RESUMEN
Neuronal development is a highly regulated process that is dependent on the correct coordination of cellular responses to extracellular cues. In response to semaphorin axon guidance proteins, the MICAL1 protein is stimulated to produce reactive oxygen species that oxidize actin on specific methionine residues, leading to filamentous actin depolymerization and consequent changes in neuronal growth cone dynamics. Crossing genetically modified mice homozygous for floxed Mical1 (Mical1fl/fl) alleles with transgenic mice expressing Cre recombinase under the control of a tyrosinase gene enhancer/promoter (Tyr::Cre) enabled conditional Mical1 deletion. Immunohistochemical analysis showed Mical1 expression in the cerebellum, which plays a prominent role in the coordination of motor movements, with reduced Mical1 expression in Mical1fl/fl mice co-expressing Tyr::Cre. Analysis of the gaits of mice running on a treadmill showed that both male and female Mical1fl/fl, Tyr::Cre mutant mice had significant alterations to their striding patterns relative to wild-type mice, although the specific aspects of their altered gaits differed between the sexes. Additional motor tests that involved movement on a rotating rod, descending a vertical pole, or crossing a balance beam did not show significant differences between the genotypes, suggesting that the effect of the Mical1fl/fl, Tyr::Cre genetic modifications was only manifested during specific highly coordinated movements that contribute to running. These findings indicate that there is a behavioral consequence in Mical1fl/fl, Tyr::Cre mutant mice that affects motor control as manifested by alterations in their gait.
Asunto(s)
Monofenol Monooxigenasa , Animales , Ratones , Femenino , Masculino , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Marcha/genética , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Ratones Transgénicos , Cerebelo/metabolismo , Carrera/fisiología , Ratones Endogámicos C57BLRESUMEN
Tracking individual cell movement during development is challenging, particularly in tissues subjected to major remodeling. Currently, most live imaging techniques in Xenopus are limited to tissue explants and/or to superficial cells. We describe here a protocol to track immature multiciliated cells (MCCs) moving within the inner epidermal layer of a whole embryo. In addition, we present a data processing protocol to uncouple the movements of individual cells from the coplanar drifts of the tissue in which they are embedded. For complete details on the use and execution of this protocol, please refer to Chuyen et al. (2021).
Asunto(s)
Movimiento Celular/fisiología , Rastreo Celular/métodos , Embrión no Mamífero/citología , Microscopía por Video/métodos , Animales , Procesamiento de Imagen Asistido por Computador , Proteínas Luminiscentes/metabolismo , Xenopus laevisRESUMEN
How global patterns emerge from individual cell behaviors is poorly understood. In the Xenopus embryonic epidermis, multiciliated cells (MCCs) are born in a random pattern within an inner mesenchymal layer and subsequently intercalate at regular intervals into an outer epithelial layer. Using video microscopy and mathematical modeling, we found that regular pattern emergence involves mutual repulsion among motile immature MCCs and affinity toward outer-layer intercellular junctions. Consistently, Arp2/3-mediated actin remodeling is required for MCC patterning. Mechanistically, we show that the Kit tyrosine kinase receptor, expressed in MCCs, and its ligand Scf, expressed in outer-layer cells, are both required for regular MCC distribution. Membrane-associated Scf behaves as a potent adhesive cue for MCCs, while its soluble form promotes their mutual repulsion. Finally, Kit expression is sufficient to confer order to a disordered heterologous cell population. This work reveals how a single signaling system can implement self-organized large-scale patterning.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Cilios/fisiología , Embrión no Mamífero/fisiología , Células Epidérmicas/fisiología , Uniones Intercelulares/fisiología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/metabolismo , Proteínas de Xenopus/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Actinas/metabolismo , Animales , Embrión no Mamífero/citología , Células Epidérmicas/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas Proto-Oncogénicas c-kit/genética , Transducción de Señal , Factor de Células Madre/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
In mammals, both sterile wounding and infection induce inflammation and activate the innate immune system, and the combination of both challenges may lead to severe health defects, revealing the importance of the balance between the intensity and resolution of the inflammatory response for the organism's fitness. Underlying mechanisms remain however elusive. Using Drosophila, we show that, upon infection with the entomopathogenic bacterium Pseudomonas entomophila (Pe), a sterile wounding induces a reduced resistance and increased host mortality. To identify the molecular mechanisms underlying the susceptibility of wounded flies to bacterial infection, we analyzed the very first steps of the process by comparing the transcriptome landscape of infected (simple hit flies, SH), wounded and infected (double hit flies, DH) and wounded (control) flies. We observed that overexpressed genes in DH flies compared to SH ones are significantly enriched in genes related to stress, including members of the JNK pathway. We demonstrated that the JNK pathway plays a central role in the DH phenotype by manipulating the Jra/dJun activity. Moreover, the CrebA/Creb3-like transcription factor (TF) and its targets were up-regulated in SH flies and we show that CrebA is required for mounting an appropriate immune response. Drosophila thus appears as a relevant model to investigate interactions between trauma and infection and allows to unravel key pathways involved.