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Genetic Control Over Biogenic Crystal Morphogenesis in Zebrafish

ABSTRACT: Organisms had to evolve mechanisms that regulate the properties of biogenic crystals to support a wide range of functions, from vision and camouflage to communication and thermal regulation. Yet, the mechanism underlying the formation of diverse intracellular crystals remains enigmatic. Here, we have unraveled the bio-chemical control over crystal morphogenesis in zebrafish iridophores. We show that the chemical composition of the crystals determines their shape, specifically by the ratio between the nucleobases guanine and hypoxanthine. Moreover, we reveal that these variations in composition are genetically controlled through tissue-specific expression of specialized paralogues, which exhibits remarkable substrate selectivity. This orchestrated combination grants the organism with the capacity to generate a broad spectrum of crystal morphologies. Overall, our findings suggest a new mechanism for the morphological and functional diversity of biogenic crystals and may thus inspire the development of genetically designed biomaterials and medical therapeutics.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Danio Rerio (zebrafish) (brachydanio Rerio)

TISSUE(S): Skin

SUBMITTER: Meital Kupervaser

LAB HEAD: Dvir Gur

PROVIDER: PXD052468 | Pride | 2024-07-31

REPOSITORIES: Pride

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Project description:Objective. To identify novel monosodium urate (MSU) crystal-induced mRNAs by transcript profiling of isolated murine air pouch membranes. Methods. Nine hours after injecting crystals into air pouches, membranes were meticulously dissected away from the adjacent soft tissues. mRNA expression differences between inflamed and control membranes were determined by oligonucleotide microarray analysis. Induction of selected mRNAs was validated by real-time relative quantitative reverse transcriptase PCR (qPCR) in pouch membranes and murine peritoneal macrophages. Results. Eleven of the 12 most highly upregulated mRNAs related to innate immunity and inflammation. They included mRNAs encoding histidine decarboxylase (the enzyme that synthesizes histamine), interleukin (IL)-6, the cell surface receptors PUMA-g and TREM-1, and the polypeptides Irg1 and PROK-2. MSU crystals induced dramatic rises in these mRNAs in the pouch membrane within 3-8 hours after the surge in pro-inflammatory cytokine (IL-6, IL-1beta and TNFalpha) and immediate early gene (Egr-1) transcription, which occurred 1h after crystal injection. MSU crystals induced these mRNAs in cultured macrophages with similar kinetics but lower fold changes. In keeping with their downregulation by MSU crystals according to the microarrays, qPCR confirmed that TREM-2 and granzyme D mRNAs decreased 79% and 94%, respectively, in MSU crystal inflamed membranes. Conclusions. This analysis disclosed several genes previously not implicated in MSU crystal inflammation. Their rise after the early surge in cytokine mRNAs suggests that they may, for instance, amplify or perpetuate inflammation. Transcript profiling of the isolated air pouch membrane promises to be a powerful tool to identify genes acting at different stages of inflammation. Experiment Overall Design: Mouse strain: female Balb/C, 6-8 weeks old Experiment Overall Design: Number of mice= 6. Experiment Overall Design: Air pouches raised on back for 7 days by injecting 2ml of sterile air. Experiment Overall Design: On day 7, injection of 1ml PBS or 2mg MSU crystals in 1ml PBS (n=3 each) Experiment Overall Design: 9 h after injection, dissection of the air pouch membranes from the adjacent soft tissues, RNA extraction with RNeasy columns. Pooling of RNA from the 3 control pouches or the 3 MSU crystal pouches. Processing and labeling of 5ug aliquots in the U Penn microarray core according to standard Affy protocols. Hybridization to Affy Mo430_2 oligonucleotide microarrays.

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Genetic Control Over Biogenic Crystal Morphogenesis in Zebrafish

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