Estudio funcional de Ric-8A/Gαq en la formación del mesodermo dorsal en Xenopus tropicalis.
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Date
2024
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Universidad de Concepción
Abstract
La cresta neural (CN) es un tejido embrionario pluripotencial, transitorio y característico en vertebrados, que es inducido en el borde de la placa neural y puede diferenciarse a distintos tejidos. Para su inducción y mantención, requiere de señales provenientes del mesodermo dorsal, ectodermo neural y no neural, que permiten su desarrollo y posterior migración a lo largo del embrión. Se ha descrito que tanto la proteína Ric-8A como Gαq se expresan en tejido de CN y mesodermo dorsal. Además, resultados previos evidencian que, tanto Ric-8A como Gαq son necesarios para la correcta inducción de la CN, así como también que Gαq se encuentra regulando a la vía Wnt en el mesodermo dorsal, tejido donde se encuentra el organizador de Spemann-Mangold, lo que nos permitió asociar todas estas proteínas con la formación del mesodermo y con ello presentar nuestra hipótesis de trabajo “La señalización mediada por Ric-8A/Gαq regula la formación del mesodermo dorsal necesaria para la correcta inducción de la cresta neural en conjunto con otras vías de señalización involucradas, en Xenopus tropicalis”. Para responder a ésta, se realizaron ensayos de pérdida y ganancia de función, tanto de Ric-8A como Gαq, en los cuales se determinó que el silenciamiento de ambas proteínas inhibe la expresión del marcador de CN snail2, por lo que ambas proteínas estarían regulando la inducción de CN. El análisis, por medio de hibridación in situ, de la expresión de los marcadores de mesodermo (chordin, goosecoid, xbra) y de ectodermo queratina xk81a1 al silenciar a Ric-8A, evidencia la inhibición de la expresión de chordin y goosecoid, sin embargo, al realizar el análisis por medio de RT-qPCR, disminuye la expresión de goosecoid, mientras que la expresión de queratina aumenta, sugiriendo que Ric-8A regula la formación del mesodermo y ectodermo no neural. Por otro lado, el silenciamiento de Gαq evidenció la inhibición de la expresión de goosecoid y xbra, al emplear ambas técnicas, sugiriendo que Gαq regula la formación del mesodermo. Ensayos de epistasis del silenciamiento de Ric-8A con Gαq, mostraron que Gαq rescata el fenotipo anormal observado, indicando que ambas proteínas estarían en la misma vía de señalización regulando la formación del mesodermo. Ensayos de recombinación de tejidos también respaldan que la vía de señalización Ric-8A/Gαq es necesaria en la inducción de CN, específicamente en la zona marginal dorso-lateral del mesodermo, ya que al silenciar estas proteínas en dicho tejido, se inhibe la inducción de la CN. Además, la expresión del marcador neural sox2 también se ve afectada al inhibir la expresión de Ric-8A y Gαq, sugiriendo que ambas proteínas, además, estarían regulando la formación del ectodermo neural. Ensayos realizados con el fin de determinar si otra subunidad Gα pudiera estar involucrada en el efecto observado con la disminución de los niveles de Ric-8A, muestran que Gαi2 no estaría involucrada. Finalmente, análisis bioinformáticos de expresión diferencial de genes empleando secuencias de ARN-seq de Zona Marginal Dorsal y CN, en conjunto con análisis de enriquecimiento de términos GO, evidencian la importancia de procesos biológicos asociados al desarrollo de tejidos, migración celular y regulación de vías de señalización como Wnt y BMP en estos tejidos. Además, empleando el programa STRING se comprobó que proteínas pertenecientes a la vía de señalización de proteína G heterotrimérica están presentes en la zona marginal dorsal, región correspondiente al organizador de Spemann-Mangold, y CN, en conjunto con proteínas de la vía Wnt, FGF, BMP, y Hedgehog. Todos estos resultados en conjunto nos permiten sugerir que la señalización de Ric-8A/Gαq regula la correcta formación del mesodermo dorsal, ectodermo neural y no neural, necesarias para la inducción de la CN, en conjunto con otras vías de señalización.
