RESUMEN
Despite the robust healing capacity of the liver, regenerative failure underlies numerous hepatic diseases, including the JAG1 haploinsufficient disorder, Alagille syndrome (ALGS). Cholestasis due to intrahepatic duct (IHD) paucity resolves in certain ALGS cases but fails in most with no clear mechanisms or therapeutic interventions. We find that modulating jag1b and jag2b allele dosage is sufficient to stratify these distinct outcomes, which can be either exacerbated or rescued with genetic manipulation of Notch signaling, demonstrating that perturbations of Jag/Notch signaling may be causal for the spectrum of ALGS liver severities. Although regenerating IHD cells proliferate, they remain clustered in mutants that fail to recover due to a blunted elevation of Notch signaling in the distal-most IHD cells. Increased Notch signaling is required for regenerating IHD cells to branch and segregate into the peripheral region of the growing liver, where biliary paucity is commonly observed in ALGS. Mosaic loss- and-gain-of-function analysis reveals Sox9b to be a key Notch transcriptional effector required cell autonomously to regulate these cellular dynamics during IHD regeneration. Treatment with a small-molecule putative Notch agonist stimulates Sox9 expression in ALGS patient fibroblasts and enhances hepatic sox9b expression, rescues IHD paucity and cholestasis, and increases survival in zebrafish mutants, thereby providing a proof-of-concept therapeutic avenue for this disorder.
Asunto(s)
Síndrome de Alagille , Conductos Biliares Intrahepáticos , Transducción de Señal , Animales , Humanos , Síndrome de Alagille/genética , Síndrome de Alagille/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Mosaicismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Regeneración , Conductos Biliares Intrahepáticos/citología , Conductos Biliares Intrahepáticos/patología , FibroblastosRESUMEN
Development of elaborate and polarized neuronal morphology requires precisely regulated transport of cellular cargos by motor proteins such as kinesin-1. Kinesin-1 has numerous cellular cargos which must be delivered to unique neuronal compartments. The process by which this motor selectively transports and delivers cargo to regulate neuronal morphogenesis is poorly understood, although the cargo-binding kinesin light chain (KLC) subunits contribute to specificity. Our work implicates one such subunit, KLC4, as an essential regulator of axon branching and arborization pattern of sensory neurons during development. Using live imaging approaches in klc4 mutant zebrafish, we show that KLC4 is required for stabilization of nascent axon branches, proper microtubule (MT) dynamics, and endosomal transport. Furthermore, KLC4 is required for proper tiling of peripheral axon arbors: in klc4 mutants, peripheral axons showed abnormal fasciculation, a behavior characteristic of central axons. This result suggests that KLC4 patterns axonal compartments and helps establish molecular differences between central and peripheral axons. Finally, we find that klc4 mutant larva are hypersensitive to touch and adults show anxiety-like behavior in a novel tank test, implicating klc4 as a new gene involved in stress response circuits.
Asunto(s)
Cinesinas , Pez Cebra , Animales , Cinesinas/genética , Axones/fisiología , Células Receptoras Sensoriales/fisiología , MorfogénesisRESUMEN
Many cells can generate complementary traveling waves of actin filaments (F-actin) and cytoskeletal regulators. This phenomenon, termed cortical excitability, results from coupled positive and negative feedback loops of cytoskeletal regulators. The nature of these feedback loops, however, remains poorly understood. We assessed the role of the Rho GAP RGA-3/4 in the cortical excitability that accompanies cytokinesis in both frog and starfish. RGA-3/4 localizes to the cytokinetic apparatus, "chases" Rho waves in an F-actin-dependent manner, and when coexpressed with the Rho GEF Ect2, is sufficient to convert the normally quiescent, immature Xenopus oocyte cortex into a dramatically excited state. Experiments and modeling show that changing the ratio of RGA-3/4 to Ect2 produces cortical behaviors ranging from pulses to complex waves of Rho activity. We conclude that RGA-3/4, Ect2, Rho, and F-actin form the core of a versatile circuit that drives a diverse range of cortical behaviors, and we demonstrate that the immature oocyte is a powerful model for characterizing these dynamics.
Asunto(s)
Actinas , Citoesqueleto , Proteínas Activadoras de GTPasa , Proteínas Proto-Oncogénicas , Proteínas de Unión al GTP rho , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Citocinesis , Citoesqueleto/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Oocitos , Proteínas Proto-Oncogénicas/metabolismo , Xenopus , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Lymphatic filariasis (LF) is a chronic debilitating neglected tropical disease (NTD) caused by mosquito-transmitted nematodes that afflicts over 60 million people. Control of LF relies on routine mass drug administration with antiparasitics that clear circulating larval parasites but are ineffective against adults. The development of effective adulticides is hampered by a poor understanding of the processes and tissues driving parasite survival in the host. The adult filariae head region contains essential tissues that control parasite feeding, sensory, secretory, and reproductive behaviors, which express promising molecular substrates for the development of antifilarial drugs, vaccines, and diagnostics. We have adapted spatial transcriptomic approaches to map gene expression patterns across these prioritized but historically intractable head tissues. Spatial and tissue-resolved data reveal distinct biases in the origins of known drug targets and secreted antigens. These data were used to identify potential new drug and vaccine targets, including putative hidden antigens expressed in the alimentary canal, and to spatially associate receptor subunits belonging to druggable families. Spatial transcriptomic approaches provide a powerful resource to aid gene function inference and seed antiparasitic discovery pipelines across helminths of relevance to human and animal health.
