RESUMO
Transcription factors (TFs) regulate cell fates, and their expression must be tightly regulated. Autoregulation is assumed to regulate many TFs' own expression to control cell fates. Here, we manipulate and quantify the (auto)regulation of PU.1, a TF controlling hematopoietic stem and progenitor cells (HSPCs), and correlate it to their future fates. We generate transgenic mice allowing both inducible activation of PU.1 and noninvasive quantification of endogenous PU.1 protein expression. The quantified HSPC PU.1 dynamics show that PU.1 up-regulation occurs as a consequence of hematopoietic differentiation independently of direct fast autoregulation. In contrast, inflammatory signaling induces fast PU.1 up-regulation, which does not require PU.1 expression or its binding to its own autoregulatory enhancer. However, the increased PU.1 levels induced by inflammatory signaling cannot be sustained via autoregulation after removal of the signaling stimulus. We conclude that PU.1 overexpression induces HSC differentiation before PU.1 up-regulation, only later generating cell types with intrinsically higher PU.1.
Assuntos
Diferenciação Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Homeostase/genética , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Regulação para Cima/genética , Animais , Células Cultivadas , Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Imagem com Lapso de Tempo/métodos , Transativadores/metabolismoRESUMO
The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors1 that delaminate and settle in the subventricular zone in enlarged brain regions2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination.
Assuntos
Centrossomo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ventrículos Laterais/citologia , Ventrículos Laterais/embriologia , Microtúbulos/metabolismo , Neurogênese , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Movimento Celular , Células Cultivadas , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Humanos , Junções Intercelulares/metabolismo , Interfase , Ventrículos Laterais/anatomia & histologia , Glândulas Mamárias Animais/citologia , Camundongos , Tamanho do Órgão , Organoides/citologiaRESUMO
The serotonin neurotransmitter system is widespread in the brain and implicated in modulation of neuronal responses to other neurotransmitters. Among 14 serotonin receptor subtypes, 5-HT2cR plays a pivotal role in controlling neuronal network excitability. Serotonergic activity conveyed through receptor 5-HT2cR is regulated post-transcriptionally via two mechanisms, alternative splicing and A-to-I RNA editing. Brain-specific small nucleolar RNA SNORD115 harbours a phylogenetically conserved 18-nucleotide antisense element with perfect complementarity to the region of 5ht2c primary transcript that undergoes post-transcriptional changes. Previous 5ht2c minigene studies have implicated SNORD115 in fine-tuning of both post-transcriptional events. We monitored post-transcriptional changes of endogenous 5ht2c transcripts during neuronal differentiation. Both SNORD115 and 5ht2c were upregulated upon neuronal commitment. We detected increased 5ht2c alternative exon Vb inclusion already at the stage of neuronal progenitors, and more extensive A-to-I editing of non-targeted sites A and B compared to adjacent adenosines at sites E, C and D throughout differentiation. As the extent of editing is known to positively correlate with exon Vb usage while it reduces receptor functionality, our data support the model where SNORD115 directly promotes alternative exon inclusion without the requirement for conversion of key adenosines to inosines, thereby favouring production of full-length receptor isoforms with higher potency.