RESUMEN
Mitophagy, the selective recycling of mitochondria through autophagy, is a crucial metabolic process induced by cellular stress, and defects are linked to aging, sarcopenia and neurodegenerative diseases. To therapeutically target mitophagy, the fundamental in vivo dynamics and molecular mechanisms must be fully understood. Here, we generated mitophagy biosensor zebrafish lines expressing mitochondrially targeted, pH-sensitive fluorescent probes, mito-Keima and mito-EGFP-mCherry, and used quantitative intravital imaging to illuminate mitophagy during physiological stresses, namely, embryonic development, fasting and hypoxia. In fasted muscle, volumetric mitolysosome size analyses documented organelle stress response dynamics, and time-lapse imaging revealed that mitochondrial filaments undergo piecemeal fragmentation and recycling rather than the wholesale turnover observed in cultured cells. Hypoxia-inducible factor (Hif) pathway activation through physiological hypoxia or chemical or genetic modulation also provoked mitophagy. Intriguingly, mutation of a single mitophagy receptor (bnip3) prevented this effect, whereas disruption of other putative hypoxia-associated mitophagy genes [bnip3la (nix), fundc1, pink1 or prkn (Parkin)] had no effect. This in vivo imaging study establishes fundamental dynamics of fasting-induced mitophagy and identifies bnip3 as the master regulator of Hif-induced mitophagy in vertebrate muscle.
Asunto(s)
Mitofagia , Pez Cebra , Animales , Microscopía Intravital , Mitocondrias , Estrés Fisiológico , Ubiquitina-Proteína Ligasas , Pez Cebra/genéticaRESUMEN
CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.
Asunto(s)
Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Edición Génica , Reparación del ADN por Recombinación , Animales , Proteína 9 Asociada a CRISPR/genética , Colorantes Fluorescentes , Proteínas Fluorescentes Verdes/genética , Mutación INDEL , Indicadores y Reactivos , Melanocitos , Nitrorreductasas/genética , ARN/química , Moldes Genéticos , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Guidance cues act during development to guide growth cones to their proper targets in both the central and peripheral nervous systems. Experiments in many species indicate that guidance molecules also play important roles after development, though less is understood about their functions in the adult. The Semaphorin family of guidance cues, signaling through Plexin receptors, influences the development of both axons and dendrites in invertebrates. Semaphorin functions have been extensively explored in Drosophila melanogaster and some other Dipteran species, but little is known about their function in hemimetabolous insects. Here, we characterize sema1a and plexA in the cricket Gryllus bimaculatus. In fact, we found two distinct predicted Sema1a proteins in this species, Sema1a.1 and Sema1a.2, which shared only 48% identity at the amino acid level. We include a phylogenetic analysis that predicted that many other insect species, both holometabolous and hemimetabolous, express two Sema1a proteins as well. Finally, we used in situ hybridization to show that sema1a.1 and sema1a.2 expression patterns were spatially distinct in the embryo, and both roughly overlap with plexA. All three transcripts were also expressed in the adult brain, mainly in the mushroom bodies, though sema1a.2 was expressed most robustly. sema1a.2 was also expressed strongly in the adult thoracic ganglia while sema1a.1 was only weakly expressed and plexA was undetectable.
Asunto(s)
Proteínas de Drosophila/biosíntesis , Proteínas de Drosophila/genética , Gryllidae/crecimiento & desarrollo , Gryllidae/genética , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Semaforinas/biosíntesis , Semaforinas/genética , Factores de Edad , Secuencia de Aminoácidos , Animales , Regulación del Desarrollo de la Expresión Génica , Gryllidae/metabolismo , FilogeniaRESUMEN
The auditory system of the cricket, Gryllus bimaculatus, demonstrates an unusual amount of anatomical plasticity in response to injury, even in adults. Unilateral removal of the ear causes deafferented auditory neurons in the prothoracic ganglion to sprout dendrites across the midline, a boundary they typically respect, and become synaptically connected to the auditory afferents of the contralateral ear. The molecular basis of this sprouting and novel synaptogenesis in the adult is not understood. We hypothesize that well-conserved developmental guidance cues may recapitulate their guidance functions in the adult in order to facilitate this compensatory growth. As a first step in testing this hypothesis, we have generated a de novo assembly of a prothoracic ganglion transcriptome derived from control and deafferented adult individuals. We have mined this transcriptome for orthologues of guidance molecules from four well-conserved signaling families: Slit, Netrin, Ephrin, and Semaphorin. Here we report that transcripts encoding putative orthologues of most of the candidate developmental ligands and receptors from these signaling families were present in the assembly, indicating expression in the adult G. bimaculatus prothoracic ganglion.