Schaaf, M. J. M. | bioRxiv Science

bioRxiv2026

Zebrafish larval nitrogen excretion is flexible and resilient to loss of rhesus glycoproteins

Nitrogenous waste excretion is essential for all developmental stages of fish. Embryonic fish excrete urea, transitioning to cutaneous and later branchial ammonia excretion. In zebrafish, ammonia excretion involves rhesus glycoproteins Rhbg and Rhcgb in keratinocytes and ionocytes, but the developmental moment they appear in the gill remains unclear. Potential redundancy between Rhbg and Rhcgb in ammonia excretion is also not fully investigated, nor is the difference in response to low pH. We hypothesized that rhesus glycoproteins are partially redundant, and that they differ in their response to low pH as ammonia excretion enables ionocytes to exchange Na+ and H+ (Rh-NHE-metabolon). We predicted that a loss of rhbg or rhcgb induces compensatory responses. We characterized the transition from urea to branchial ammonia excretion from 0 to 8 days-post fertilization (dpf) and the response to pH 5.0 on the expression and localization of rhesus glycoproteins in control zebrafish and rhbg or rhcgb-crispants. Effects of high external ammonia (HEA, 500 M NH4Cl) and 10 mM HEPES-buffering were further characterized in rhcgb-crispants. Rhag and Rhbg appeared in the gill at 5 dpf, while Rhcgb appeared at 6 dpf. A loss of rhbg or rhcgb did not impact baseline N-excretion, illustrating that zebrafish can maintain ammonia excretion without the full complement of rhesus glycoproteins. We observed no compensatory increase in rhesus glycoproteins, but expression of the transporter hippocampus-abundant transcript 1b increased. HEA-exposed rhcgb-crispants switched to urea as primary nitrogen waste. Together, these findings underline the plasticity of the larval in dealing with nitrogenous waste.

physiology↗

bioRxiv2018

Glucocorticoids inhibit macrophage differentiation towards a pro-inflammatory phenotype upon wounding without affecting their migration

Glucocorticoid drugs are widely used to treat immune-related diseases, but their use is limited by side effects and by resistance, which especially occurs in macrophage-dominated diseases. In order to improve glucocorticoid therapies, more research is required into the mechanisms of glucocorticoid action. In the present study, we have used a zebrafish model for inflammation to study glucocorticoid effects on the innate immune response. In zebrafish larvae, the migration of neutrophils towards a site of injury is inhibited by the synthetic glucocorticoid beclomethasone, while migration of macrophages is glucocorticoid resistant. We show that wounding-induced increases in expression of genes encoding neutrophil-specific chemoattractants (Il8 and Cxcl18b) are attenuated by beclomethasone, but that beclomethasone does not attenuate the induction of the genes encoding Ccl2 and Cxcl11aa, which we show to be required for macrophage recruitment. RNA sequencing on Fluorescence-Activated Cell Sorting (FACS)-sorted macrophages showed that the vast majority of the wounding-induced transcriptional changes in these cells are inhibited by beclomethasone, whereas a small subset is glucocorticoid-insensitive. As a result, beclomethasone decreases the number of macrophages that differentiate towards a pro-inflammatory (M1) phenotype, which we demonstrated using a tnfa:eGFP-F reporter line and analysis of macrophage morphology. We conclude that the glucocorticoid resistance of the wounding-induced macrophage migration is due to the insensitivity of the induction of macrophage-specific chemoattractants to glucocorticoid inhibition, which may explain the relative resistance of macrophage-dominated diseases to glucocorticoid therapy. However, the induction of pro-inflammatory genes in macrophages is strongly attenuated, which inhibits their differentiation to an M1 phenotype.\n\nSummary statementIn a zebrafish model for inflammation, glucocorticoids do not affect the migration of macrophages, but inhibit differentiation towards an M1 phenotype, by strongly attenuating transcriptional responses in these cells.

immunology↗