Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3’ vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models.

Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible, modular system. Here, we established several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene-regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2, and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3' vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Lastly, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker active prior to hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish, and other models.

Background: Fetal Alcohol Spectrum Disorders (FASD) describe a continuum of ethanol-induced developmental defects including commonly observed craniofacial malformations. While ethanol-sensitive genetic mutations are a major contributor to facial malformations, the impacted cellular mechanisms underlying these facial anomalies remain unknown. The Bone Morphogenetic Protein (Bmp) signaling pathway is a key regulator of epithelial morphogenesis driving facial development, providing a possible ethanol-sensitive mechanism to malformations to the facial skeleton. Methods: Using zebrafish, we tested several mutants for Bmp pathway components for ethanol-induced facial malformations. Mutant embryos were exposed to ethanol in the media from 10-18 hours post-fertilization (hpf). Exposed zebrafish were fixed at 36 hpf to analyze anterior pharyngeal endoderm size and shape by immunofluorescence or at 5 days post-fertilization (dpf) to quantitatively examine shape of the facial skeleton stained with Alcian Blue/Alizarin Red staining. Integrating human genetic data, we screened for Bmp-ethanol associations in jaw volume of ethanol-exposed children. Results: We found that mutations in the Bmp pathway sensitize zebrafish embryos to ethanol-induced malformations to anterior pharyngeal endoderm shape, leading to altered expression of fgf8a in the oral ectoderm. These changes correlate with shape changes in the viscerocranium, suggesting that ethanol-induced malformations of the anterior pharyngeal endoderm lead to facial malformations. Variants in the Bmp receptor gene, BMPR1B were associated with ethanol-related differences in jaw volume in humans. Conclusions: For the first time, we show that ethanol exposure disrupts proper morphogenesis of, and tissue interactions between, the facial epithelia. These shape changes in the anterior pharyngeal endoderm-oral ectoderm-signaling axis during early zebrafish development mirror the overall shape changes observed in the viscerocranium and were predictive for Bmp-ethanol associations in jaw development in human. Collectively, our work provides a mechanistic paradigm linking the impact of ethanol to the epithelial cell behaviors that underlie facial defects in FASD.

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