RESUMEN
BACKGROUND: To understand the basis of nervous system development, we must learn how multipotent progenitors generate diverse neuronal and glial lineages. We addressed this issue in the zebrafish enteric nervous system (ENS), a complex neuronal and glial network that regulates essential intestinal functions. Little is currently known about how ENS progenitor subpopulations generate enteric neuronal and glial diversity. RESULTS: We identified temporally and spatially dependent progenitor subpopulations based on coexpression of three genes essential for normal ENS development: phox2bb, sox10, and ret. Our data suggest that combinatorial expression of these genes delineates three major ENS progenitor subpopulations, (1) phox2bb + /ret- /sox10-, (2) phox2bb + /ret + /sox10-, and (3) phox2bb + /ret + /sox10+, that reflect temporal progression of progenitor maturation during migration. We also found that differentiating zebrafish neurons maintain phox2bb and ret expression, and lose sox10 expression. CONCLUSIONS: Our data show that zebrafish enteric progenitors constitute a heterogeneous population at both early and late stages of ENS development and suggest that marker gene expression is indicative of a progenitor's fate. We propose that a progenitor's expression profile reveals its developmental state: "younger" wave front progenitors express all three genes, whereas more mature progenitors behind the wave front selectively lose sox10 and/or ret expression, which may indicate developmental restriction. Developmental Dynamics 245:1081-1096, 2016. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Sistema Nervioso Entérico/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Sistema Nervioso Entérico/citología , Sistema Nervioso Entérico/embriología , Regulación del Desarrollo de la Expresión Génica , Cresta Neural/citología , Cresta Neural/enzimología , Cresta Neural/metabolismo , ARN Mensajero/genética , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
The sorting of signaling receptors within the endocytic system is important for appropriate cellular responses. After activation, receptors are trafficked to early endosomes and either recycled or sorted to lysosomes and degraded. Most receptors trafficked to lysosomes are modified with ubiquitin and recruited into an endosomal subdomain enriched in hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a ubiquitin-binding component of the endosomal-sorting complex required for transport (ESCRT) machinery, and then sorted into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs)/lysosomes. However, not all receptors use ubiquitin or the canonical ESCRT machinery to sort to MVBs/lysosomes. This is exemplified by protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, which sorts to lysosomes independent of ubiquitination and HRS. We recently showed that the adaptor protein ALIX binds to PAR1, recruits ESCRT-III, and mediates receptor sorting to ILVs of MVBs. However, the mechanism that initiates PAR1 sorting at the early endosome is not known. We now report that the adaptor protein complex-3 (AP-3) regulates PAR1 ubiquitin-independent sorting to MVBs through an ALIX-dependent pathway. AP-3 binds to a PAR1 cytoplasmic tail-localized tyrosine-based motif and mediates PAR1 lysosomal degradation independent of ubiquitination. Moreover, AP-3 facilitates PAR1 interaction with ALIX, suggesting that AP-3 functions before PAR1 engagement of ALIX and MVB/lysosomal sorting.
Asunto(s)
Complejo 3 de Proteína Adaptadora/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Lisosomas/metabolismo , Cuerpos Multivesiculares/metabolismo , Receptor PAR-1/metabolismo , Complejo 3 de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/genética , Subunidades delta de Complexo de Proteína Adaptadora/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Ciclo Celular/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Células HeLa , Humanos , Immunoblotting , Microscopía Confocal , Modelos Biológicos , Unión Proteica , Transporte de Proteínas , Interferencia de ARN , Receptor PAR-1/genética , Transducción de Señal , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
The sorting of signaling receptors to lysosomes is an essential regulatory process in mammalian cells. During degradation, receptors are modified with ubiquitin and sorted by endosomal sorting complex required for transport (ESCRT)-0, -I, -II, and -III complexes into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). However, it remains unclear whether a single universal mechanism mediates MVB sorting of all receptors. We previously showed that protease-activated receptor 1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is internalized after activation and sorted to lysosomes independent of ubiquitination and the ubiquitin-binding ESCRT components hepatocyte growth factor-regulated tyrosine kinase substrate and Tsg101. In this paper, we report that PAR1 sorted to ILVs of MVBs through an ESCRT-III-dependent pathway independent of ubiquitination. We further demonstrate that ALIX, a charged MVB protein 4-ESCRT-III interacting protein, bound to a YPX(3)L motif of PAR1 via its central V domain to mediate lysosomal degradation. This study reveals a novel MVB/lysosomal sorting pathway for signaling receptors that bypasses the requirement for ubiquitination and ubiquitin-binding ESCRTs and may be applicable to a subset of GPCRs containing YPX(n)L motifs.
