RESUMEN
Genetic differences between pluripotent stem cell lines cause variable activity of extracellular signaling pathways, limiting reproducibility of directed differentiation protocols. Here we used human embryonic stem cells (hESCs) to interrogate how exogenous factors modulate endogenous signaling events during specification of foregut endoderm lineages. We find that transforming growth factor ß1 (TGF-ß1) activates a putative human OTX2/LHX1 gene regulatory network which promotes anterior fate by antagonizing endogenous Wnt signaling. In contrast to Porcupine inhibition, TGF-ß1 effects cannot be reversed by exogenous Wnt ligands, suggesting that induction of SHISA proteins and intracellular accumulation of Fzd receptors render TGF-ß1-treated cells refractory to Wnt signaling. Subsequently, TGF-ß1-mediated inhibition of BMP and Wnt signaling suppresses liver fate and promotes pancreas fate. Furthermore, combined TGF-ß1 treatment and Wnt inhibition during pancreatic specification reproducibly and robustly enhance INSULIN+ cell yield across hESC lines. This modification of widely used differentiation protocols will enhance pancreatic ß cell yield for cell-based therapeutic applications.
Asunto(s)
Proteínas Morfogenéticas Óseas , Diferenciación Celular , Endodermo , Células Madre Embrionarias Humanas , Vía de Señalización Wnt , Humanos , Endodermo/citología , Endodermo/metabolismo , Diferenciación Celular/efectos de los fármacos , Vía de Señalización Wnt/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Proteínas Morfogenéticas Óseas/metabolismo , Linaje de la Célula/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismo , Línea Celular , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
The Notch ligands Jag1 and Dll1 guide differentiation of multipotent pancreatic progenitor cells (MPCs) into unipotent pro-acinar cells (PACs) and bipotent duct/endocrine progenitors (BPs). Ligand-mediated trans-activation of Notch receptors induces oscillating expression of the transcription factor Hes1, while ligand-receptor cis-interaction indirectly represses Hes1 activation. Despite Dll1 and Jag1 both displaying cis- and trans-interactions, the two mutants have different phenotypes for reasons not fully understood. Here, we present a mathematical model that recapitulates the spatiotemporal differentiation of MPCs into PACs and BPs. The model correctly captures cell fate changes in Notch pathway knockout mice and small molecule inhibitor studies, and a requirement for oscillatory Hes1 expression to maintain the multipotent state. Crucially, the model entails cell-autonomous attenuation of Notch signaling by Jag1-mediated cis-inhibition in MPC differentiation. The model sheds light on the underlying mechanisms, suggesting that cis-interaction is crucial for exiting the multipotent state, while trans-interaction is required for adopting the bipotent fate.
Asunto(s)
Organogénesis , Receptores Notch , Animales , Ratones , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular/fisiología , Ligandos , Ratones Noqueados , Receptores Notch/genética , Receptores Notch/metabolismoRESUMEN
Notch signaling controls proliferation of multipotent pancreatic progenitor cells (MPCs) and their segregation into bipotent progenitors (BPs) and unipotent pro-acinar cells (PACs). Here, we showed that fast ultradian oscillations of the ligand Dll1 and the transcriptional effector Hes1 were crucial for MPC expansion, and changes in Hes1 oscillation parameters were associated with selective adoption of BP or PAC fate. Conversely, Jag1, a uniformly expressed ligand, restrained MPC growth. However, when its expression later segregated to PACs, Jag1 became critical for the specification of all but the most proximal BPs, and BPs were entirely lost in Jag1; Dll1 double mutants. Anatomically, ductal morphogenesis and organ architecture are minimally perturbed in Jag1 mutants until later stages, when ductal remodeling fails, and signs of acinar-to-ductal metaplasia appear. Our study thus uncovers that oscillating Notch activity in the developing pancreas, modulated by Jag1, is required to coordinate MPC growth and fate.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/metabolismo , Proteína Jagged-1/metabolismo , Páncreas/citología , Transducción de Señal , Animales , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Linaje de la Célula , Células Madre Embrionarias/citología , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteína Jagged-1/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Páncreas/embriología , Páncreas/metabolismo , Periodicidad , Receptores Notch/genética , Receptores Notch/metabolismo , Factor de Transcripción HES-1/genética , Factor de Transcripción HES-1/metabolismoRESUMEN
Expression of the basic helix-loop-helix factor Hairy and Enhancer of Split-1 (Hes1) is required for normal development of a number of tissues during embryonic development. Depending on context, Hes1 may act as a Notch signalling effector which promotes the undifferentiated and proliferative state of progenitor cells, but increasing evidence also points to Notch independent regulation of Hes1 expression. Here we use high resolution confocal scanning of EGFP in a novel BAC transgenic mouse reporter line, Tg(Hes1-EGFP)(1Hri), to analyse Hes1 expression from embryonic day 7.0 (e7.0). Our data recapitulates some previous observations on Hes1 expression and suggests new, hitherto unrecognised expression domains including expression in the definitive endoderm at early somite stages before gut tube closure and thus preceding organogenesis. This mouse line will be a valuable tool for studies addressing the role of Hes1 in a number of different research areas including organ specification, development and regeneration.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cromosomas Artificiales Bacterianos/genética , Endodermo/citología , Endodermo/embriología , Endodermo/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Transgénicos , Organogénesis/genética , Somitos/citología , Somitos/embriología , Somitos/metabolismo , Factor de Transcripción HES-1RESUMEN
Pancreas morphogenesis and cell differentiation are highly conserved among vertebrates during fetal development. The pancreas develops through simple budlike structures on the primitive gut tube to a highly branched organ containing many specialized cell types. This review presents an overview of key molecular components and important signaling sources illustrated by an extensive three-dimensional (3D) imaging of the developing mouse pancreas at single cell resolution. The 3D documentation covers the time window between embryonic days 8.5 and 14.5 in which all the pancreatic cell types become specified and therefore includes gene expression patterns of pancreatic endocrine hormones, exocrine gene products, and essential transcription factors. The 3D perspective provides valuable insight into how a complex organ like the pancreas is formed and a perception of ventral and dorsal pancreatic growth that is otherwise difficult to uncover. We further discuss how this global analysis of the developing pancreas confirms and extends previous studies, and we envisage that this type of analysis can be instrumental for evaluating mutant phenotypes in the future.