Ras controls melanocyte expansion during zebrafish fin stripe regeneration.

Regenerative medicine for complex tissues like limbs will require the provision or activation of precursors for different cell types, in the correct number, and with the appropriate instructions. These strategies can be guided by what is learned from spectacular events of natural limb or fin regeneration in urodele amphibians and teleost fish. Following zebrafish fin amputation, melanocyte stripes faithfully regenerate in tandem with complex fin structures. Distinct populations of melanocyte precursors emerge and differentiate to pigment regenerating fins, yet the regulation of their proliferation and patterning is incompletely understood. Here, we found that transgenic increases in active Ras dose-dependently hyperpigmented regenerating zebrafish fins. Lineage tracing and marker analysis indicated that increases in active Ras stimulated the in situ amplification of undifferentiated melanocyte precursors expressing mitfa and kita. Active Ras also hyperpigmented early fin regenerates of kita mutants, which are normally devoid of primary regeneration melanocytes, suppressing defects in precursor function and survival. By contrast, this protocol had no noticeable impact on pigmentation by secondary regulatory melanocyte precursors in late-stage kita regenerates. Our results provide evidence that Ras activity levels control the repopulation and expansion of adult melanocyte precursors after tissue loss, enabling the recovery of patterned melanocyte stripes during zebrafish appendage regeneration.

Development of the morpholino gene knockdown technique in Fundulus heteroclitus: a tool for studying molecular mechanisms in an established environmental model.

A significant challenge in environmental toxicology is that many genetic and genomic tools available in laboratory models are not developed for commonly used environmental models. The Atlantic killifish (Fundulus heteroclitus) is one of the most studied teleost environmental models, yet few genetic or genomic tools have been developed for use in this species. The advancement of genetic and evolutionary toxicology will require that many of the tools developed in laboratory models be transferred into species more applicable to environmental toxicology. Antisense morpholino oligonucleotide (MO) gene knockdown technology has been widely utilized to study development in zebrafish and has been proven to be a powerful tool in toxicological investigations through direct manipulation of molecular pathways. To expand the utility of killifish as an environmental model, MO gene knockdown technology was adapted for use in Fundulus. Morpholino microinjection methods were altered to overcome the significant differences between these two species. Morpholino efficacy and functional duration were evaluated with molecular and phenotypic methods. A cytochrome P450-1A (CYP1A) MO was used to confirm effectiveness of the methodology. For CYP1A MO-injected embryos, a 70% reduction in CYP1A activity, a 86% reduction in total CYP1A protein, a significant increase in beta-naphthoflavone-induced teratogenicity, and estimates of functional duration (50% reduction in activity 10 dpf, and 86% reduction in total protein 12 dpf) conclusively demonstrated that MO technologies can be used effectively in killifish and will likely be just as informative as they have been in zebrafish.

Evolutionary characters, phenotypes and ontologies: curating data from the systematic biology literature.

BACKGROUND: The wealth of phenotypic descriptions documented in the published articles, monographs, and dissertations of phylogenetic systematics is traditionally reported in a free-text format, and it is therefore largely inaccessible for linkage to biological databases for genetics, development, and phenotypes, and difficult to manage for large-scale integrative work. The Phenoscape project aims to represent these complex and detailed descriptions with rich and formal semantics that are amenable to computation and integration with phenotype data from other fields of biology. This entails reconceptualizing the traditional free-text characters into the computable Entity-Quality (EQ) formalism using ontologies. METHODOLOGY/PRINCIPAL FINDINGS: We used ontologies and the EQ formalism to curate a collection of 47 phylogenetic studies on ostariophysan fishes (including catfishes, characins, minnows, knifefishes) and their relatives with the goal of integrating these complex phenotype descriptions with information from an existing model organism database (zebrafish, http://zfin.org). We developed a curation workflow for the collection of character, taxonomic and specimen data from these publications. A total of 4,617 phenotypic characters (10,512 states) for 3,449 taxa, primarily species, were curated into EQ formalism (for a total of 12,861 EQ statements) using anatomical and taxonomic terms from teleost-specific ontologies (Teleost Anatomy Ontology and Teleost Taxonomy Ontology) in combination with terms from a quality ontology (Phenotype and Trait Ontology). Standards and guidelines for consistently and accurately representing phenotypes were developed in response to the challenges that were evident from two annotation experiments and from feedback from curators. CONCLUSIONS/SIGNIFICANCE: The challenges we encountered and many of the curation standards and methods for improving consistency that we developed are generally applicable to any effort to represent phenotypes using ontologies. This is because an ontological representation of the detailed variations in phenotype, whether between mutant or wildtype, among individual humans, or across the diversity of species, requires a process by which a precise combination of terms from domain ontologies are selected and organized according to logical relations. The efficiencies that we have developed in this process will be useful for any attempt to annotate complex phenotypic descriptions using ontologies. We also discuss some ramifications of EQ representation for the domain of systematics.

