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Zygotic genome activation has been extensively studied in a variety of systems including flies, frogs, and mammals. However, there is comparatively little known about the precise timings of gene induction during the earliest phases of embryogenesis. Here we employ high-resolution in situ detection methods, along with genetic and experimental manipulations, to study the timing of zygotic activation in the simple model chordate, Ciona intestinalis with minute-scale temporal precision. We found that two Prdm1 homologs in Ciona are the earliest genes that respond to FGF signaling. We present evidence for a FGF timing mechanism that is driven by derepression of the ERF repressor by ERK activity, which works in concert with localized activators such as Foxa.a. Absence of ERF results in derepression of target genes throughout the embryo. A highlight of this timer is the sharp transition in FGF responsiveness between the 8- and 16-cell stages of development. We propose that this timer is an innovation of chordates that is also employed by other vertebrates.

When challenged by hypertonicity, dehydrated cells must defend their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless droplets, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to rescue volume. The formation of WNK1 condensates is driven by its intrinsically disordered C-terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows WNK1 to bypass a strengthened ionic milieu that favors kinase inactivity and reclaim cell volume through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

Previous work has shown that hepatic levels of human glutathione transferase zeta 1 (GSTZ1) protein, involved in tyrosine catabolism and responsible for metabolism of the investigational drug dichloroacetate, increase in cytosol after birth before reaching a plateau around age 7. However, the mechanism regulating this change of expression is still unknown, and previous studies showed that GSTZ1 mRNA levels did not correlate with GSTZ1 protein expression. In this study, we addressed the hypothesis that microRNAs (miRNAs) could regulate expression of GSTZ1. We obtained liver samples from donors aged less than 1 year or older than 13 years and isolated total RNA for use in a microarray to identify miRNAs that were downregulated in the livers of adults compared with children. From a total of 2578 human miRNAs tested, 63 miRNAs were more than 2-fold down-regulated in adults, of which miR-376c-3p was predicted to bind to the 3' untranslated region of GSTZ1 mRNA. There was an inverse correlation of miR-376c-3p and GSTZ1 protein expression in the liver samples. Using cell culture, we confirmed that miR-376c-3p could downregulate GSTZ1 protein expression. Our findings suggest that miR-376c-3p prevents production of GSTZ1 through inhibition of translation. These experiments further our understanding of GSTZ1 regulation. Furthermore, our array results provide a database resource for future studies on mechanisms regulating human hepatic developmental expression. SIGNIFICANCE STATEMENT: Hepatic glutathione transferase zeta 1 (GSTZ1) is responsible for metabolism of the tyrosine catabolite maleylacetoacetate as well as the investigational drug dichloroacetate. Through examination of microRNA (miRNA) expression in liver from infants and adults and studies in cells, we showed that expression of GSTZ1 is controlled by miRNA. This finding has application to the dosing regimen of the drug dichloroacetate. The miRNA expression profiles are provided and will prove useful for future studies of drug-metabolizing enzymes in infants and adults.

Methoxylated bromodiphenyl ethers (MeO-BDEs), marine natural products, can be demethylated by cytochrome P450 to produce hydroxylated bromodiphenyl ethers (OH-BDEs), potentially toxic metabolites that are also formed by hydroxylation of BDE flame retardants. The OH-BDEs may be detoxified by glucuronidation and sulfonation. This study examined the demethylation of 6-MeO-BDE47, 2′-MeO-BDE68 and 4′-MeO-BDE68, in hepatic microsomes from the red snapper, Lutjanus campechanus, a marine fish likely to be exposed naturally to MeO-BDEs, and the channel catfish, Ictalurus punctatus, a freshwater fish in which pathways of xenobiotic biotransformation have been studied. We further studied the glucuronidation and sulfonation of the resulting OH-BDEs as well as of 6-OH-2′-MeO-BDE68 in hepatic microsomes and cytosol fractions of these fish. The three studied biotransformation pathways were active in both species, with high individual variability. The range of activities overlapped in the two species. Demethylation of MeO-BDEs, studied in the concentration range 10–500 μM, followed Michaelis-Menten kinetics in both fish species, however enzyme efficiencies were low, ranging from 0.024 to 0.334 μL min.mg protein. Conjugation of the studied OH-BDEs followed Michaelis-Menten kinetics in the concentration ranges 1–50 μM (glucuronidation) or 2.5–100 μM (sulfonation). These OH-BDEs were readily glucuronidated and sulfonated in the fish livers of both species, with enzyme efficiencies one to three orders of magnitude higher than for demethylation of the precursor MeO-BDEs. The relatively low efficiencies of demethylation of the MeO-BDEs, compared with higher efficiencies for OH-BDE conjugation, suggests that MeO-BDEs are more likely than OH-BDEs to bioaccumulate in tissues of exposed fish.