RESUMEN
Neuromesodermal (NM) stem cells generate neural and paraxial presomitic mesoderm (PSM) cells, which are the respective progenitors of the spinal cord and musculoskeleton of the trunk and tail. The Wnt-regulated basic helix-loop-helix (bHLH) transcription factor mesogenin 1 (Msgn1) has been implicated as a cooperative regulator working in concert with T-box genes to control PSM formation in zebrafish, although the mechanism is unknown. We show here that, in mice, Msgn1 alone controls PSM differentiation by directly activating the transcriptional programs that define PSM identity, epithelial-mesenchymal transition, motility and segmentation. Forced expression of Msgn1 in NM stem cells in vivo reduced the contribution of their progeny to the neural tube, and dramatically expanded the unsegmented mesenchymal PSM while blocking somitogenesis and notochord differentiation. Expression of Msgn1 was sufficient to partially rescue PSM differentiation in Wnt3a(-/-) embryos, demonstrating that Msgn1 functions downstream of Wnt3a as the master regulator of PSM differentiation. Our data provide new insights into how cell fate decisions are imposed by the expression of a single transcriptional regulator.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Mesodermo/embriología , Músculo Esquelético/embriología , Sistema Nervioso/embriología , Animales , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Inmunohistoquímica , Hibridación in Situ , Luciferasas , Mesodermo/citología , Ratones , Ratones Noqueados , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Wnt3A/genéticaRESUMEN
Neuromesodermal (NM) stem cells reside in the primitive streak (PS) of gastrulating vertebrate embryos and generate precursors of the spinal cord and musculoskeletal system. Although Wnt3a/ß-catenin signaling is crucial for NM stem cell maintenance and differentiation, few key transcriptional effectors have been identified. Through a concerted transcriptional profiling and genetic approach we have determined that two Zn(2+)-finger transcription factors, Sp5 and Sp8, are regulated by Wnt3a in the PS, and are essential for neural and musculoskeletal patterning. These results identify Sp5 and Sp8 as pivotal downstream effectors of Wnt3a, and suggest that they are essential for the self-renewal and differentiation of NM stem cells.
Asunto(s)
Proteínas de Unión al ADN/genética , Mesodermo/metabolismo , Células-Madre Neurales/metabolismo , Línea Primitiva/metabolismo , Factores de Transcripción/genética , Proteína Wnt3A/genética , beta Catenina/genética , Animales , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Gastrulación/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Mesodermo/citología , Ratones , Ratones Transgénicos , Mutación , Células-Madre Neurales/citología , Línea Primitiva/citología , Factores de Transcripción/metabolismo , Transcripción Genética , Proteína Wnt3A/metabolismo , beta Catenina/metabolismoRESUMEN
Segmentation is an organizing principle of body plans. The segmentation clock, a molecular oscillator best illustrated by the cyclic expression of Notch signalling genes, controls the periodic cleavage of somites from unsegmented presomitic mesoderm during vertebrate segmentation. Wnt3a controls the spatiotemporal expression of cyclic Notch genes; however, the underlying mechanisms remain obscure. Here we show by transcriptional profiling of Wnt3a (-/-) embryos that the bHLH transcription factor, Mesogenin1 (Msgn1), is a direct target gene of Wnt3a. To identify Msgn1 targets, we conducted genome-wide studies of Msgn1 activity in embryonic stem cells. We show that Msgn1 is a major transcriptional activator of a Notch signalling program and synergizes with Notch to trigger clock gene expression. Msgn1 also indirectly regulates cyclic genes in the Fgf and Wnt pathways. Thus, Msgn1 is a central component of a transcriptional cascade that translates a spatial Wnt3a gradient into a temporal pattern of clock gene expression.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Relojes Biológicos/fisiología , Tipificación del Cuerpo/fisiología , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Animales , Diferenciación Celular , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Células Madre Embrionarias , Perfilación de la Expresión Génica , Hibridación in Situ , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/metabolismoRESUMEN
Somitogenesis is thought to be controlled by a segmentation clock, which consists of molecular oscillators in the Wnt3a, Fgf8 and Notch pathways. Using conditional alleles of Ctnnb1 (beta-catenin), we show that the canonical Wnt3a/beta-catenin pathway is necessary for molecular oscillations in all three signaling pathways but does not function as an integral component of the oscillator. Small, irregular somites persist in abnormally posterior locations in the absence of beta-catenin and cycling clock gene expression. Conversely, Notch pathway genes continue to oscillate in the presence of stabilized beta-catenin but boundary formation is delayed and anteriorized. Together, these results suggest that the Wnt3a/beta-catenin pathway is permissive but not instructive for oscillating clock genes and that it controls the anterior-posterior positioning of boundary formation in the presomitic mesoderm (PSM). The Wnt3a/beta-catenin pathway does so by regulating the activation of the segment boundary determination genes Mesp2 and Ripply2 in the PSM through the activation of the Notch ligand Dll1 and the mesodermal transcription factors T and Tbx6. Spatial restriction of Ripply2 to the anterior PSM is ensured by the Wnt3a/beta-catenin-mediated repression of Ripply2 in posterior PSM. Thus, Wnt3a regulates somitogenesis by activating a network of interacting target genes that promote mesodermal fates, activate the segmentation clock, and position boundary determination genes in the anterior PSM.
