Genetic duplication of tissue factor reveals subfunctionalization in venous and arterial hemostasis.

Tissue factor (TF) is an evolutionarily conserved protein necessary for initiation of hemostasis. Zebrafish have two copies of the tissue factor gene (f3a and f3b) as the result of an ancestral teleost fish duplication event (so called ohnologs). In vivo physiologic studies of TF function have been difficult given early lethality of TF knockout in the mouse. We used genome editing to produce knockouts of both f3a and f3b in zebrafish. Since ohnologs arose through sub- or neofunctionalization, they can unmask unknown functions of non-teleost genes and could reveal whether mammalian TF has developmental functions distinct from coagulation. Here we show that a single copy of either f3a or f3b is necessary and sufficient for normal lifespan. Complete loss of TF results in lethal hemorrhage by 2-4 months despite normal embryonic and vascular development. Larval vascular endothelial injury reveals predominant roles for TFa in venous circulation and TFb in arterial circulation. Finally, we demonstrate that loss of TF predisposes to a stress-induced cardiac tamponade independent of its role in fibrin formation. Overall, our data suggest partial subfunctionalization of TFa and TFb. This multigenic zebrafish model has the potential to facilitate study of the role of TF in different vascular beds.

A genetic modifier of venous thrombosis in zebrafish reveals a functional role for fibrinogen AαE in early hemostasis.

Plasma fibrinogen molecules comprise 2 copies of Aα, Bß, and γ chains folded into a hexameric protein. A minor fibrinogen isoform with an extended Aα chain (AαE) is more abundant in newborn human blood than in adults. Larval zebrafish produce predominantly AαE-containing fibrinogen, but its functional significance is unclear. In 3-day-old zebrafish, when hemostasis is reliant on fibrinogen and erythrocyte-rich clotting but is largely thrombocyte-independent, we measured the time to occlusion (TTO) in a laser-induced venous thrombosis assay in 3 zebrafish strains (AB, TU, and AB × TL hybrids). AB larvae showed delayed TTO compared with the TU and AB × TL strains. Mating AB with TU or TL produced larvae with a TU-like TTO. In contrast to TU, AB larvae failed to produce fibrinogen AαE, due to a mutation in the AαE-specific coding region of fibrinogen α-chain gene (fga). We investigated whether the lack of AαE explained the delayed AB TTO. Transgenic expression of AαE, but not Aα, shortened the AB TTO to that of TU. AαE rescued venous occlusion in fibrinogen mutants or larvae with morpholino-targeted fibrinogen α-chain messenger RNA, but Aα was less effective. In 5-day-old larvae, circulating thrombocytes contribute to hemostasis, as visualized in Tg(itga2b:EGFP) transgenics. Laser-induced venous thrombocyte adhesion and aggregation is reduced in fibrinogen mutants, but transgenic expression of Aα or AαE restored similar thrombocyte accumulation at the injury site. Our data demonstrate a genetic modifier of venous thrombosis and a role for fibrinogen AαE in early developmental blood coagulation, and suggest a link between differentially expressed fibrinogen isoforms and the cell types available for clotting.

Disruption of the kringle 1 domain of prothrombin leads to late onset mortality in zebrafish.

The ability to prevent blood loss in response to injury is a conserved function of all vertebrates. Complete deficiency of the central clotting enzyme prothrombin has never been observed in humans and is incompatible with postnatal life in mice, thus limiting the ability to study its role in vivo. Zebrafish are able to tolerate severe hemostatic deficiencies that are lethal in mammals. We have generated a targeted genetic deletion in the kringle 1 domain of zebrafish prothrombin. Homozygous mutant embryos develop normally into the mid-juvenile stage but demonstrate complete mortality by 2 months of age primarily due to internal hemorrhage. Mutants are unable to form occlusive venous and arterial thrombi in response to endothelial injury, a defect that was phenocopied using direct oral anticoagulants. Human prothrombin engineered with the equivalent mutation exhibits a severe reduction in secretion, thrombin generation, and fibrinogen cleavage. Together, these data demonstrate the conserved function of thrombin in zebrafish and provide insight into the role of kringle 1 in prothrombin maturation and activity. Understanding how zebrafish are able to develop normally and survive into early adulthood without thrombin activity will provide important insight into its pleiotropic functions as well as the management of patients with bleeding disorders.

Zebrafish otolith biomineralization requires polyketide synthase.

