ABSTRACT
Vertebrate Hox clusters are comprised of multiple Hox genes that control morphology and developmental timing along multiple body axes. Although results of genetic analyses using Hox-knockout mice have been accumulating, genetic studies in other vertebrates have not been sufficient for functional comparisons of vertebrate Hox genes. In this study, we isolated all of the seven hox cluster loss-of-function alleles in zebrafish using the CRISPR-Cas9 system. Comprehensive analysis of the embryonic phenotype and X-ray micro-computed tomography scan analysis of adult fish revealed several species-specific functional contributions of homologous Hox clusters along the appendicular axis, whereas important shared general principles were also confirmed, as exemplified by serial anterior vertebral transformations along the main body axis, observed in fish for the first time. Our results provide insights into discrete sub/neofunctionalization of vertebrate Hox clusters after quadruplication of the ancient Hox cluster. This set of seven complete hox cluster loss-of-function alleles provide a formidable resource for future developmental genetic analysis of the Hox patterning system in zebrafish.
Subject(s)
Genes, Homeobox/genetics , Multigene Family , Zebrafish/genetics , Zebrafish/physiology , Animals , CRISPR-Cas Systems , Embryonic Development/genetics , Evolution, Molecular , Female , Gene Duplication , Gene Expression Regulation, Developmental , Male , Mutation , Skeleton/anatomy & histology , Skeleton/growth & development , Species Specificity , X-Ray Microtomography , Zebrafish/embryologyABSTRACT
Narcolepsy-cataplexy is a debilitating disorder of sleep/wakefulness caused by a loss of orexin-producing neurons in the lateroposterior hypothalamus. Genetic or pharmacologic orexin replacement ameliorates symptoms in mouse models of narcolepsy-cataplexy. We have recently discovered a potent, nonpeptide OX2R-selective agonist, YNT-185. This study validates the pharmacological activity of this compound in OX2R-transfected cells and in OX2R-expressing neurons in brain slice preparations. Intraperitoneal, and intracerebroventricular, administration of YNT-185 suppressed cataplexy-like episodes in orexin knockout and orexin neuron-ablated mice, but not in orexin receptor-deficient mice. Peripherally administered YNT-185 also promotes wakefulness without affecting body temperature in wild-type mice. Further, there was no immediate rebound sleep after YNT-185 administration in active phase in wild-type and orexin-deficient mice. No desensitization was observed after repeated administration of YNT-185 with respect to the suppression of cataplexy-like episodes. These results provide a proof-of-concept for a mechanistic therapy of narcolepsy-cataplexy by OX2R agonists.
Subject(s)
Aniline Compounds/pharmacology , Benzamides/pharmacology , Cataplexy/drug therapy , Narcolepsy/drug therapy , Orexin Receptors/agonists , Orexins/metabolism , Sleep Disorders, Circadian Rhythm/drug therapy , Wakefulness-Promoting Agents/therapeutic use , Wakefulness/drug effects , Aniline Compounds/chemistry , Animals , Benzamides/chemistry , Disease Models, Animal , Hypothalamus/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Orexin Receptors/genetics , Orexins/genetics , Patch-Clamp Techniques , Sleep/drug effectsABSTRACT
Photostabilities of some dihydropyridine calcium-channel blockers in pulverized pharmaceutical tablets were studied. Powdered tablets including amlodipine, nifedipine, or nilvadipine were exposed to D65 daylight lamp radiation according to an ICH guideline (ICH Q1B). The photodegradation of pharmaceutical components and their degradation products were monitored by HPLC using a reversed phase column with UV detection; their peak components were identified using MS analysis. Photochemical reactions involved in the photodegradation of these pharmaceuticals include aromatization of the dihydropyridine moiety and conversion to nitroso group from the nitro group in benzene rings. Chemical stability studies of these drugs indicate that nifedipine is the most photosensitive. The rate constant of nifedipine is indicated as seven times higher than those of the other two drugs.
