RESUMEN
Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. Here, we introduce morphogen-age measurements as a novel approach to test models of morphogen gradient formation. Using a tandem fluorescent timer as a protein age sensor, we find a gradient of increasing age of Bicoid along the anterior-posterior axis in the early Drosophila embryo. Quantitative analysis of the protein age distribution across the embryo reveals that the synthesis-diffusion-degradation model is the most likely model underlying Bicoid gradient formation, and rules out other hypotheses for gradient formation. Moreover, we show that the timer can detect transitions in the dynamics associated with syncytial cellularization. Our results provide new insight into Bicoid gradient formation and demonstrate how morphogen-age information can complement knowledge about movement, abundance, and distribution, which should be widely applicable to other systems.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrión no Mamífero/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Imagen Óptica/métodos , Transactivadores/genética , Animales , Tipificación del Cuerpo/genética , Proteínas de Drosophila/biosíntesis , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/diagnóstico por imagen , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/biosíntesis , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Estabilidad Proteica , Transporte de Proteínas , Proteolisis , Transducción de Señal , Transactivadores/biosíntesis , Proteína Fluorescente RojaRESUMEN
Control of cell proliferation is a fundamental aspect of tissue formation in development and regeneration. Cells experience various spatial and mechanical constraints depending on their environmental context in the body, but we do not fully understand if and how such constraints influence cell cycle progression and thereby proliferation patterns in tissues. Here, we study the impact of mechanical manipulations on the cell cycle of individual cells within a mammalian model epithelium. By monitoring the response to experimentally applied forces, we find a checkpoint at the G1-S boundary that, in response to spatial constraints, controls cell cycle progression. This checkpoint prevents cells from entering S phase if the available space remains below a characteristic threshold because of crowding. Stretching the tissue results in fast cell cycle reactivation, whereas compression rapidly leads to cell cycle arrest. Our kinetic analysis of this response shows that cells have no memory of past constraints and allows us to formulate a biophysical model that predicts tissue growth in response to changes in spatial constraints in the environment. This characteristic biomechanical cell cycle response likely serves as a fundamental control mechanism to maintain tissue integrity and to ensure control of tissue growth during development and regeneration.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Proliferación Celular , Inhibición de Contacto/fisiología , Modelos Biológicos , Animales , Fenómenos Biomecánicos , Biofisica , Perros , Cinética , Células de Riñón Canino Madin DarbyRESUMEN
Serotonergic neurotransmission plays an important role during neural development. Serotonergic dysfunction is observed in various psychiatric disorders and many psychoactive drugs target proteins on serotonergic neurons. Serotonergic neurons are located in the raphé nuclei and densely innervate the whole brain. The low number and the intricate accessibility of these neurons do not allow to culture them and therefore to date it was impossible to study drug-target interactions on bona fide serotonergic neurons. In order to circumvent such problems we have developed a protocol that allows the rapid and efficient generation of serotonergic neurons from mouse embryonic stem cells. Neuronal precursors were obtained by neuronal stem sphere formation in floating culture in the presence of various mitogens. Differentiation into neurons was induced by withdrawal of the mitogens. About 90% of the resulting neurons exhibited a serotonergic phenotype as judged by immunostaining against serotonin, its synthesising enzyme tryptophan hydroxylase 2, the serotonin transporter as well as 5-HT1(A) and 5-HT1(B) autoreceptors. In addition, we found expression of the vesicular monoamine transporter vMAT2 and the presynaptic protein Bassoon, which is involved in organizing the assembly of the presynaptic active zone. Depolarisation-induced calcium influx was visualised by Fluo-4, and accompanying exocytotic events by FM dye staining. Proteins involved in 5-HT release and re-uptake as well as depolarisation evoked exocytosis were evenly co-distributed on neurites and cell bodies suggesting that ES cell-derived serotonergic neurons also exhibit somatodendritic release comparable to serotonergic neurons in the raphé nuclei.