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Abstract
The foregut endoderm gives rise to several organs including liver, pancreas, lung and thyroid with important roles in human physiology. Understanding which genes and signalling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to develop a rapid and scalable CRISPR/Cas-based mutagenesis strategy aiming at the identification of genes involved in morphogenesis and function of the thyroid. Core elements of the mutagenesis assay comprise bi-allelic gene invalidation in somatic mutants, a non-invasive monitoring of thyroid development in live transgenic fish, complementary analyses of thyroid function in fixed specimens and quantitative analyses of mutagenesis efficiency by Illumina sequencing of individual fish. We successfully validated our mutagenesis-phenotyping strategy in experiments targeting genes with known functions in early thyroid morphogenesis (pax2a, nkx2.4b) and thyroid functional differentiation (duox, duoxa, tshr). We also demonstrate that duox and duoxa crispants phenocopy thyroid phenotypes previously observed in human patients with bi-allelic DUOX2 and DUOXA2 mutations. The proposed combination of efficient mutagenesis protocols, rapid non-invasive phenotyping and sensitive genotyping holds great potential to systematically characterize the function of larger candidate gene panels during thyroid development and is applicable to other organs and tissues.
Highlights
At the molecular level, one important resource for the understanding of organogenesis and developmental disorders is knowledge about organ- and cell type-specific gene expression programs during critical developmental www.nature.com/scientificreports/
We developed a reverse genetic screening strategy to identify genes involved in thyroid morphogenesis and thyroid function
Our approach capitalizes on the high mutagenic efficiency and sequence-specificity of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, the possibility to monitor the evolution of organ abnormalities in transgenic zebrafish and the sensitive analysis of mutagenesis efficiency by Illumina HiSeq sequencing
Summary
One important resource for the understanding of organogenesis and developmental disorders is knowledge about organ- and cell type-specific gene expression programs during critical developmental www.nature.com/scientificreports/. One has to appreciate that information-rich analyses based on NGS techniques created new bottlenecks for developmental biologists including the need to identify rapid and scalable approaches to characterize the biological function for larger panels of candidate genes with hitherto unknown function during organ development. Recent transcriptomic studies have disclosed gene expression patterns in foregut-derived organ buds including the thyroid[8,23], little progress has been made in assigning specific functions to most of the newly identified genes during thyroid morphogenesis. This holds true for processes including thyroid precursor cell specification, thyroid bud formation, detachment and relocalization, early thyroid growth and functional maturation[6]. Most of our current knowledge about the development of functional thyroid tissue from endodermal precursors stems from studies in murine models but recent studies in zebrafish revealed that many aspects of thyroid morphogenesis are well conserved from fish to men[6,24]
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