RESUMO
Although kidneys of equal size can vary 10-fold in nephron number at birth, discovering what regulates such variation has been hampered by a lack of quantitative parameters defining kidney development. Here we report a comprehensive, quantitative, multiscale analysis of mammalian kidney development in which we measure changes in cell number, compartment volumes, and cellular dynamics across the entirety of organogenesis, focusing on two key nephrogenic progenitor populations: the ureteric epithelium and the cap mesenchyme. In doing so, we describe a discontinuous developmental program governed by dynamic changes in interactions between these key cellular populations occurring within a previously unappreciated structurally stereotypic organ architecture. We also illustrate the application of this approach to the detection of a subtle mutant phenotype. This baseline program of kidney morphogenesis provides a framework for assessing genetic and environmental developmental perturbation and will serve as a gold standard for the analysis of other organs.
Assuntos
Rim/embriologia , Néfrons/embriologia , Ureter/embriologia , Urotélio/embriologia , Animais , Contagem de Células , Células-Tronco Embrionárias/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Rim/citologia , Rim/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Néfrons/citologia , Néfrons/fisiologia , Fenótipo , Gravidez , Ureter/citologia , Ureter/fisiologia , Urotélio/citologia , Urotélio/fisiologiaRESUMO
Rab GTPases including Rab27a, Rab38 and Rab32 function in melanosome maturation or trafficking in melanocytes. A screen to identify additional Rabs involved in these processes revealed the localization of GFP-Rab17 on recycling endosomes (REs) and melanosomes in melanocytic cells. Rab17 mRNA expression is regulated by microphthalmia transcription factor (MITF), a characteristic of known pigmentation genes. Rab17 siRNA knockdown in melanoma cells quantitatively increased melanosome concentration at the cell periphery. Rab17 knockdown did not inhibit melanosome maturation nor movement, but it caused accumulation of melanin inside cells. Double knockdown of Rab17 and Rab27a indicated that Rab17 acts on melanosomes downstream of Rab27a. Filopodia are known to play a role in melanosome transfer, and in Rab17 knockdown cells filopodia formation was inhibited. Furthermore, we show that stimulation of melanoma cells with α-melanocyte-stimulating hormone induces filopodia formation, supporting a role for filopodia in melanosome release. Cell stimulation also caused redistribution of REs to the periphery, and knockdown of additional RE-associated Rabs 11a and 11b produced a similar accumulation of melanosomes and melanin to that seen after loss of Rab17. Our findings reveal new functions for RE and Rab17 in pigmentation through a distal step in the process of melanosome release via filopodia.
Assuntos
Endossomos/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Melanossomas/metabolismo , Pseudópodes/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Melaninas/metabolismo , Melanócitos/efeitos dos fármacos , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Pseudópodes/ultraestrutura , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , alfa-MSH/farmacologia , Proteínas rab de Ligação ao GTP/genéticaRESUMO
UNLABELLED: New high-resolution approaches for mapping ultrastructure of cells in 3D are leading to unprecedented quantities of spatial data. Here we present Illoura, a software tool for the integrated management, analysis and visualization of these data within a semantic context, and illustrate its capability by analysis of spatial relationships in mammalian beta cells. AVAILABILITY: http://www.visiblecell.com/illoura. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
