Project description:Maternally expressed proteins function in vertebrates to establish the major body axes of the embryo, and to establish a pre-pattern that sets the stage for later acting zygotic signals. This pre-pattern drives the propensity of Xenopus animal cap cells to adopt neural fates under various experimental conditions. Previous studies found that the maternally expressed transcription factor, encoded by the Xenopus achaete-scute like gene ascl1, is enriched at the animal pole. Asc1l is a bHLH protein involved in neural development, but its maternal function has not been studied. In this study, we have performed a series of gain and loss of function experiments on maternal ascl1, and present three novel findings. First, Ascl1 is a repressor of mesendoderm induced by VegT, but not of Nodal induced mesendoderm. Secondly, a previously uncharacterized N-terminal domain of Ascl1 interacts with HDAC1 to inhibit mesendoderm gene expression. This N-terminal domain is dispensable for its neurogenic function, indicating that Ascl1 has acts by different mechanisms at different times. Ascl1-mediated repression of mesendoderm genes was dependent on HDAC activity and accompanied by histone deacetylation in the promoter regions of VegT targets. Finally, maternal Ascl1 is required for animal cap cells to retain their competence to adopt neural fates. These results establish maternal Asc1l as a key factor in establishing the pre-pattern of the early embryo, acting in opposition to VegT and biasing the animal pole to adopt neural fates. The data presented here significantly extend our understanding of early embryonic pattern formation. Examination of genes expression in control (cMO) and Ascl1 MO knockdown (AMOs) embryos by deep sequencing.
Project description:Maternally expressed proteins function in vertebrates to establish the major body axes of the embryo, and to establish a pre-pattern that sets the stage for later acting zygotic signals. This pre-pattern drives the propensity of Xenopus animal cap cells to adopt neural fates under various experimental conditions. Previous studies found that the maternally expressed transcription factor, encoded by the Xenopus achaete-scute like gene ascl1, is enriched at the animal pole. Asc1l is a bHLH protein involved in neural development, but its maternal function has not been studied. In this study, we have performed a series of gain and loss of function experiments on maternal ascl1, and present three novel findings. First, Ascl1 is a repressor of mesendoderm induced by VegT, but not of Nodal induced mesendoderm. Secondly, a previously uncharacterized N-terminal domain of Ascl1 interacts with HDAC1 to inhibit mesendoderm gene expression. This N-terminal domain is dispensable for its neurogenic function, indicating that Ascl1 has acts by different mechanisms at different times. Ascl1-mediated repression of mesendoderm genes was dependent on HDAC activity and accompanied by histone deacetylation in the promoter regions of VegT targets. Finally, maternal Ascl1 is required for animal cap cells to retain their competence to adopt neural fates. These results establish maternal Asc1l as a key factor in establishing the pre-pattern of the early embryo, acting in opposition to VegT and biasing the animal pole to adopt neural fates. The data presented here significantly extend our understanding of early embryonic pattern formation.
Project description:A conserved molecular pathway has emerged controlling endoderm formation in Xenopus zebrafish and mice. Key genes in this pathway include Nodal ligands and transcription factors of the Mix-like paired homeodomain class, Gata4-6 zinc finger factors and Sox17 HMG domain proteins. While a linear epistatic pathway has been proposed, the precise hierarchical relationships between these factors and their downstream targets are largely unresolved. Here we used a combination of microarray analysis and loss-of-function experiments to examine the global regulatory network controlling Xenopus endoderm formation. We identified over 300 transcripts enriched in the gastrula endoderm, including most of the known endoderm regulators as well as over a hundred uncharacterized genes. Surprisingly only 10% of the endoderm transcriptome is regulated as predicted by the current linear model. We find that Nodals, Mixer and Sox17 have both shared and distinct sets of downstream targets and that a number of unexpected autoregulatory loops exist between Sox17 and Gata4-6, Sox17 and Bix1, 2, 4 and between Sox17 and Xnr4. We find that Mixer does not function primarily via Sox17 as previously proposed. This data provides a new insight into the complexity of endoderm formation and will serve as valuable resource for establishing a complete endoderm gene regulatory network. Experiment Overall Design: Define a set of transcripts with enriched expression in the gastrula endoderm of the Xenopus laevis embryo and determine how these are regulated by nodal signaling, Mixer and Sox17 using loss-of-function experiments. For more specific details see Sinner et al., (2006) Global analysis of the transcriptional network controlling Xenopus endoderm formation.
Project description:Centromeres play an essential role in cell division by specifying the site of kinetochore formation on each chromosome so that chromosomes can attach to the mitotic spindle for segregation. Centromeres are defined epigenetically by the histone H3 variant CEntromere Protein A (CENP-A). Dividing cells maintain the centromere by depositing new CENP-A each cell cycle to replenish CENP-A diluted by replication. The CENP-A nucleosome serves as the primary signal to the machinery responsible for its replenishment. Vertebrate centromeres are frequently built on repetitive sequences organized in tandem arrays. Repetitive centromeric DNA has been suggested to play a role in centromere maintenance and in de novo centromere formation, but this has been difficult to dissect because of the difficulty in manipulating centromere in cells. Extracts from Xenopus laevis eggs are able to assemble centromeres and kinetochores in vitro and thus provide a useful system for studying the role of centromeric DNA in centromere formation. However centromeric sequences in X. laevis have not been extensively characterized.. In this study we characterize repeat sequences found at X. laevis centromeres. We utilize a k-mer based approach in order to uncover the previously unknown diversity of X. laevis centromeric sequences. We validate centromere localization of repeat sequences by in situ hybridization and identify the location of the centromeric repetitive array on each chromosome by mapping the distribution of centromere enriched k-mers on the Xenopus genome. Our identification of X. laevis centromere sequences enables previously unapproachable genomic studies of centromeres. The k-mer based approach that we used to investigate centromeric repetitive DNA is suitable for the analysis of other repetitive sequences found across the genome or the study of repeats in other organisms.