RESUMEN
During vertebrate development, extracellular signal-regulated kinase (ERK) is activated by growth factors such as fibroblast growth factor (FGF), and it regulates the formation of tissues/organs including eyes, brains, somites, limbs, and inner ears. However, an experimental system to monitor ERK activity dynamics in the entire body of the vertebrate embryo is lacking. We recently studied ERK activity dynamics in the pre-somitic mesoderm of living zebrafish embryos injected with mRNAs encoding a Förster resonance energy transfer (FRET)-based ERK biosensor. In this study, transgenic zebrafish stably and ubiquitously expressing the ERK biosensor were generated to monitor ERK activity dynamics throughout embryonic development. The system allowed the identification of ERK activation domains in embryos from the late blastula to the late segmentation stage, consistent with immunostaining patterns obtained using anti-phosphorylated ERK antibody. A spatiotemporal map of ERK activity in the entire body during zebrafish embryogenesis was generated, and previously unidentified activation dynamics and ERK domains were identified. The proposed system is the first reported method to monitor ERK activity dynamics during vertebrate embryogenesis, providing insight into the role of ERK activity in normal and abnormal development in living vertebrate embryos.
Asunto(s)
Desarrollo Embrionario , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Técnicas Biosensibles , Quinasas MAP Reguladas por Señal Extracelular/química , Estadios del Ciclo de Vida , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Pez Cebra/genéticaRESUMEN
Macrophage migration inhibitory factor (MIF) plays versatile roles in the immune system. MIF is also widely expressed during embryonic development, particularly in the nervous system, although its roles in neural development are only beginning to be understood. Evidence from frogs, mice and zebrafish suggests that MIF has a major role as a neurotrophin in the early development of sensory systems, including the auditory system. Here we show that the zebrafish mif pathway is required for both sensory hair cell (HC) and sensory neuronal cell survival in the ear, for HC differentiation, semicircular canal formation, statoacoustic ganglion (SAG) development, and lateral line HC differentiation. This is consistent with our findings that MIF is expressed in the developing mammalian and avian auditory systems and promotes mouse and chick SAG neurite outgrowth and neuronal survival, demonstrating key instructional roles for MIF in vertebrate otic development.
Asunto(s)
Oído Interno/metabolismo , Factores Inhibidores de la Migración de Macrófagos/genética , Factores de Crecimiento Nervioso/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Diferenciación Celular/genética , Oído Interno/embriología , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Ganglios Sensoriales/embriología , Ganglios Sensoriales/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células Ciliadas Auditivas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Pirimidinas/farmacología , Receptores Inmunológicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales Semicirculares/embriología , Canales Semicirculares/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Factores de Tiempo , Pez Cebra/embriologíaRESUMEN
There is very little information on the capacity of freshwater carnivorous fish to biosynthesize highly unsaturated fatty acids (HUFA). The striped snakehead fish (Channa striata) is a carnivorous species cultured inland of several Southeast Asian countries due to its pharmaceutical properties in wound healing enhancement. We described here the full-length cDNA cloning of a striped snakehead fatty acid desaturase (fads), which is responsible for desaturation of unsaturated fatty acids in the HUFA biosynthesis. Bioinformatics analysis reveals a protein coding region with length of 445 amino acids containing all characteristic features of desaturase enzyme, including a cytochrome b5-domain with the heme-binding motif, two transmembrane domains and three histidine-rich regions. The striped snakehead fads amino acid sequence shares high similarity with known fads of other teleosts. The mRNA expression of striped snakehead fads also showed an ontogenic-related increase in expression in 0-20 days after hatch larva. Using ISH, we localized the presence of fads in larva brain, liver and intestinal tissues.