Asunto(s)
Antiinfecciosos , Brugia Malayi , Filariasis Linfática , Parásitos , Vacunas , Animales , Antiinfecciosos/farmacología , Antiparasitarios/farmacología , Brugia Malayi/genética , Humanos , Parásitos/genética , TranscriptomaRESUMEN
BACKGROUND AND AIMS: Alagille Syndrome (ALGS) is a congenital disorder caused by mutations in the Notch ligand gene JAGGED1, leading to neonatal loss of intrahepatic duct (IHD) cells and cholestasis. Cholestasis can resolve in certain patients with ALGS, suggesting regeneration of IHD cells. However, the mechanisms driving IHD cell regeneration following Jagged loss remains unclear. Here, we show that cholestasis due to developmental loss of IHD cells can be consistently phenocopied in zebrafish with compound jagged1b and jagged2b mutations or knockdown. APPROACH AND RESULTS: Leveraging the transience of jagged knockdown in juvenile zebrafish, we find that resumption of Jagged expression leads to robust regeneration of IHD cells through a Notch-dependent mechanism. Combining multiple lineage tracing strategies with whole-liver three-dimensional imaging, we demonstrate that the extrahepatic duct (EHD) is the primary source of multipotent progenitors that contribute to the regeneration, but not to the development, of IHD cells. Hepatocyte-to-IHD cell transdifferentiation is possible but rarely detected. Progenitors in the EHD proliferate and migrate into the liver with Notch signaling loss and differentiate into IHD cells if Notch signaling increases. Tissue-specific mosaic analysis with an inducible dominant-negative Fgf receptor suggests that Fgf signaling from the surrounding mesenchymal cells maintains this extrahepatic niche by directly preventing premature differentiation and allocation of EHD progenitors to the liver. Indeed, transcriptional profiling and functional analysis of adult mouse EHD organoids uncover their distinct differentiation and proliferative potential relative to IHD organoids. CONCLUSIONS: Our data show that IHD cells regenerate upon resumption of Jagged/Notch signaling, from multipotent progenitors originating from an Fgf-dependent extrahepatic stem cell niche. We posit that if Jagged/Notch signaling is augmented, through normal stochastic variation, gene therapy, or a Notch agonist, regeneration of IHD cells in patients with ALGS may be enhanced.
Asunto(s)
Síndrome de Alagille , Conductos Biliares Extrahepáticos , Conductos Biliares Intrahepáticos , Proteínas de Unión al Calcio , Proteína Jagged-1 , Regeneración Hepática/fisiología , Receptores Notch/metabolismo , Proteínas de Pez Cebra , Síndrome de Alagille/genética , Síndrome de Alagille/metabolismo , Animales , Conductos Biliares Extrahepáticos/crecimiento & desarrollo , Conductos Biliares Extrahepáticos/fisiología , Conductos Biliares Intrahepáticos/crecimiento & desarrollo , Conductos Biliares Intrahepáticos/fisiología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Transdiferenciación Celular , Modelos Animales de Enfermedad , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
High-throughput drug screening of patient-derived organoids offers an attractive platform to determine cancer treatment efficacy. Here, selective plane illumination microscopy (SPIM) was used to determine treatment response in organoids with endogenous fluorescence from the metabolic coenzymes NAD(P)H and FAD. Rapid 3-D autofluorescence imaging of colorectal cancer organoids was achieved. A quantitative image analysis approach was developed to segment each organoid and quantify changes in endogenous fluorescence caused by treatment. Quantitative analysis of SPIM volumes confirmed the sensitivity of patient-derived organoids to standard therapies. This proof-of-principle study demonstrates that SPIM is a powerful tool for high-throughput screening of organoid treatment response.
RESUMEN
Fluorescence microscopy is an essential tool for biological discoveries. There is a constant demand for better spatial resolution across a larger field of view. Although strides have been made to improve the theoretical resolution and speed of the optical instruments, in mesoscopic samples, image quality is still largely limited by the optical properties of the sample. In Selective Plane Illumination Microscopy (SPIM), the achievable optical performance is hampered by optical degradations encountered in both the illumination and detection. Multi-view imaging, either through sample rotation or additional optical paths, is a popular strategy to improve sample coverage. In this work, we introduce a smart rotation workflow that utilizes on-the-fly image analysis to identify the optimal light sheet imaging orientations. The smart rotation workflow outperforms the conventional approach without additional hardware and achieves a better sample coverage using the same number of angles or less and thereby reduces data volume and phototoxicity.
Asunto(s)
Aumento de la Imagen/métodos , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
Antifungal therapy can fail in a remarkable number of patients with invasive fungal disease, resulting in significant morbidity worldwide. A major contributor to this failure is that while these drugs have high potency in vitro, we do not fully understand how they work inside infected hosts. Here, we used a transparent larval zebrafish model of Aspergillus fumigatus infection amenable to real-time imaging of invasive disease as an in vivo intermediate vertebrate model to investigate the efficacy and mechanism of the antifungal drug voriconazole. We found that the ability of voriconazole to protect against A. fumigatus infection depends on host innate immune cells and, specifically, on the presence of macrophages. While voriconazole inhibits fungal spore germination and growth in vitro, it does not do so in larval zebrafish. Instead, live imaging of whole, intact larvae over a multiday course of infection revealed that macrophages slow down initial fungal growth, allowing voriconazole time to target and kill A. fumigatus hyphae postgermination. These findings shed light on how antifungal drugs such as voriconazole may synergize with the immune response in living hosts.