Integrating CRISPR-Cas9 Technology into Undergraduate Courses: Perspectives from a National Science Foundation (NSF) Workshop for Undergraduate Faculty, June 2018.

As CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 technology becomes more mainstream in life science research, it becomes critical for undergraduate instructors to devise engaging ways to bring the technology into their classrooms. To help meet this challenge, the National Science Foundation sponsored a workshop for undergraduate instructors in June 2018 at The Ohio State University in conjunction with the annual Association of Biology Laboratory Educators meeting based on a workflow developed by the workshop's facilitators. Over the course of two and a half days, participants worked through a modular workflow for the use of CRISPR-Cas9 in a course-based (undergraduate) research experience (CURE) setting while discussing the barriers each of their institutions had to implementing such work, and how such barriers could be overcome. The result of the workshop was a team with newfound energy and confidence to implement CRISPR-Cas9 technology in their courses and the development of a community of undergraduate educators dedicated to supporting each other in the implementation of the workflow either in a CURE or modular format. In this article, we review the activities and discussions from the workshop that helped each participant devise their own tailored approaches of how best to bring this exciting new technology into their classes.

Quantifying cardiac functions in embryonic and adult zebrafish.

Zebrafish embryos have been extensively used to study heart development and cardiac function, mainly due to the unique embryology and genetics of this model organism. Since most human heart disease occurs during adulthood, adult zebrafish models of heart disease are being created to dissect mechanisms of the disease and discover novel therapies. However, due to its small heart size, the use of cardiac functional assays in the adult zebrafish has been limited. To address this bottleneck, the transparent fish line casper;Tg(cmlc2:nuDsRed) that has a red fluorescent heart can be used to document beating hearts in vivo and to quantify cardiac functions in adult zebrafish. Here, we describe our methods for quantifying shortening fraction and heart rate in embryonic zebrafish, as well as in the juvenile and adult casper;Tg(cmlc2:nuDsRed) fish. In addition, we describe the red blood cell flow rate assay that can be used to reflect cardiac function indirectly in zebrafish at any stage.

Functions of the Wnt/ß-catenin pathway in an anemia-induced zebrafish model of cardiomyopathy are location dependent.

Recent evidence that the heart is not a terminally-differentiated organ has provided more credence to investigations of pathways involved in inducing cardiomyocyte (CM) hyperplasia as a therapy for heart disease. Here, we leveraged zebrafish as a novel vertebrate model of cardiomyopathy to explore the therapeutic potential based on the Wnt/ß-catenin signaling. In the anemia-induced zebrafish model of cardiomyopathy (tr265), we detected differently regulated CM hyperplasia and CM hypertrophy in the compact region and the trabecular region. To assess the effects of the Wnt/ß-catenin pathway on these two regions, the anemia line was crossed with heat shock-inducible transgenic fish to upregulate or downregulate the pathway. Upregulation resulted in increased cardiomyocyte hyperplasia in the heart and increased cardiomyocyte hypertrophy in the trabecular region, while downregulation resulted in reduced cardiomyocyte hyperplasia in the heart and reduced cardiomyocyte hypertrophy in the trabecular region. Importantly, upregulation of the pathway resulted in improved fish survival, while downregulation decreased it. In summary, our data suggested that (1) the compact region and the trabecular region respond differently during cardiac remodeling; (2) activation of the Wnt/ß-catenin pathway might exert a cardioprotective function via promoting cardiomyocyte hyperplasia.

Haploinsufficiency of target of rapamycin attenuates cardiomyopathies in adult zebrafish.

