Specification of the primitive myeloid precursor pool requires signaling through Alk8 in zebrafish.

In the zebrafish embryo, primitive hematopoiesis initiates in two spatially distinct regions. Rostrally, the cells of the anterior lateral plate mesoderm (ALPM) give rise exclusively to cells of the myeloid lineage in a pu.1-dependent manner. Caudally, in the posterior lateral plate mesoderm (PLPM), the expression of gata1 defines a precursor pool that gives rise predominantly to the embryonic erythrocytes. The transcription factor scl acts upstream of both gata1 and pu.1 in these precursor pools, activating a series of conserved transcription factors that cell-autonomously specify either myeloid or erythroid fates. However, the mechanisms underlying the spatial separation of the hematopoietic precursor pools and the induction of differential gene expression within these pools are not well understood. We show here that the Bmp receptor lost-a-fin/alk8 is required for rostral pu.1 expression and myelopoiesis, identifying an early genetic event that distinguishes between the induction of anterior and posterior hematopoiesis. Introducing a constitutively active version of the Alk8 receptor led to increased pu.1 expression, but the role of alk8 was independent of the scl-dependent cell-fate pathway. Furthermore, the role of Alk8 in myelopoiesis was genetically separable from its earlier role in dorsal-ventral embryonic patterning.

Toxicological assessment of additively manufactured methacrylates for medical devices in dentistry.

The paucity of information on the biological risks of photopolymers in additive manufacturing is a major challenge for the uptake of the technology in the construction of medical devices in dentistry. In this paper, the biocompatibility of methacrylates for denture bases, splints, retainers and surgical guides were evaluated using the innovative zebrafish embryo model, which is providing a high potential for toxicity profiling of photopolymers and has high genetic similarity to humans. Toxicological data obtained confirmed gradations of toxicity influenced by ethanol treatment, exposure scenarios and extraction vehicles. In direct exposure tests, juvenile fish exposed to non-treated methacrylates in ultrapure water showed accelerated toxicity endpoints compared to fish in transparent E3 medium. Similarly, toxic extracts induced mostly acute responses (embryonic mortality) in contrast to cumulative chronic (sublethal and teratogenic effects) in direct exposure. Methacrylates composed of >60% Ethoxylated bisphenol A dimethacrylate produced a relatively lower conversion rate in FTIR spectroscopy, but were safe in zebrafish bioassays after ethanol treatment. The study affirms that biocompatibility was influenced primarily by physico-chemical characteristics of the materials, which subsequently influenced their residual monomer content before and after immersion in ethanol. Given the precautionary implications of the study, we propose a 3-tiered approach i.e. using approved materials, apposite manufacturing parameters and post-processing techniques that together guarantee optimal results for medical devices. STATEMENT OF SIGNIFICANCE: This study is timely and relevant since there is limited published literature that precisely describes the toxicological properties of additively manufactured methacrylates despite their increased popularity for medical devices. While it is generally accepted that the zebrafish excels as a model system for developmental toxicity, a further examination of its utility in this study using different protocols provides basis for its consideration and adoption at a crucial time when there is a lack of consensus regarding the most suited biological assessment methods for medical devices.

In vivo mutation of pre-mRNA processing factor 8 (Prpf8) affects transcript splicing, cell survival and myeloid differentiation.

Mutated spliceosome components are recurrently being associated with perturbed tissue development and disease pathogenesis. Cephalophonus (cph), is a zebrafish mutant carrying an early premature STOP codon in the spliceosome component Prpf8 (pre-mRNA processing factor 8). Cph initially develops normally, but then develops widespread cell death, especially in neurons, and is embryonic lethal. Cph mutants accumulate aberrantly spliced transcripts retaining both U2- and U12-type introns. Within early haematopoiesis, myeloid differentiation is impaired, suggesting Prpf8 is required for haematopoietic development. Cph provides an animal model for zygotic PRPF8 dysfunction diseases and for evaluating therapeutic interventions.

Neutrophil-delivered myeloperoxidase dampens the hydrogen peroxide burst after tissue wounding in zebrafish.

Prompt neutrophil arrival is critical for host defense immediately after injury [1-3]. Following wounding, a hydrogen peroxide (H(2)O(2)) burst generated in injured tissues is the earliest known leukocyte chemoattractant [4]. Generating this tissue-scale H(2)O(2) gradient uses dual oxidase [4] and neutrophils sense H(2)O(2) by a mechanism involving the LYN Src-family kinase [5], but the molecular mechanisms responsible for H(2)O(2) clearance are unknown [6]. Neutrophils carry abundant amounts of myeloperoxidase, an enzyme catalyzing an H(2)O(2)-consuming reaction [7, 8]. We hypothesized that this neutrophil-delivered myeloperoxidase downregulates the high tissue H(2)O(2) concentrations that follow wounding. This was tested in zebrafish using simultaneous fluorophore-based imaging of H(2)O(2) concentrations and leukocytes [4, 9-11] and a new neutrophil-replete but myeloperoxidase-deficient mutant (durif). Leukocyte-depleted zebrafish had an abnormally sustained wound H(2)O(2) burst, indicating that leukocytes themselves were required for H(2)O(2) downregulation. Myeloperoxidase-deficient zebrafish also had abnormally sustained high wound H(2)O(2) concentrations despite similar numbers of arriving neutrophils. A local H(2)O(2)/myeloperoxidase interaction within wound-recruited neutrophils was demonstrated. These data demonstrate that leukocyte-delivered myeloperoxidase cell-autonomously downregulates tissue-generated wound H(2)O(2) gradients in vivo, defining a new requirement for myeloperoxidase during inflammation. Durif provides a new animal model of myeloperoxidase deficiency closely phenocopying the prevalent human disorder [7, 12, 13], offering unique possibilities for investigating its clinical consequences.

The zebrafish spi1 promoter drives myeloid-specific expression in stable transgenic fish.

The spi1 (pu.1) gene has recently been identified as a useful marker of early myeloid cells in zebrafish. To enhance the versatility of this organism as a model for studying myeloid development, the promoter of this gene has been isolated and characterized. Transient transgenesis revealed that a 5.3 kilobase promoter fragment immediately upstream of the spi1 coding sequence was sufficient to drive expression of enhanced green fluorescent protein (EGFP) in injected embryos in a manner that largely recapitulated the native spi1 gene expression pattern. This fragment was successfully used to produce a germ line transgenic line of zebrafish with EGFP-expressing myeloid cells. These TG(spi1:EGFP)pA301 transgenic zebrafish represent a valuable tool for further studies of myeloid development and its perturbation.