Trails to Research: an Inquiry-Based Course Using Zebrafish To Provide Research Experience to Tribal College Students.

Embryonic development is fascinating to follow and highly engaging and, therefore, lends itself for undergraduate students' first steps in experimental science. We developed the "Trails to Research" inquiry-based course, which exposes students to life science research using zebrafish as model organism. Zebrafish are ideal in the classroom: they are easy to maintain, their embryos develop rapidly, and they are easily manipulated. Further, they lend themselves to teach about embryo development and experimental design. We developed the course for undergraduates at 2-year colleges and, therefore, for students with little or no research experience. In this 5-day intensive course (which is taught during summers as a stand-alone course), students design treatment experiments for zebrafish embryos with known teratogens and with substances they select. The course comprises three modules that overlap over the 5 days: (i) introduction to developmental biology, model organisms, toxicology, and experimental design, (ii) zebrafish embryo experimental setup, and (iii) collecting, analyzing, and presenting data. Student learning was significant in the areas of experimental design, working with model systems, working with zebrafish embryos, using laboratory equipment, and presenting the results of their experiments using effective methods.

Amphibian Zic Genes.

Studies in Xenopus laevis have greatly contributed to understanding the roles that the Zic family of zinc finger transcription factors play as essential drivers of early development. Explant systems that are not readily available in other organisms give Xenopus embryos a unique place in these studies, facilitated by the recent sequencing of the Xenopus laevis genome. A number of upstream regulators of zic gene expression have been identified, such as inhibition of BMP signaling, as well as calcium, FGF, and canonical Wnt signaling. Screens using induced ectodermal explants have identified genes that are direct targets of Zic proteins during early neural development and neural crest specification. These direct targets include Xfeb (also called glipr2; hindbrain development), aqp3b (dorsal marginal zone in gastrula embryos and neural folds), snail family members (premigratory neural crest), genes that play roles in retinoic acid signaling, noncanonical Wnt signaling, and mesoderm development, in addition to a variety of genes some with and many without known roles during neural or neural crest development. Functional experiments in Xenopus embryos demonstrated the involvement of Zic family members in left-right determination, early neural patterning, formation of the midbrain-hindbrain boundary, and neural crest specification. The role of zic genes in cell proliferation vs. differentiation remains unclear, and the activities of Zic factors as inhibitors or activators of canonical Wnt signaling may be dependent on developmental context. Overall, Xenopus has contributed much to our understanding of how Zic transcriptional activities shape the development of the embryo and contribute to disease.

Roles for Xenopus aquaporin-3b (aqp3.L) during gastrulation: Fibrillar fibronectin and tissue boundary establishment in the dorsal margin.

Aquaporins and aquaglyceroporins are a large family of membrane channel proteins that allow rapid movement of water and small, uncharged solutes into and out of cells along concentration gradients. Recently, aquaporins have been gaining recognition for more complex biological roles than the regulation of cellular osmotic homeostasis. We have identified a specific expression pattern for Xenopus aqp3b (also called aqp3.L) during gastrulation, where it is localized to the sensorial (deep) layer of the blastocoel roof and dorsal margin. Interference with aqp3b expression resulted in loss of fibrillar fibronectin matrix in Brachet's cleft at the dorsal marginal zone, but not on the free surface of the blastocoel. Detailed observation showed that the absence of fibronectin matrix correlated with compromised border integrities between involuted mesendoderm and noninvoluted ectoderm in the marginal zone. Knockdown of aqp3b also led to delayed closure of the blastopore, suggesting defects in gastrulation movements. Radial intercalation was not affected in aqp3b morphants, while the data presented are consistent with impeded convergent extension movements of the dorsal mesoderm in response to loss of aqp3b. Our emerging model suggests that aqp3b is part of a mechanism that promotes proper interaction between cells and the extracellular matrix, thereby playing a critical role in gastrulation.

Gain-of-Function Mutations in ZIC1 Are Associated with Coronal Craniosynostosis and Learning Disability.

Human ZIC1 (zinc finger protein of cerebellum 1), one of five homologs of the Drosophila pair-rule gene odd-paired, encodes a transcription factor previously implicated in vertebrate brain development. Heterozygous deletions of ZIC1 and its nearby paralog ZIC4 on chromosome 3q25.1 are associated with Dandy-Walker malformation of the cerebellum, and loss of the orthologous Zic1 gene in the mouse causes cerebellar hypoplasia and vertebral defects. We describe individuals from five families with heterozygous mutations located in the final (third) exon of ZIC1 (encoding four nonsense and one missense change) who have a distinct phenotype in which severe craniosynostosis, specifically involving the coronal sutures, and variable learning disability are the most characteristic features. The location of the nonsense mutations predicts escape of mutant ZIC1 transcripts from nonsense-mediated decay, which was confirmed in a cell line from an affected individual. Both nonsense and missense mutations are associated with altered and/or enhanced expression of a target gene, engrailed-2, in a Xenopus embryo assay. Analysis of mouse embryos revealed a localized domain of Zic1 expression at embryonic days 11.5-12.5 in a region overlapping the supraorbital regulatory center, which patterns the coronal suture. We conclude that the human mutations uncover a previously unsuspected role for Zic1 in early cranial suture development, potentially by regulating engrailed 1, which was previously shown to be critical for positioning of the murine coronal suture. The diagnosis of a ZIC1 mutation has significant implications for prognosis and we recommend genetic testing when common causes of coronal synostosis have been excluded.

Exploring differential gene expression in zebrafish to teach basic molecular biology skills.

In an effort to engage students in original research while teaching them basic molecular biology skills, we have designed a course for upper level undergraduate students and beginning graduate students that employs in situ hybridization in whole-mount zebrafish embryos to explore the concept of differential gene regulation. The course was taught in a workshop format during a break between the normal fall and spring semesters, which allowed students to immerse themselves in the concepts and techniques full time over a 13-day period. Overall, the course was successful in exposing students to a variety of techniques in the context of an ongoing research project in our laboratory, which provided beneficial outcomes for students and instructors alike. Here we provide a detailed account of the course organization and preparation, as well as an analysis of learning outcomes achieved by the students.