Meeting the COVID Challenge to a Research-intensive Pre-college Science Education Program.

The objective of our program is to foster and facilitate authentic research experiences in middle and high school classrooms. We achieve this directly by providing students with a complete experience in scientific experimentation and communication. The centerpiece is a set of experiment modules which students use to investigate the effects of toxic chemicals on living organisms through the use of model organisms such as the earthworm, fathead minnow, and the zebrafish, and chemical contaminants commonly found in the environment. In parallel, we partner with the University of Wisconsin-Milwaukee science teacher certification program to prepare pre-service teachers to offer real research experiences in their future classrooms. With the COVID virus restricting or eliminating in-person learning, the program's challenge was (i) to create new ways to conduct experiments virtually that retain elements of the authentic research experience and (ii) to move all of the accompanying facets of the program to online formats. This paper will describe the new online materials and activities that were introduced this past year as well as the challenges they presented and the opportunities that they offer for the future.

Swimming Against the Current: Zebrafish Help Address Educational Challenges.

Zebrafish can be important tools for learning and authentic student research. The broad zebrafish community is rich with examples to improve education for learners of all ages and geographical locales. This special collection of articles is presented with the hope of encouraging readers to reflect on the educational outcomes reported here and to consider new ways zebrafish may engage others to learn and grow.

Education Platform at ZDM8.

Interest among the zebrafish community in education and science accessibility for all ages has increased. At the 8th Annual Zebrafish Disease Models Conference (ZDM8), a specifically designed session enabled professional scientists, educators, and students to have a venue to present their science, discuss ideas in education, and partner to navigate a scientific meeting as an educational experience. This meeting report describes the format of the Platform Session as well as challenges and future plans to leverage impact of conferences on the local communities.

Whole exome sequencing identifies a POLRID mutation segregating in a father and two daughters with findings of Klippel-Feil and Treacher Collins syndromes.

We report on a father and his two daughters diagnosed with Klippel-Feil syndrome (KFS) but with craniofacial differences (zygomatic and mandibular hypoplasia and cleft palate) and external ear abnormalities suggestive of Treacher Collins syndrome (TCS). The diagnosis of KFS was favored, given that the neck anomalies were the predominant manifestations, and that the diagnosis predated later recognition of the association between spinal segmentation abnormalities and TCS. Genetic heterogeneity and the rarity of large families with KFS have limited the ability to identify mutations by traditional methods. Whole exome sequencing identified a nonsynonymous mutation in POLR1D (subunit of RNA polymerase I and II): exon2:c.T332C:p.L111P. Mutations in POLR1D are present in about 5% of individuals diagnosed with TCS. We propose that this mutation is causal in this family, suggesting a pathogenetic link between KFS and TCS.

Zebrafish approaches enhance the translational research tackle box.

During the past few decades, zebrafish (Danio rerio) have been a workhorse for developmental biology and genetics. Concurrently, zebrafish have proved highly accessible and effective for translational research by providing a vertebrate animal model useful for gene discovery, disease modeling, chemical genetic screening, and other medically relevant studies. Key resources such as an annotated and complete genome sequence, and diverse tools for genetic manipulation continue to spur broad use of zebrafish. Thus, the purpose of this introductory review is to provide a window into the unique characteristics and diverse uses of zebrafish and to highlight in particular the increasing relevance of zebrafish as a translational animal model. This is accomplished by reviewing broad considerations of anatomic and physiological conservation, approaches for disease modeling and creation, general laboratory methods, genetic tools, genome conservation, and diverse opportunities for functional validation. Additional commentary throughout the review also guides the reader to the 4 new reviews found elsewhere in this special issue that showcase the many unique ways the zebrafish is improving understanding of renal regeneration, mitochondrial disease, dyslipidemia, and aging, for example. With many other possible approaches and a rapidly increasing number of medically relevant reports, zebrafish approaches enhance significantly the tools available for translational research and are actively improving the understanding of human disease.

Making a difference: education at the 10th International Conference on Zebrafish Development and Genetics.

Scientists, educators, and students met at the 10th International Conference on Zebrafish Development and Genetics during the 2-day Education Workshop, chaired by Dr. Jennifer Liang and supported in part by the Genetics Society of America. The goal of the workshop was to share expertise, to discuss the challenges faced when using zebrafish in the classroom, and to articulate goals for expanding the impact of zebrafish in education.

Gene-breaking transposon mutagenesis reveals an essential role for histone H2afza in zebrafish larval development.

We report a novel gene tagging, identification and mutagenicity ('gene-breaking') method for the zebrafish, Danio rerio. This modular approach consists of two distinct and separable molecular cassettes. The first is a gene-finding cassette. In this study, we employed a 3' gene-tagging approach that selectively 'traps' transcripts regardless of expression status, and we show that this cassette identifies both known and novel endogenous transcripts in transgenic zebrafish. The second is a transcriptional termination mutagenicity cassette assembled from a combination of a splice acceptor and polyadenylation signal to disrupt tagged transcripts upon integration into intronic sequence. We identified both novel and conserved loci as linked phenotypic mutations using this gene-breaking strategy, generating molecularly null mutations in both larval lethal and adult viable loci. We show that the Histone 2a family member z (H2afza) variant is essential for larval development through the generation of a lethal locus with a truncation of conserved carboxy-terminal residues in the protein. In principle this gene-breaking strategy is scalable for functional genomics screens and can be used in Sleeping Beauty transposon and other gene delivery systems in the zebrafish.