RATIONALE: Although a cardioprotective function of target of rapamycin (TOR) signaling inhibition has been suggested by pharmacological studies using rapamycin, genetic evidences are still lacking. We explored adult zebrafish as a novel vertebrate model for dissecting signaling pathways in cardiomyopathy. OBJECTIVE: We generated the second adult zebrafish cardiomyopathy model induced by doxorubicin. By genetically analyzing both the doxorubicin and our previous established anemia-induced cardiomyopathy models, we decipher the functions of TOR signaling in cardiomyopathies of different etiology. METHODS AND RESULTS: Along the progression of both cardiomyopathy models, we detected dynamic TOR activity at different stages of pathogenesis as well as distinct effects of TOR signaling inhibition. Nevertheless, cardiac enlargement in both models can be effectively attenuated by inhibition of TOR signaling through short-term rapamycin treatment. To assess the long-term effects of TOR reduction, we used a zebrafish target of rapamycin (ztor) mutant identified from an insertional mutagenesis screen. We show that TOR haploinsufficiency in the ztor heterozygous fish improved cardiac function, prevented pathological remodeling events, and ultimately reduced mortality in both adult fish models of cardiomyopathy. Mechanistically, these cardioprotective effects are conveyed by the antihypertrophy, antiapoptosis, and proautophagy function of TOR signaling inhibition. CONCLUSIONS: Our results prove adult zebrafish as a conserved novel vertebrate model for human cardiomyopathies. Moreover, we provide the first genetic evidence to demonstrate a long-term cardioprotective effect of TOR signaling inhibition on at least 2 cardiomyopathies of distinct etiology, despite dynamic TOR activities during their pathogenesis.

Cardiac hypertrophy involves both myocyte hypertrophy and hyperplasia in anemic zebrafish.

BACKGROUND: An adult zebrafish heart possesses a high capacity of regeneration. However, it has been unclear whether and how myocyte hyperplasia contributes to cardiac remodeling in response to biomechanical stress and whether myocyte hypertrophy exists in the zebrafish. To address these questions, we characterized the zebrafish mutant tr265/tr265, whose Band 3 mutation disrupts erythrocyte formation and results in anemia. Although Band 3 does not express and function in the heart, the chronic anemia imposes a sequential biomechanical stress towards the heart. METHODOLOGY/PRINCIPAL FINDINGS: Hearts of the tr265/tr265 Danio rerio mutant become larger than those of the sibling by week 4 post fertilization and gradually exhibit characteristics of human cardiomyopathy, such as muscular disarray, re-activated fetal gene expression, and severe arrhythmia. At the cellular level, we found both increased individual cardiomyocyte size and increased myocyte proliferation can be detected in week 4 to week 12 tr265/tr265 fish. Interestingly, all tr265/tr265 fish that survive after week-12 have many more cardiomyocytes of smaller size than those in the sibling, suggesting that myocyte hyperplasia allows the long-term survival of these fish. We also show the cardiac hypertrophy process can be recapitulated in wild-type fish using the anemia-inducing drug phenylhydrazine (PHZ). CONCLUSIONS/SIGNIFICANCE: The anemia-induced cardiac hypertrophy models reported here are the first adult zebrafish cardiac hypertrophy models characterized. Unlike mammalian models, both cardiomyocyte hypertrophy and hyperplasia contribute to the cardiac remodeling process in these models, thus allowing the effects of cardiomyocyte hyperplasia on cardiac remodeling to be studied. However, since anemia can induce effects on the heart other than biomechanical, non-anemic zebrafish cardiac hypertrophy models shall be generated and characterized.

Long, abundantly expressed non-coding transcripts are altered in cancer.

Recent studies with tiling arrays have revealed more genomic transcription than previously anticipated. Whole new groups of non-coding transcripts (NCTs) have been detected. Some of these NCTs, including miRNAs, can regulate gene expression. To date, most known NCTs studied have been relatively short, but several important regulatory NCTs, including XIST, MALAT-1, BC1 and BC200, are considerably larger in length and represent a novel class of long, non-coding RNA species. Whole-genome tiling arrays were utilized to identify novel long NCTs across the entire human genome. Our results have identified a new group of long (>400 nt), abundantly expressed NCTs and have found that a subset of these are also highly evolutionarily conserved. In this report, we have begun to characterize 15 long, conserved NCTs. Quantitative real-time RT-PCR was used to analyze their expression in different normal human tissue and also in breast and ovarian cancers. We found altered expression of many of these NCTs in both cancer types. In addition, several of these NCTs have consistent mutations when sequences of normal samples were compared with a panel of cancer-derived cell lines. One NCT was found to be consistently mutated in a panel of endometrial cancers compared with matched normal blood. These NCTs were among the most abundantly expressed transcripts detected. There are probably many long, conserved NCTs, albeit with lower levels of expression. Although the function of these NCTs is currently unknown, our study indicates that they may play an important function in both normal cells and in cancer development.