RESUMEN
Neuronal development requires a complex choreography of transcriptional decisions to obtain specific cellular identities. Realizing the ultimate goal of identifying genome-wide signatures that define and drive specific neuronal fates has been hampered by enormous complexity in both time and space during development. Here, we have paired high-throughput purification of pyramidal neuron subclasses with deep profiling of spatiotemporal transcriptional dynamics during corticogenesis to resolve lineage choice decisions. We identified numerous features ranging from spatial and temporal usage of alternative mRNA isoforms and promoters to a host of mRNA genes modulated during fate specification. Notably, we uncovered numerous long noncoding RNAs with restricted temporal and cell-type-specific expression. To facilitate future exploration, we provide an interactive online database to enable multidimensional data mining and dissemination. This multifaceted study generates a powerful resource and informs understanding of the transcriptional regulation underlying pyramidal neuron diversity in the neocortex.
Asunto(s)
Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica/genética , Neocórtex/metabolismo , Células Piramidales/metabolismo , ARN Largo no Codificante/genética , ARN Mensajero/genética , Transcriptoma , Animales , Secuencia de Bases , Cuerpo Calloso/citología , Citometría de Flujo , Perfilación de la Expresión Génica , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Ratones , Datos de Secuencia Molecular , Neuronas Motoras , Neurogénesis/genética , Neuronas/metabolismo , Tractos Piramidales/citología , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
We report here the first attempt to produce and use whole acellular (AC) lung as a matrix to support development of engineered lung tissue from murine embryonic stem cells (mESCs). We compared the influence of AC lung, Gelfoam, Matrigel, and a collagen I hydrogel matrix on the mESC attachment, differentiation, and subsequent formation of complex tissue. We found that AC lung allowed for better retention of cells with more differentiation of mESCs into epithelial and endothelial lineages. In constructs produced on whole AC lung, we saw indications of organization of differentiating ESC into three-dimensional structures reminiscent of complex tissues. We also saw expression of thyroid transcription factor-1, an immature lung epithelial cell marker; pro-surfactant protein C, a type II pneumocyte marker; PECAM-1/CD31, an endothelial cell marker; cytokeratin 18; alpha-actin, a smooth muscle marker; CD140a or platelet-derived growth factor receptor-alpha; and Clara cell protein 10. There was also evidence of site-specific differentiation in the trachea with the formation of sheets of cytokeratin-positive cells and Clara cell protein 10-expressing Clara cells. Our findings support the utility of AC lung as a matrix for engineering lung tissue and highlight the critical role played by matrix or scaffold-associated cues in guiding ESC differentiation toward lung-specific lineages.