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TissUExM enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy

ABSTRACT: Summary Super-resolution microscopy reveals the molecular organization of biological structures down to the nanoscale. While it allows the study of protein complexes in single cells, small organisms, or thin tissue sections, there is currently no versatile approach for ultrastructural analysis compatible with whole vertebrate embryos. Here, we present tissue ultrastructure expansion microscopy (TissUExM), a method to expand millimeter-scale and mechanically heterogeneous whole embryonic tissues, including Drosophila wing discs, whole zebrafish, and mouse embryos. TissUExM is designed for the observation of endogenous proteins. It permits quantitative characterization of protein complexes in various organelles at super-resolution in a range of ∼3 mm-sized tissues using conventional microscopes. We demonstrate its strength by investigating tissue-specific ciliary architecture heterogeneity and ultrastructural defects observed upon ciliary protein overexpression. Overall, TissUExM is ideal for performing ultrastructural studies and molecular mapping in situ in whole embryos. Graphical abstract Highlights • We developed TissUExM for expansion microscopy of mechanically heterogeneous tissues• TissUExM relies on post-expansion labeling for localization of endogenous proteins• TissUExM facilitates quantitative ultrastructural analysis in situ in whole embryos• TissUExM can be applied to whole zebrafish and mouse embryos or to fly wings Motivation Super-resolution microscopy is challenging in whole embryos, which limits structure-function analysis during developmental processes. We developed a method allowing quantitative ultrastructural analysis in mechanically heterogeneous millimeter-scaled embryos, optimized for post-expansion labeling, and the localization of endogenous proteins in high-density environments. Super-resolution microscopy is challenging in whole embryos, limiting structure-function analysis during developmental processes. Steib et al. developed TissUExM to physically expand heterogeneous tissues and label endogenous protein complexes. This approach can be applied to fly wings or to whole zebrafish and mouse embryos, enabling quantitative ultrastructural analysis at the nanometer scale.

SUBMITTER: Steib E

PROVIDER: S-EPMC9606133 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Project description:BackgroundGATA transcription factors influence many developmental processes, including the specification of embryonic germ layers. The GATA gene family has significantly expanded in many animal lineages: whereas diverse cnidarians have only one GATA transcription factor, six GATA genes have been identified in many vertebrates, five in many insects, and eleven to thirteen in Caenorhabditis nematodes. All bilaterian animal genomes have at least one member each of two classes, GATA123 and GATA456.ResultsWe have identified one GATA123 gene and one GATA456 gene from the genomic sequence of two invertebrate deuterostomes, a cephalochordate (Branchiostoma floridae) and a hemichordate (Saccoglossus kowalevskii). We also have confirmed the presence of six GATA genes in all vertebrate genomes, as well as additional GATA genes in teleost fish. Analyses of conserved sequence motifs and of changes to the exon-intron structure, and molecular phylogenetic analyses of these deuterostome GATA genes support their origin from two ancestral deuterostome genes, one GATA 123 and one GATA456. Comparison of the conserved genomic organization across vertebrates identified eighteen paralogous gene families linked to multiple vertebrate GATA genes (GATA paralogons), providing the strongest evidence yet for expansion of vertebrate GATA gene families via genome duplication events.ConclusionFrom our analysis, we infer the evolutionary birth order and relationships among vertebrate GATA transcription factors, and define their expansion via multiple rounds of whole genome duplication events. As the genomes of four independent invertebrate deuterostome lineages contain single copy GATA123 and GATA456 genes, we infer that the 0R (pre-genome duplication) invertebrate deuterostome ancestor also had two GATA genes, one of each class. Synteny analyses identify duplications of paralogous chromosomal regions (paralogons), from single ancestral vertebrate GATA123 and GATA456 chromosomes to four paralogons after the first round of vertebrate genome duplication, to seven paralogons after the second round of vertebrate genome duplication, and to fourteen paralogons after the fish-specific 3R genome duplication. The evolutionary analysis of GATA gene origins and relationships may inform understanding vertebrate GATA factor redundancies and specializations.

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TissUExM enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy

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