptional repression specifies muscle fibre type in the zebrafish embryo.

The zebrafish u-boot (ubo) gene encodes the transcription factor Prdm1, which is essential for the specification of the primary slow-twitch muscle fibres that derive from adaxial cells. Here, we show that Prdm1 functions by acting as a transcriptional repressor and that slow-twitch-specific muscle gene expression is activated by Prdm1-mediated repression of the transcriptional repressor Sox6. Genes encoding fast-specific isoforms of sarcomeric proteins are ectopically expressed in the adaxial cells of ubo(tp39) mutant embryos.

Expression of multiple slow myosin heavy chain genes reveals a diversity of zebrafish slow twitch muscle fibres with differing requirements for Hedgehog and Prdm1 activity.

The zebrafish embryo develops a series of anatomically distinct slow twitch muscle fibres that characteristically express genes encoding lineage-specific isoforms of sarcomeric proteins such as MyHC and troponin. We show here that different subsets of these slow fibres express distinct members of a tandem array of slow MyHC genes. The first slow twitch muscle fibres to differentiate, which are specified by the activity of the transcription factor Prdm1 (also called Ubo or Blimp1) in response to Hedgehog (Hh) signalling, express the smyhc1 gene.

Modeling inflammation in the zebrafish: how a fish can help us understand lung disease.

Neutrophilic inflammation is responsible for much of the tissue damage seen in many lung diseases. For resolution of inflammation to occur, neutrophils must die by apoptosis, allowing their recognition and removal by macrophages. The molecular events controlling this important regulatory step are poorly understood, in large part due to the genetic intractability of the human neutrophil granulocyte.

Sdf1a patterns zebrafish melanophores and links the somite and melanophore pattern defects in choker mutants.

Pigment pattern formation in zebrafish presents a tractable model system for studying the morphogenesis of neural crest derivatives. Embryos mutant for choker manifest a unique pigment pattern phenotype that combines a loss of lateral stripe melanophores with an ectopic melanophore ;collar' at the head-trunk border. We find that defects in neural crest migration are largely restricted to the lateral migration pathway, affecting both xanthophores (lost) and melanophores (gained) in choker mutants.

A transgenic zebrafish model of neutrophilic inflammation.

We have established an in vivo model for genetic analysis of the inflammatory response by generating a transgenic zebrafish line that expresses GFP under the neutrophil-specific myeloperoxidase promoter. We show that inflammation is induced after transection of the tail of zebrafish larvae and that this inflammation subsequently resolves over a similar time course to mammalian systems. Quantitative data can be generated from this model by counting of fluorescent cells or by digital image analysis.

Phox2b function in the enteric nervous system is conserved in zebrafish and is sox10-dependent.

Zebrafish lacking functional sox10 have defects in non-ectomesenchymal neural crest derivatives including the enteric nervous system (ENS) and as such provide an animal model for human Waardenburg Syndrome IV. Here, we characterize zebrafish phox2b as a functionally conserved marker of the developing ENS. We show that morpholino-mediated knockdown of Phox2b generates fish modeling Hirschsprung disease.

Roles for GFRalpha1 receptors in zebrafish enteric nervous system development.

Components of the zebrafish GDNF receptor complex are expressed very early in the development of enteric nervous system precursors, and are already present as these cells begin to enter the gut and migrate caudally along its length. Both gfra1a and gfra1b as well as ret are expressed at this time, while gfra2 expression, the receptor component that binds the GDNF-related ligand neurturin, is not detected until the precursors have migrated along the gut. Gfra genes are also expressed in regions of the zebrafish brain and peripheral ganglia, expression domains conserved with other species.

Transcriptional regulation of mitfa accounts for the sox10 requirement in zebrafish melanophore development.

The transcription factor Sox10 is required for the specification, migration and survival of all nonectomesenchymal neural crest derivatives including melanophores. sox10(-/-) zebrafish lack expression of the transcription factor mitfa, which itself is required for melanophore development. We demonstrate that the zebrafish mitfa promoter has sox10 binding sites necessary for activity in vitro, consistent with studies using mammalian cell cultures that have shown that Sox10 directly regulates Mitf expression.

Structural and functional studies of an intermediate on the pathway to operator binding by Escherichia coli MetJ.

We present the results of in vitro DNA-binding assays for a mutant protein (Q44K) of the E. coli methionine repressor, MetJ, as well as the crystal structure at 2.2 A resolution of the apo-mutant bound to a 10-mer oligonucleotide encompassing an 8 bp met-box sequence.