Jason P. Breves

Regenerating complex tissues requires coordination between local cellular responses and body-wide systemic signals. As a central endocrine regulator, the pituitary is well positioned to coordinate this process. Here, we identify the pituitary as an injury-responsive regulator of axolotl limb and tilapia fin regeneration. Limb loss triggered sustained transcriptional responses in the axolotl pituitary. Single-cell transcriptomics linked pituitary loss to altered gene expression in prolactin- and growth hormone-responsive limb cells. Local disruption of the prolactin receptor impaired digit formation, whereas combined hormone treatment partially restored regeneration in hypophysectomized axolotls. In tilapia, pituitary removal impaired fin regeneration and prolactin-related signaling. Mouse digits differed from axolotls and tilapia in how and when they received pituitary signals during regeneration, suggesting pituitary-dependent regrowth is evolutionarily divergent among highly regenerative vertebrates.

Euryhaline fishes tolerate wide variations in salinity through complex physiological mechanisms. Some species inhabit estuaries, where they encounter frequently changing salinities that challenge homeostasis. Located at a critical interface with the aquatic environment, the gills simultaneously support hydromineral balance and mount immune responses against environmental pathogens. In this study, Mozambique tilapia (Oreochromis mossambicus) were exposed to a tidal regime (TR) that entailed oscillations between freshwater (FW) and seawater (SW) conditions every 6 h. We observed branchial interlamellar thickening in fish exposed to a TR, suggesting activation of immune, stress, hyperplasia, and cellular differentiation responses. RNA-Seq analysis was performed to probe gene expression patterns underlying these apparent responses. Comparative analysis between salinity groups highlighted enriched biological processes and signaling pathways related to immune regulation, cellular stress responses, hyperplasia/proliferation, and epithelial-mesenchymal transition (EMT). The greatest number of differentially expressed genes was found when comparing fish held in a TR with those held continuously in SW. The most abundant and differentially expressed transcripts associated with immune and stress responses (ccl20, il1b, gsto1, gstr, and hsp40) and with hyperplasia and EMT (pcna, npcc, mki67, fndc7a, and ets1) were selected for qPCR analysis. While il1b, gstr, gsto1, hsp40, and pcna were differentially expressed between the FW and SW phases of the TR, ccl20, nppc, mki67, fndc7a, and ets1 were downregulated in TR compared with SW. Collectively, these results indicate that tilapia thicken their branchial epithelium, exhibit immune and stress responses, and activate cellular proliferation and differentiation pathways to sustain osmoregulatory balance under dynamic tidal conditions.

Objective: Euryhaline fishes can inhabit salinities ranging from fresh water (FW) to seawater (SW), in part because their endocrine system aligns the ion-transporting capacities of branchial ionocytes with the external environment. Ionocytes also directly sense extracellular osmolality and adjust their functions accordingly; therefore, this study investigated the interplay between osmotic conditions and osmoregulatory hormones in Mozambique tilapia ( Oreochromis mossambicus ) to further elucidate the basis of euryhalinity. We sought to determine whether prolactin (Prl) supports FW acclimation by counteracting osmotic and hormonal signals that initiate branchial responses to SW environments. Methods: We first combined hypophysectomy, hormone replacement, RNA-Seq, and qPCR to identify cystic fibrosis transmembrane conductance regulator (cftr) , osmotic stress transcription factor 1 (ostf1) , and serum- and glucocorticoid-inducible kinase 1 (sgk1) as Prl-repressed genes. We then leveraged a series of in vivo and in vitro experimental paradigms to characterize their regulation by environmental salinity, Prl, cortisol, and extracellular osmolality. Results: Our findings indicate that 1) Prl’s capacity to antagonize cortisol-stimulated cftr expression depends on extracellular osmotic conditions; 2) Prl and cortisol play opposing roles in regulating branchial ostf1 expression; 3) Sgk1 is expressed in ‘SW-type’ ionocytes, where interactions among Prl, cortisol, and osmotic conditions influence its expression; and 4) Prl promotes the expression of Na ⁺ /Cl ^- cotransporter 2 and Clc family Cl ⁻ channel 2c , as shown previously. Conclusions: The combined actions of osmotic stimuli, Prl, and cortisol shape the branchial expression of cftr , ostf1 , and sgk1 . While hyperosmotic extracellular conditions and cortisol promote the activation of these genes during SW acclimation, Prl supports FW adaptation by suppressing these genes and promoting processes underlying active ion uptake. Thus, euryhaline tilapia adjust the ion-transporting activity of their branchial ionocytes to meet environmental demands by integrating multiple regulatory cues.