The neural crest (NC) is a pluripotent, transient embryonic tissue unique to vertebrates. It forms at the edge of the neural plate and can differentiate into various cell types and tissues. For its induction and maintenance, the NC requires signals from the dorsal mesoderm, neural and non-neural ectoderm, which support its development and subsequent migration throughout the embryo. Previous studies have shown that both Ric-8A and Gαq proteins are expressed in NC tissue and dorsal mesoderm. Furthermore, prior results show that both Ric 8A and Gαq are necessary for proper NC, with Gαq also regulating the Wnt pathway within the dorsal mesoderm, a region associated with the Spemann Mangold organizer. Based on this, our hypothesis is: “Ric-8A/Gαq-mediated signaling regulates dorsal mesoderm formation required for proper NC induction in coordination with other signaling pathways in Xenopus tropicalis”. To test this hypothesis, we performed gain- and loss-of-function experiments for both Ric-8A and Gαq. Knocking down either protein inhibited the expression of the NC marker snail2, suggesting both proteins play a role in NC induction. Additionally, in situ hybridization analysis of mesodermal markers (chordin, goosecoid, xbra) and ectodermal marker keratin xk81a1 upon Ric-8A knockdown revealed inhibited expression of chordin and goosecoid. Quantitative RT-qPCR analysis further showed decreased goosecoid expression and increased keratin expression, suggesting Ric-8A role in regulating dorsal mesoderm and non-neural ectoderm formation. On the other hand, knockdown of Gαq inhibited goosecoid and xbra expression across both methods, indicating Gαq involvement in mesoderm formation. Epistasis assays of Ric-8A knockdown with Gαq showed that Gαq could rescue the abnormal phenotype observed, indicating both proteins act within the same signaling pathway to regulate mesoderm formation. Tissue recombination assays further confirmed that Ric-8A/Gαq signaling is essential for NC induction, specifically in the dorsolateral marginal zone of the mesoderm, thus knocking down these proteins in this region inhibited NC induction. Additionally, the expression of the neural marker sox2 was disrupted by Ric-8A and Gαq knocking down, suggesting these proteins also regulate neural ectoderm formation. Further assays ruled out the involvement of another Gα subunit, Gαi2, in the effect observed with Ric-8A knockdown. Finally, bioinformatics analysis of differential gene expression using RNA-seq data from the dorsal marginal zone and NC, along with GO term enrichment analysis, showed the importance of biological processes associated with tissue development, cell migration and the regulation of Wnt and BMP signaling pathways in these regions. STRING analysis confirmed that proteins belonging to the heterotrimeric G protein signaling pathway are present in the dorsal marginal zone, a region corresponding to the Spemann-Mangold organizer, and NC, along with proteins from the Wnt, FGF, BMP, and Hedgehog pathways. Taken together, these findings suggest that Ric 8A/Gαq signaling regulates proper formation of the dorsal mesoderm, neural and non-neural ectoderm, all of which are essential for NC induction, in coordination with other signaling pathways.
The neural crest (NC) is a pluripotent, transient embryonic tissue unique to vertebrates. It forms at the edge of the neural plate and can differentiate into various cell types and tissues. For its induction and maintenance, the NC requires signals from the dorsal mesoderm, neural and non-neural ectoderm, which support its development and subsequent migration throughout the embryo. Previous studies have shown that both Ric-8A and Gαq proteins are expressed in NC tissue and dorsal mesoderm. Furthermore, prior results show that both Ric 8A and Gαq are necessary for proper NC, with Gαq also regulating the Wnt pathway within the dorsal mesoderm, a region associated with the Spemann Mangold organizer. Based on this, our hypothesis is: “Ric-8A/Gαq-mediated signaling regulates dorsal mesoderm formation required for proper NC induction in coordination with other signaling pathways in Xenopus tropicalis”. To test this hypothesis, we performed gain- and loss-of-function experiments for both Ric-8A and Gαq. Knocking down either protein inhibited the expression of the NC marker snail2, suggesting both proteins play a role in NC induction. Additionally, in situ hybridization analysis of mesodermal markers (chordin, goosecoid, xbra) and ectodermal marker keratin xk81a1 upon Ric-8A knockdown revealed inhibited expression of chordin and goosecoid. Quantitative RT-qPCR analysis further showed decreased goosecoid expression and increased keratin expression, suggesting Ric-8A role in regulating dorsal mesoderm and non-neural ectoderm formation. On the other hand, knockdown of Gαq inhibited goosecoid and xbra expression across both methods, indicating Gαq involvement in mesoderm formation. Epistasis assays of Ric-8A knockdown with Gαq showed that Gαq could rescue the abnormal phenotype observed, indicating both proteins act within the same signaling pathway to regulate mesoderm formation. Tissue recombination assays further confirmed that Ric-8A/Gαq signaling is essential for NC induction, specifically in the dorsolateral marginal zone of the mesoderm, thus knocking down these proteins in this region inhibited NC induction. Additionally, the expression of the neural marker sox2 was disrupted by Ric-8A and Gαq knocking down, suggesting these proteins also regulate neural ectoderm formation. Further assays ruled out the involvement of another Gα subunit, Gαi2, in the effect observed with Ric-8A knockdown. Finally, bioinformatics analysis of differential gene expression using RNA-seq data from the dorsal marginal zone and NC, along with GO term enrichment analysis, showed the importance of biological processes associated with tissue development, cell migration and the regulation of Wnt and BMP signaling pathways in these regions. STRING analysis confirmed that proteins belonging to the heterotrimeric G protein signaling pathway are present in the dorsal marginal zone, a region corresponding to the Spemann-Mangold organizer, and NC, along with proteins from the Wnt, FGF, BMP, and Hedgehog pathways. Taken together, these findings suggest that Ric 8A/Gαq signaling regulates proper formation of the dorsal mesoderm, neural and non-neural ectoderm, all of which are essential for NC induction, in coordination with other signaling pathways.
Description
Tesis presentada para optar al grado académico de Magíster en Bioquímica y Bioinformática
Keywords
Anfibios, Desarrollo embrionario, Diferenciación celular