A unified anatomy ontology of the vertebrate skeletal system.

The skeleton is of fundamental importance in research in comparative vertebrate morphology, paleontology, biomechanics, developmental biology, and systematics. Motivated by research questions that require computational access to and comparative reasoning across the diverse skeletal phenotypes of vertebrates, we developed a module of anatomical concepts for the skeletal system, the Vertebrate Skeletal Anatomy Ontology (VSAO), to accommodate and unify the existing skeletal terminologies for the species-specific (mouse, the frog Xenopus, zebrafish) and multispecies (teleost, amphibian) vertebrate anatomy ontologies. Previous differences between these terminologies prevented even simple queries across databases pertaining to vertebrate morphology. This module of upper-level and specific skeletal terms currently includes 223 defined terms and 179 synonyms that integrate skeletal cells, tissues, biological processes, organs (skeletal elements such as bones and cartilages), and subdivisions of the skeletal system. The VSAO is designed to integrate with other ontologies, including the Common Anatomy Reference Ontology (CARO), Gene Ontology (GO), Uberon, and Cell Ontology (CL), and it is freely available to the community to be updated with additional terms required for research. Its structure accommodates anatomical variation among vertebrate species in development, structure, and composition. Annotation of diverse vertebrate phenotypes with this ontology will enable novel inquiries across the full spectrum of phenotypic diversity.

Developmental exposure to a complex PAH mixture causes persistent behavioral effects in naive Fundulus heteroclitus (killifish) but not in a population of PAH-adapted killifish.

Acute exposures to some individual polycyclic aromatic hydrocarbons (PAHs) and complex PAH mixtures are known to cause cardiac malformations and edema in the developing fish embryo. However, the heart is not the only organ impacted by developmental PAH exposure. The developing brain is also affected, resulting in lasting behavioral dysfunction. While acute exposures to some PAHs are teratogenically lethal in fish, little is known about the later life consequences of early life, lower dose subteratogenic PAH exposures. We sought to determine and characterize the long-term behavioral consequences of subteratogenic developmental PAH mixture exposure in both naive killifish and PAH-adapted killifish using sediment pore water derived from the Atlantic Wood Industries Superfund Site. Killifish offspring were embryonically treated with two low-level PAH mixture dilutions of Elizabeth River sediment extract (ERSE) (TPAH 5.04 μg/L and 50.4 μg/L) at 24h post fertilization. Following exposure, killifish were raised to larval, juvenile, and adult life stages and subjected to a series of behavioral tests including: a locomotor activity test (4 days post-hatch), a sensorimotor response tap/habituation test (3 months post hatch), and a novel tank diving and exploration test (3months post hatch). Killifish were also monitored for survival at 1, 2, and 5 months over 5-month rearing period. Developmental PAH exposure caused short-term as well as persistent behavioral impairments in naive killifish. In contrast, the PAH-adapted killifish did not show behavioral alterations following PAH exposure. PAH mixture exposure caused increased mortality in reference killifish over time; yet, the PAH-adapted killifish, while demonstrating long-term rearing mortality, had no significant changes in mortality associated with ERSE exposure. This study demonstrated that early embryonic exposure to PAH-contaminated sediment pore water caused long-term locomotor and behavioral alterations in killifish, and that locomotor alterations could be observed in early larval stages. Additionally, our study highlights the resistance to behavioral alterations caused by low-level PAH mixture exposure in the adapted killifish population. Furthermore, this is the first longitudinal behavioral study to use killifish, an environmentally important estuarine teleost fish, and this testing framework can be used for future contaminant assessment.