RESUMEN
Wnt signalling drives many processes in development, homeostasis and disease; however, the role and mechanism of individual ligand-receptor (Wnt-Frizzled (Fzd)) interactions in specific biological processes remain poorly understood. Wnt9a is specifically required for the amplification of blood progenitor cells during development. Using genetic studies in zebrafish and human embryonic stem cells, paired with in vitro cell biology and biochemistry, we determined that Wnt9a signals specifically through Fzd9b to elicit ß-catenin-dependent Wnt signalling that regulates haematopoietic stem and progenitor cell emergence. We demonstrate that the epidermal growth factor receptor (EGFR) is required as a cofactor for Wnt9a-Fzd9b signalling. EGFR-mediated phosphorylation of one tyrosine residue on the Fzd9b intracellular tail in response to Wnt9a promotes internalization of the Wnt9a-Fzd9b-LRP signalosome and subsequent signal transduction. These findings provide mechanistic insights for specific Wnt-Fzd signals, which will be crucial for specific therapeutic targeting and regenerative medicine.
Asunto(s)
Células Madre Hematopoyéticas/citología , Receptores de Neurotransmisores/genética , Proteínas Wnt/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Receptores ErbB/genética , Humanos , Fosforilación , Vía de Señalización Wnt , Pez Cebra/crecimiento & desarrollo , beta Catenina/genéticaRESUMEN
The WNT/ß-catenin signaling pathway is a prominent player in many developmental processes, including gastrulation, anterior-posterior axis specification, organ and tissue development, and homeostasis. Here, we use human pluripotent stem cells (hPSCs) to study the dynamics of the transcriptional response to exogenous activation of the WNT pathway. We describe a mechanism involving the WNT target gene SP5 that leads to termination of the transcriptional program initiated by WNT signaling. Integration of gene expression profiles of wild-type and SP5 mutant cells with genome-wide SP5 binding events reveals that SP5 acts to diminish expression of genes previously activated by the WNT pathway. Furthermore, we show that activation of SP5 by WNT signaling is most robust in cells with developmental potential, such as stem cells. These findings indicate a mechanism by which the developmental WNT signaling pathway reins in expression of transcriptional programs.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismo , Proteína Wnt3A/metabolismo , Línea Celular , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes/citología , Factores de Transcripción/genética , Vía de Señalización Wnt , Proteína Wnt3A/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has been applied to edit genomes in a wide variety of model systems. Although this process can be quite efficient, editing at precise locations in the genome remains difficult without a suitable single guide RNA (sgRNA). We have developed a method for screening sgRNA function in vitro, using reagents that most zebrafish laboratories are already using. The results from our in vitro assay correlate with function in vivo in every sgRNA that we have examined so far. When combined with endonucleases with alternative protospacer adjacent motif site specificities and alternative sgRNAs, this method will streamline genome editing at almost any locus.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , ARN Guía de Kinetoplastida/genética , Pez Cebra/genética , Animales , Endonucleasas/metabolismo , Marcación de Gen , Técnicas In Vitro , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/genéticaRESUMEN
All mature blood cell types in the adult animal arise from hematopoietic stem and progenitor cells (HSPCs). However, the developmental cues regulating HSPC ontogeny are incompletely understood. In particular, the details surrounding a requirement for Wnt/ß-catenin signaling in the development of mature HSPCs are controversial and difficult to consolidate. Using zebrafish, we demonstrate that Wnt signaling is required to direct an amplification of HSPCs in the aorta. Wnt9a is specifically required for this process and cannot be replaced by Wnt9b or Wnt3a. This proliferative event occurs independently of initial HSPC fate specification, and the Wnt9a input is required prior to aorta formation. HSPC arterial amplification occurs prior to seeding of secondary hematopoietic tissues and proceeds, in part, through the cell cycle regulator myca (c-myc). Our results support a general paradigm, in which early signaling events, including Wnt, direct later HSPC developmental processes.