To reside in marine habitats, marine fishes must actively eliminate the excess Na+ and Cl− they acquire from their environment. These ions are passively absorbed across body surfaces and through the ingestion of seawater (SW), which is necessary to maintain water balance. Salt secretion occurs through the actions of mitochondrion-rich ‘SW-type ionocytes’ in the gills or other specialized salt-secreting organs. For nearly 50 years, the SW-type ionocyte model proposed by Patricio Silva and colleagues has proven remarkably enduring. The Silva model guided researchers toward identifying key molecular components of SW-type ionocytes, such as cystic fibrosis transmembrane conductance regulator 1 (Cftr1), Na+/K+/2Cl− cotransporter 1 (Nkcc1) and the inwardly rectifying K+ channel (Kir). However, emerging findings indicate that alternative molecular mechanisms may complement, or in some cases, operate in lieu of those included in the Silva model. In this Commentary, we argue that it is time to critically evaluate whether ionocyte-based strategies for salt secretion are more diverse than currently recognized. We highlight recent developments regarding the operation of Cftr-independent ionocytes in lamprey, the emerging role of anoctamin 1 (Ano1) in lamprey and bony fishes, and various ‘new’ pathways for Cl− and K+ to enter and exit ionocytes. Additionally, we propose future research directions for identifying novel salt-secretory mechanisms in various lineages of marine fishes. We conclude this Commentary by presenting three hypotheses for the divergence of ionocyte-mediated salt secretion in marine fishes and outlining a conceptual framework for considering the evolutionary homology of salt-secreting ionocytes across vertebrates.

In response to changes in environmental salinity, euryhaline fishes mobilize energy to support the active transport of ions across osmoregulatory epithelia. Glycogen synthase (GS) and glycogen phosphorylase (GP) are key controllers of carbohydrate metabolism due to their roles in promoting glycogenesis and glycogenolysis, respectively. However, the coordinated responses of GS, GP and glucose transporters (GLUTs) in the liver and gill to changes in salinity remain unresolved. In this study, we initially subjected Mozambique tilapia (Oreochromis mossambicus) to unidirectional transfers, either from fresh water (FW) to seawater (SW) or from SW to FW. We then transferred FW‐ and SW‐acclimated tilapia to a tidal regime (TR) where salinity alternated between FW and SW every 6 h. Our goal was to characterize how carbohydrate metabolism is affected by unidirectional and tidal changes in salinity. Whether fish were transferred from SW to FW in a unidirectional manner or to a TR, glucose levels increased after transfer to FW or during the FW phase of the TR (TF). Conversely, hepatic glycogen levels were higher in fish in SW and the SW phase of the TR (TS) than in TF. In both FW and TF, branchial expression of the gill‐specific isoform of GP (gpg) was downregulated, while gs was upregulated. Branchial gpg was upregulated in fish unidirectionally transferred from FW to SW or sampled during TS. Hepatic gp and gs expression increased following transfer from SW to FW. Thus, we consistently found that increases in salinity promoted branchial glycogen breakdown, while decreases in salinity led to hyperglycaemia. Moreover, while branchial glucose transporter 1 (glut 1) expression was downregulated after transfer from FW to SW, glut1 was transiently upregulated in the liver. In both liver and gill, glut1 expression was higher in fish in TF compared to TS. Gill filament explants incubated with cortisol exhibited reduced glut1 expression regardless of medium osmolality. Our collective data indicate that salinity differentially regulates hepatic and branchial carbohydrate metabolism.

In this study, we provide evidence in a teleost fish that the Ca²⁺-activated Cl⁻ channel, anoctamin 1 may provide an additional path for Cl⁻ secretion by seawater-type ionocytes. Not only is this the first report of a Cftr-independent Cl⁻-secreting pathway conferring survival in seawater but also the first description of its regulation by the pituitary hormone prolactin.

The endocrine system is an essential regulator of the osmoregulatory organs that enable euryhaline fishes to maintain hydromineral balance in a broad range of environmental salinities. Because branchial ionocytes are the primary site for the active exchange of Na⁺, Cl⁻, and Ca²⁺ with the external environment, their functional regulation is inextricably linked with adaptive responses to changes in salinity. Here, we review the molecular-level processes that connect osmoregulatory hormones with branchial ion transport. We focus on how factors such as prolactin, growth hormone, cortisol, and insulin-like growth-factors operate through their cognate receptors to direct the expression of specific ion transporters/channels, Na⁺/K⁺-ATPases, tight-junction proteins, and aquaporins in ion-absorptive (freshwater-type) and ion-secretory (seawater-type) ionocytes. While these connections have historically been deduced in teleost models, more recently, increased attention has been given to understanding the nature of these connections in basal lineages. We conclude our review by proposing areas for future investigation that aim to fill gaps in the collective understanding of how hormonal signaling underlies ionocyte-based processes.