Abstract
Recent developments in tissue clearing methods have significantly advanced the three-dimensional analysis of biological structures in whole, intact tissue, providing a greater understanding of spatial relationships and biological circuits. Nonetheless, studies have reported issues with maintaining structural integrity and preventing tissue disintegration, limiting the wide application of these techniques to fragile tissues such as developing embryos. Here, we present an optimized passive tissue clearing technique (PACT)-based embryo clearing method, initial embedding PACT (IMPACT)-Basic, that improves tissue rigidity without compromising optical transparency. We also present IMPACT-Advance, which is specifically optimized for thin slices of mouse embryos past E13.5. We demonstrate proof-of-concept by investigating the expression of two relatively understudied PR domain (PRDM) proteins, PRDM10 and PRDM13, in intact cleared mouse embryos at various stages of development. We observed strong PRDM10 and PRDM13 expression in the developing nervous system and skeletal cartilage, suggesting a functional role for these proteins in these tissues throughout embryogenesis.
Highlights
Significant recent advancements in the field of tissue clearing have allowed for the appreciation of molecular patterns and cellular circuits in various biological tissues in threedimensional space
To demonstrate proof-of-concept, we investigated the expression of PR domain 10 (PRDM10) and PRDM13, two members of the PRDM (PRDI-BF1 and RIZ homology domain-containing) family [8,9], in mouse embryos cleared via initial embedding passive tissue clearing techniques (PACTs) (IMPACT)-Basic and IMPACTAdvance
In mPACT and mPACT-A, samples are fixed in paraformaldehyde (PFA), treated with A4P0 (4% acrylamide in phosphate-buffered saline (PBS)) and 0.25% 2,2 azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044), and they are embedded with vacuum and nitrogen gas followed by either a PBS wash or A4P0, respectively, prior to clearing
Summary
Significant recent advancements in the field of tissue clearing have allowed for the appreciation of molecular patterns and cellular circuits in various biological tissues in threedimensional space. PRDMs have emerged as important transcriptional regulators that control the development of numerous organ systems throughout embryogenesis While both PRDM10 and PRDM13 have been implicated in key developmental processes, such as cell fate specification, and in various cancer [10,11,12,13,14,15,16,17,18,19], they remain significantly less wellcharacterized compared to their counterparts in the PRDM family [8,9,20]. Our studies demonstrate the applicability and efficacy of the IMPACT methods for clearing embryonic tissue and provide the first three-dimensional survey of PRDM10 and PRDM13 expression in the developing mouse embryo
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