Asunto(s)
Mesodermo/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas de Dominio T Box , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3A , beta Catenina/genéticaRESUMEN
Somites are blocks of mesoderm that form when segment boundaries are periodically generated in the anterior presomitic mesoderm (PSM). Periodicity is thought to be driven by an oscillating Notch-centered segmentation clock, whereas boundaries are spatially positioned by the secreted signaling molecules Wnt3a and Fgf8. We identified the putative transcriptional corepressor Ripply2 as a differentially expressed gene in wild-type and Wnt3a(-/-) embryos. Here, we show that Ripply2 is expressed in the anterior PSM and that it indeed lies downstream of Wnt3a. Dynamic Ripply2 expression in prospective somites S0 and S-I overlaps with the rostral expression of cycling genes in the Notch pathway, suggesting that Ripply2 may be controlled by the segmentation clock. Continued expression of Ripply2 in embryos lacking Hes7, a molecular oscillator in the Notch clock, indicates that Hes7 is not a major regulator of Ripply2. Our data are consistent with Ripply2 functioning as a segment boundary determination gene during mammalian embryogenesis. Developmental
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Mesodermo/embriología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Somitos/embriología , Proteínas Wnt/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Desarrollo Embrionario , Proteínas de la Matriz Extracelular/metabolismo , Glicosiltransferasas/metabolismo , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Mesodermo/metabolismo , Ratones , Receptores Notch/metabolismo , Somitos/metabolismo , Proteína Wnt3 , Proteína Wnt3ARESUMEN
During vertebrate embryogenesis, secreted Wnt molecules regulate cell fates by signaling through the canonical pathway mediated by beta-catenin, and regulate planar cell polarity (PCP) and convergent extension movements through alternative pathways. The phosphoprotein Dishevelled (Dsh/Dvl) is a Wnt signal transducer thought to function in all Wnt signaling pathways. A recently identified member of the Formin family, Daam (Dishevelled--associated activator of morphogenesis), regulates the morphogenetic movements of vertebrate gastrulation in a Wnt-dependent manner through direct interactions with Dsh/Dvl and RhoA. We describe two mouse Daam cDNAs, mDaam1 and mDaam2, which encode proteins characterized by highly conserved formin homology domains and which are expressed in complementary patterns during mouse development. Cross-species comparisons indicate that the expression domains of Xenopus Daam1 (XDaam1) mirror mDaam1 expression. Our results demonstrate that Daams are expressed in tissues known to require Wnts and are consistent with Daams being effectors of Wnt signaling during vertebrate development.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Microfilamentos/genética , Transducción de Señal/fisiología , Proteínas de Unión al GTP rho/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Extremidades/embriología , Extremidades/fisiología , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Mucosa Gástrica/metabolismo , Hibridación in Situ , Hígado/embriología , Hígado/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Ratones , Proteínas de Microfilamentos/biosíntesis , Proteínas de Microfilamentos/aislamiento & purificación , Médula Espinal/embriología , Médula Espinal/metabolismo , Estómago/embriología , Proteínas Wnt , Xenopus , Proteínas de Xenopus/biosíntesis , Proteínas de Xenopus/genética , Proteínas de Xenopus/aislamiento & purificación , Proteínas de Unión al GTP rho/biosíntesis , Proteínas de Unión al GTP rho/aislamiento & purificaciónRESUMEN
Acute exposure of mouse embryos to ethanol during stages of hindbrain segmentation results in excessive cell death in specific cell populations. This study details the ethanol-induced cell loss and defines the subsequent effects of this early insult on rhombomere and cranial nerve development. Ethanol at a teratogenic dosage (2.9 g/kg) or a comparable volume of vehicle was administered in each of two intraperitoneal injections to pregnant C57BL/6J mice on gestational day (GD) 8, 8 h, and GD 8, 12 h (defined hereafter as GD 8.5). Ethanol-exposed GD 9 embryos, visualized in three dimensions using laser scanning confocal microscopy of LysoTracker Red fluorescence or Nile blue sulphate vital staining, displayed excessive apoptosis in the rostral hindbrain, specifically within rhombomeres 1-3, as well as in cranial neural crest cells and ectodermal placodes. Comparably treated embryos examined on GD 10.5-11 illustrated a disproportionate reduction in the length of the rostral hindbrain. Examination of plastic histological sections of GD 9 embryos and via scanning electron microscopy on GD 10 revealed deficiencies in the hindbrain, with a phenotype including abnormal rhombomere segmentation and an extremely small fourth ventricular roofplate. Whole-mount antineurofilament immunohistochemistry on GD 10.5 and GD 11 illustrated a variety of cranial nerve abnormalities ranging from fused or absent ganglia to ectopic or disorganized fibers. In addition, a delay in the development of the glossopharyngeal (IX) nerve/ganglia complex was observed. These hindbrain and cranial nerve abnormalities are discussed in the context of the genesis of human alcohol-related birth defects and neurodevelopmental disorder.