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1.
Development ; 146(24)2019 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-31822478

RESUMEN

A Wnt signaling network governs early anterior-posterior (AP) specification and patterning of the deuterostome sea urchin embryo. We have previously shown that non-canonical Fzl1/2/7 signaling antagonizes the progressive posterior-to-anterior downregulation of the anterior neuroectoderm (ANE) gene regulatory network (GRN) by canonical Wnt/ß-catenin and non-canonical Wnt1/Wnt8-Fzl5/8-JNK signaling. This study focuses on the non-canonical function of the Wnt16 ligand during early AP specification and patterning. Maternally supplied wnt16 is expressed ubiquitously during cleavage and zygotic wnt16 expression is concentrated in the endoderm/mesoderm beginning at mid-blastula stage. Wnt16 antagonizes the ANE restriction mechanism and this activity depends on a functional Fzl1/2/7 receptor. Our results also show that zygotic wnt16 expression depends on both Fzl5/8 and Wnt/ß-catenin signaling. Furthermore, Wnt16 is necessary for the activation and/or maintenance of key regulatory endoderm/mesoderm genes and is essential for gastrulation. Together, our data show that Wnt16 has two functions during early AP specification and patterning: (1) an initial role activating the Fzl1/2/7 pathway that antagonizes the ANE restriction mechanism; and (2) a subsequent function in activating key endoderm GRN factors and the morphogenetic movements of gastrulation.


Asunto(s)
Tipificación del Cuerpo/genética , Morfogénesis/genética , Erizos de Mar , Proteínas Wnt/fisiología , Animales , Embrión no Mamífero , Receptores Frizzled/genética , Receptores Frizzled/fisiología , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo/embriología , Mesodermo/metabolismo , Placa Neural/embriología , Placa Neural/metabolismo , Erizos de Mar/embriología , Erizos de Mar/genética , Proteínas Wnt/genética , Vía de Señalización Wnt/fisiología
2.
Methods Cell Biol ; 151: 177-196, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30948007

RESUMEN

A critical process in embryonic development is the activation and spatial localization of mRNAs to specific cells and territories of the embryo. Revealing the spatial distribution of mRNAs and how it changes during development is a vital piece of information that aids in understanding the signaling and regulatory genes driving specific gene regulatory networks. In the laboratory, a cost-efficient, reliable method to determine the spatial distribution of mRNAs in embryos is in situ hybridization. This sensitive and straightforward method employs exogenous antisense RNA probes to find specific and complementary sequences in fixed embryos. Antigenic moieties conjugated to the ribonucleotides incorporated in the probe cross-react with antibodies, and numerous staining methods can be subsequently employed to reveal the spatial distribution of the targeted mRNA. The quality of the data produced by this method is equivalent to the experience of the researcher, and thus a thorough understanding of the numerous steps comprising this method is important for obtaining high quality data. Here we compile and summarize several protocols that have been employed chiefly on five sea urchin species in numerous laboratories around the world. Whereas the protocols can vary for the different species, the overarching steps are similar and can be readily mastered. When properly and carefully undertaken, in situ hybridization is a powerful tool providing unambiguous data for which there currently is no comparable substitute and will continue to be an important method in the era of big data and beyond.


Asunto(s)
Desarrollo Embrionario/genética , Redes Reguladoras de Genes/genética , Hibridación in Situ/métodos , Erizos de Mar/genética , Animales , Embrión no Mamífero/citología , Regulación del Desarrollo de la Expresión Génica/genética , Larva/genética , Larva/crecimiento & desarrollo , ARN Mensajero/genética , Erizos de Mar/crecimiento & desarrollo
3.
Evodevo ; 9: 1, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29387332

RESUMEN

The anterior neuroectoderm (ANE) in many deuterostome embryos (echinoderms, hemichordates, urochordates, cephalochordates, and vertebrates) is progressively restricted along the anterior-posterior axis to a domain around the anterior pole. In the sea urchin embryo, three integrated Wnt signaling branches (Wnt/ß-catenin, Wnt/JNK, and Wnt/PKC) govern this progressive restriction process, which begins around the 32- to 60-cell stage and terminates by the early gastrula stage. We previously have established that several secreted Wnt modulators of the Dickkopf and secreted Frizzled-related protein families (Dkk1, Dkk3, and sFRP-1/5) are expressed within the ANE and play important roles in modulating the Wnt signaling network during this process. In this study, we use morpholino and dominant-negative interference approaches to characterize the function of a novel Frizzled-related protein, secreted Frizzled-related protein 1 (sFRP-1), during ANE restriction. sFRP-1 appears to be related to a secreted Wnt modulator, sFRP3/4, that is essential to block Wnt signaling and establish the ANE in vertebrates. Here, we show that the sea urchin sFRP3/4 orthologue is not expressed during ANE restriction in the sea urchin embryo. Instead, our results indicate that ubiquitously expressed maternal sFRP-1 and Fzl1/2/7 signaling act together as early as the 32- to 60-cell stage to antagonize the ANE restriction mechanism mediated by Wnt/ß-catenin and Wnt/JNK signaling. Then, starting from the blastula stage, Fzl5/8 signaling activates zygotic sFRP-1 within the ANE territory, where it works with the secreted Wnt antagonist Dkk1 (also activated by Fzl5/8 signaling) to antagonize Wnt1/Wnt8-Fzl5/8-JNK signaling in a negative feedback mechanism that defines the outer ANE territory boundary. Together, these data indicate that maternal and zygotic sFRP-1 protects the ANE territory by antagonizing the Wnt1/Wnt8-Fzl5/8-JNK signaling pathway throughout ANE restriction, providing precise spatiotemporal control of the mechanism responsible for the establishment of the ANE territory around the anterior pole of the sea urchin embryo.

4.
J Vis Exp ; (120)2017 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-28287557

RESUMEN

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.


Asunto(s)
Comunicación Celular/fisiología , Embrión no Mamífero/fisiología , Desarrollo Embrionario/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Erizos de Mar/embriología , Transducción de Señal/fisiología , Animales , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica/fisiología , Modelos Animales , Modelos Biológicos
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