Deflecting biomineralized crystals attached to vestibular hair cells are necessary for maintaining balance. Zebrafish (Danio rerio) are useful organisms to study these biomineralized crystals called otoliths, as many required genes are homologous to human otoconial development. We sought to identify and characterize the causative gene in a trio of homozygous recessive mutants, no content (nco) and corkscrew (csr), and vanished (vns), which fail to develop otoliths during early ear development. We show that nco, csr, and vns have potentially deleterious mutations in polyketide synthase (pks1), a multi-modular protein that has been previously implicated in biomineralization events in chordates and echinoderms. We found that Otoconin-90 (Oc90) expression within the otocyst is diffuse in nco and csr; therefore, it is not sufficient for otolith biomineralization in zebrafish. Similarly, normal localization of Otogelin, a protein required for otolith tethering in the otolithic membrane, is not sufficient for Oc90 attachment. Furthermore, eNOS signaling and Endothelin-1 signaling were the most up- and down-regulated pathways during otolith agenesis in nco, respectively. Our results demonstrate distinct processes for otolith nucleation and biomineralization in vertebrates and will be a starting point for models that are independent of Oc90-mediated seeding. This study will serve as a basis for investigating the role of eNOS signaling and Endothelin-1 signaling during otolith formation.

Autophagy regulates cytoplasmic remodeling during cell reprogramming in a zebrafish model of muscle regeneration.

Cell identity involves both selective gene activity and specialization of cytoplasmic architecture and protein machinery. Similarly, reprogramming differentiated cells requires both genetic program alterations and remodeling of the cellular architecture. While changes in genetic and epigenetic programs have been well documented in dedifferentiating cells, the pathways responsible for remodeling the cellular architecture and eliminating specialized protein complexes are not as well understood. Here, we utilize a zebrafish model of adult muscle regeneration to study cytoplasmic remodeling during cell dedifferentiation. We describe activation of autophagy early in the regenerative response to muscle injury, while blocking autophagy using chloroquine or Atg5 and Becn1 knockdown reduced the rate of regeneration with accumulation of sarcomeric and nuclear debris. We further identify Casp3/caspase 3 as a candidate mediator of cellular reprogramming and Fgf signaling as an important activator of autophagy in dedifferentiating myocytes. We conclude that autophagy plays a critical role in cell reprogramming by regulating cytoplasmic remodeling, facilitating the transition to a less differentiated cell identity.

Myocyte Dedifferentiation Drives Extraocular Muscle Regeneration in Adult Zebrafish.

PURPOSE: The purpose of this study was to characterize the injury response of extraocular muscles (EOMs) in adult zebrafish. METHODS: Adult zebrafish underwent lateral rectus (LR) muscle myectomy surgery to remove 50% of the muscle, followed by molecular and cellular characterization of the tissue response to the injury. RESULTS: Following myectomy, the LR muscle regenerated an anatomically correct and functional muscle within 7 to 10 days post injury (DPI). Following injury, the residual muscle stump was replaced by a mesenchymal cell population that lost cell polarity and expressed mesenchymal markers. Next, a robust proliferative burst repopulated the area of the regenerating muscle. Regenerating cells expressed myod, identifying them as myoblasts. However, both immunofluorescence and electron microscopy failed to identify classic Pax7-positive satellite cells in control or injured EOMs. Instead, some proliferating nuclei were noted to express mef2c at the very earliest point in the proliferative burst, suggesting myonuclear reprogramming and dedifferentiation. Bromodeoxyuridine (BrdU) labeling of regenerating cells followed by a second myectomy without repeat labeling resulted in a twice-regenerated muscle broadly populated by BrdU-labeled nuclei with minimal apparent dilution of the BrdU signal. A double-pulse experiment using BrdU and 5-ethynyl-2'-deoxyuridine (EdU) identified double-labeled nuclei, confirming the shared progenitor lineage. Rapid regeneration occurred despite a cell cycle length of 19.1 hours, whereas 72% of the regenerating muscle nuclei entered the cell cycle by 48 hours post injury (HPI). Dextran lineage tracing revealed that residual myocytes were responsible for muscle regeneration. CONCLUSIONS: EOM regeneration in adult zebrafish occurs by dedifferentiation of residual myocytes involving a muscle-to-mesenchyme transition. A mechanistic understanding of myocyte reprogramming may facilitate novel approaches to the development of molecular tools for targeted therapeutic regeneration in skeletal muscle disorders and beyond.

Natural variability of Kozak sequences correlates with function in a zebrafish model.

In eukaryotes, targeting the small ribosomal subunit to the mRNA transcript requires a Kozak sequence at the translation initiation site. Despite the critical importance of the Kozak sequence to regulation of gene expression, there have been no correlation studies between its natural variance and efficiency of translation. Combining bioinformatics analysis with molecular biology techniques, and using zebrafish as a test case, we identify Kozak sequences based on their natural variance and characterize their function in vivo. Our data reveal that while the canonical Kozak sequence is efficient, in zebrafish it is neither the most common nor the most efficient translation initiation sequence. Rather, the most frequent natural variation of the Kozak sequence is almost twice as efficient. We conclude that the canonical Kozak sequence is a poor predictor of translation efficiency in different model organisms. Furthermore, our results provide an experimental approach to testing and optimizing an important tool for molecular biology.