RESUMEN
The canonical Wnt signalling pathway has been implicated in organogenesis and self-renewal of essentially all stem cell systems. In vivo reporter systems are crucial to assess the role of Wnt signalling in the biology and pathology of stem cell systems. We set out to develop a Turquoise (TQ) fluorescent protein based Wnt reporter. We used a CRISPR-Cas9 approach to insert a TQ fluorescent protein encoding gene into the general Wnt target gene Axin2, thereby establishing a Wnt reporter mouse similar to previously generated Wnt reporter mice but with the mTurquoise2 gene instead of E. coli-ß-galactosidase (LacZ). The use of mTurquoise2 is especially important in organ systems in which cells need to a be alive for further experimentation such as in vitro activation or transplantation studies. We here report successful generation of Axin2-TQ mice and show that cells from these mice faithfully respond to Wnt signals. High Wnt signals were detected in the intestinal crypts, a classical Wnt signalling site in vivo, and by flow cytometry in the thymus. These mice are an improved tool to further elucidate the role of Wnt signalling in vivo.
Asunto(s)
Proteína Axina/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Vía de Señalización Wnt , Animales , Proteína Axina/genética , Sistemas CRISPR-Cas , Marcación de Gen/métodos , Proteínas Fluorescentes Verdes/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Timo/citología , Timo/metabolismoAsunto(s)
Células Madre Pluripotentes Inducidas/fisiología , Timo/fisiología , Animales , Diferenciación Celular , Células Cultivadas , Factores de Transcripción Forkhead/genética , Humanos , Ratones , Ratones Noqueados , Ratones Desnudos , Morfogénesis , Técnicas de Cultivo de Órganos , Trasplante de Órganos , Timo/trasplante , Trasplante HeterólogoRESUMEN
The development of T lymphocytes in the thymus and their stem cell precursors in the bone marrow is controlled by Wnt signaling in strictly regulated, cell-type specific dosages. In this study, we investigated levels of canonical Wnt signaling during hematopoiesis and T cell development within the Axin2-mTurquoise2 reporter. We demonstrate active Wnt signaling in hematopoietic stem cells (HSCs) and early thymocytes, but also in more mature thymic subsets and peripheral T lymphocytes. Thymic epithelial cells displayed particularly high Wnt signaling, suggesting an interesting crosstalk between thymocytes and thymic epithelial cells (TECs). Additionally, reporter mice allowed us to investigate the loss of Axin2 function, demonstrating decreased HSC repopulation upon transplantation and the partial arrest of early thymocyte development in Axin2Tg/Tg full mutant mice. Mechanistically, loss of Axin2 leads to supraphysiological Wnt levels that disrupt HSC differentiation and thymocyte development.
Asunto(s)
Proteína Axina , Hematopoyesis , Linfopoyesis , Animales , Proteína Axina/genética , Proteína Axina/metabolismo , Diferenciación Celular , Hematopoyesis/genética , Células Madre Hematopoyéticas , Linfopoyesis/genética , Ratones , Vía de Señalización WntRESUMEN
RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development, from early CD7-CD5- to CD4+CD8+. The impaired differentiation was accompanied by an increase in CD7-CD56+CD33+ natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells, whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements, normalized T cell development, and corrected the NK cell-like phenotype. In conclusion, we succeeded in generating an iPSC-based RAG2-SCID model, which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/patología , Linfocitos T/inmunología , Animales , Antígenos CD/metabolismo , Diferenciación Celular , Linaje de la Célula , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Hematopoyesis , Humanos , Células Asesinas Naturales/inmunología , Ratones SCIDRESUMEN
The Wnt signaling pathway is an evolutionary conserved pathway that is involved in the development of almost every organ system in the body and provides self-renewal signals for most, if not all, adult stem cell systems. In recent years, this pathway has been studied by various research groups working on hematopoietic stem cells, resulting in contradicting conclusions. Here, we discuss and interpret the results of these studies and propose that Wnt dosage, the source of hematopoietic stem cells, and interactions with other pathways explain these disparate results.
Asunto(s)
Hematopoyesis , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Animales , Células Sanguíneas/citología , Células Sanguíneas/metabolismo , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Proteínas Wnt/genéticaRESUMEN
Kirsten RAS (KRAS) is a small GTPase that plays a key role in Ras/mitogen-activated protein kinase signaling; somatic mutations in KRAS are frequently found in many cancers. The most common KRAS mutations result in a constitutively active protein. Accurate detection of KRAS mutations is pivotal to the molecular diagnosis of cancer and may guide proper treatment selection. Here, we describe a two-step KRAS mutation screening protocol that combines whole-genome amplification (WGA), high-resolution melting analysis (HRM) as a prescreen method for mutation carrying samples, and direct Sanger sequencing of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue, from which limited amounts of DNA are available. We developed target-specific primers, thereby avoiding amplification of homologous KRAS sequences. The addition of herring sperm DNA facilitated WGA in DNA samples isolated from as few as 100 cells. KRAS mutation screening using high-resolution melting analysis on wgaDNA from formalin-fixed, paraffin-embedded tissue is highly sensitive and specific; additionally, this method is feasible for screening of clinical specimens, as illustrated by our analysis of pancreatic cancers. Furthermore, PCR on wgaDNA does not introduce genotypic changes, as opposed to unamplified genomic DNA. This method can, after validation, be applied to virtually any potentially mutated region in the genome.