Genome-wide reverse genetics framework to identify novel functions of the vertebrate secretome.

BACKGROUND: Understanding the functional role(s) of the more than 20,000 proteins of the vertebrate genome is a major next step in the post-genome era. The approximately 4,000 co-translationally translocated (CTT) proteins - representing the vertebrate secretome - are important for such vertebrate-critical processes as organogenesis. However, the role(s) for most of these genes is currently unknown. RESULTS: We identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. We further investigated 150 of these genes (Figure 1) for unique function using morpholino-based analysis in zebrafish embryos. 12% of the CTT protein-deficient embryos resulted in specific developmental defects, a notably higher rate of gene function annotation than the 2%-3% estimate from random gene mutagenesis studies. CONCLUSION: This initial collection includes novel genes required for the development of vascular, hematopoietic, pigmentation, and craniofacial tissues, as well as lipid metabolism, and organogenesis. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome.

Functional analysis of human hematopoietic stem cell gene expression using zebrafish.

Although several reports have characterized the hematopoietic stem cell (HSC) transcriptome, the roles of HSC-specific genes in hematopoiesis remain elusive. To identify candidate regulators of HSC fate decisions, we compared the transcriptome of human umbilical cord blood and bone marrow (CD34+)(CD33-)(CD38-)Rho(lo)(c-kit+) cells, enriched for hematopoietic stem/progenitor cells with (CD34+)(CD33-)(CD38-)Rho(hi) cells, enriched in committed progenitors. We identified 277 differentially expressed transcripts conserved in these ontogenically distinct cell sources. We next performed a morpholino antisense oligonucleotide (MO)-based functional screen in zebrafish to determine the hematopoietic function of 61 genes that had no previously known function in HSC biology and for which a likely zebrafish ortholog could be identified. MO knock down of 14/61 (23%) of the differentially expressed transcripts resulted in hematopoietic defects in developing zebrafish embryos, as demonstrated by altered levels of circulating blood cells at 30 and 48 h postfertilization and subsequently confirmed by quantitative RT-PCR for erythroid-specific hbae1 and myeloid-specific lcp1 transcripts. Recapitulating the knockdown phenotype using a second MO of independent sequence, absence of the phenotype using a mismatched MO sequence, and rescue of the phenotype by cDNA-based overexpression of the targeted transcript for zebrafish spry4 confirmed the specificity of MO targeting in this system. Further characterization of the spry4-deficient zebrafish embryos demonstrated that hematopoietic defects were not due to more widespread defects in the mesodermal development, and therefore represented primary defects in HSC specification, proliferation, and/or differentiation. Overall, this high-throughput screen for the functional validation of differentially expressed genes using a zebrafish model of hematopoiesis represents a major step toward obtaining meaningful information from global gene profiling of HSCs.

AMOD: a morpholino oligonucleotide selection tool.

AMOD is a web-based program that aids in the functional evaluation of nucleotide sequences through sequence characterization and antisense morpholino oligonucleotide (target site) selection. Submitted sequences are analyzed by translation initiation site prediction algorithms and sequence-to-sequence comparisons; results are used to characterize sequence features required for morpholino design. Within a defined subsequence, base composition and homodimerization values are computed for all putative morpholino oligonucleotides. Using these properties, morpholino candidates are selected and compared with genomic and transcriptome databases with the goal to identify target-specific enriched morpholinos. AMOD has been used at the University of Minnesota to design approximately 200 morpholinos for a functional genomics screen in zebrafish. The AMOD web server and a tutorial are freely available to both academic and commercial users at http://www.secretomes.umn.edu/AMOD/.

Functional genomics tools for the analysis of zebrafish pigment.

Genetic model organisms are increasingly valuable in the post-genomics era to provide a basis for comparative analysis of the human genome. For higher order processes of vertebrate pigment cell biology and development, the mouse has historically been the model of choice. A complementary organism, the zebrafish (Danio rerio), shares many of the signaling and biological processes of vertebrates, e.g. neural crest development. The zebrafish has a number of characteristics that make it an especially valuable model for the study of pigment cell biology and disease. Large-scale genetic screens have identified a collection of pigmentation mutants that have already made valuable contributions to pigment research. An increasing repertoire of genomic resources such as an expressed sequence tag-based Gene Index (The Institute for Genomic Research) and improving methods of mutagenesis, transgenesis, and gene targeting make zebrafish a particularly attractive model. Morpholino phosphorodiamidate oligonucleotide (MO) 'knockdown' of pigment gene expression provides a non-conventional antisense tool for the analysis of genes involved in pigment cell biology and disease. In addition, an ongoing, reverse-genetic, MO-based screen for the rapid identification of gene function promises to be a valuable complement to other high-throughput microarray and proteomic approaches for understanding pigment cell biology. Novel reagents for zebrafish transgenesis, such as the Sleeping Beauty transposon system, continue to improve the capacity for genetic analysis in this system and ensure that the zebrafish will be a valuable genetic model for understanding a variety of biological processes and human diseases for years to come.