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1.
Nat Commun ; 14(1): 298, 2023 01 18.
Article in English | MEDLINE | ID: mdl-36653403

ABSTRACT

For successful regeneration, the identity of the missing tissue must be specified according to the pre-existing tissue. Planarians are ideal for the study of the mechanisms underlying this process; the same field of cells can regrow a head or a tail according to the missing body part. After amputation, the differential activation of the Wnt/ß-catenin signal specifies anterior versus posterior identity. Initially, both wnt1 and notum (Wnt inhibitor) are expressed in all wounds, but 48 hours later they are restricted to posterior or anterior facing wounds, respectively, by an unknown mechanism. Here we show that 12 hours after amputation, the chromatin accessibility of cells in the wound region changes according to the polarity of the pre-existing tissue in a Wnt/ß-catenin-dependent manner. Genomic analyses suggest that homeobox transcription factors and chromatin-remodeling proteins are direct Wnt/ß-catenin targets, which trigger the expression of posterior effectors. Finally, we identify FoxG as a wnt1 up-stream regulator, probably via binding to its first intron enhancer region.


Subject(s)
Planarians , Animals , Planarians/physiology , Wnt Proteins/genetics , Wnt Proteins/metabolism , Chromatin Assembly and Disassembly , beta Catenin/genetics , beta Catenin/metabolism , Body Patterning/genetics
2.
Sci Adv ; 8(48): eabn2258, 2022 12 02.
Article in English | MEDLINE | ID: mdl-36459547

ABSTRACT

DNA methylation [5-methylcytosine (5mC)] is a repressive gene-regulatory mark required for vertebrate embryogenesis. Genomic 5mC is tightly regulated through the action of DNA methyltransferases, which deposit 5mC, and ten-eleven translocation (TET) enzymes, which participate in its active removal through the formation of 5-hydroxymethylcytosine (5hmC). TET enzymes are essential for mammalian gastrulation and activation of vertebrate developmental enhancers; however, to date, a clear picture of 5hmC function, abundance, and genomic distribution in nonvertebrate lineages is lacking. By using base-resolution 5mC and 5hmC quantification during sea urchin and lancelet embryogenesis, we shed light on the roles of nonvertebrate 5hmC and TET enzymes. We find that these invertebrate deuterostomes use TET enzymes for targeted demethylation of regulatory regions associated with developmental genes and show that the complement of identified 5hmC-regulated genes is conserved to vertebrates. This work demonstrates that active 5mC removal from regulatory regions is a common feature of deuterostome embryogenesis suggestive of an unexpected deep conservation of a major gene-regulatory module.


Subject(s)
DNA Demethylation , Vertebrates , Animals , Vertebrates/genetics , Gene Regulatory Networks , Embryonic Development/genetics , DNA Methylation , Mammals
3.
Dev Biol ; 448(2): 71-87, 2019 04 15.
Article in English | MEDLINE | ID: mdl-30661644

ABSTRACT

Ascidian species of the Phallusia and Ciona genera are distantly related, their last common ancestor dating several hundred million years ago. Although their genome sequences have extensively diverged since this radiation, Phallusia and Ciona species share almost identical early morphogenesis and stereotyped cell lineages. Here, we explored the evolution of transcriptional control between P. mammillata and C. robusta. We combined genome-wide mapping of open chromatin regions in both species with a comparative analysis of the regulatory sequences of a test set of 10 pairs of orthologous early regulatory genes with conserved expression patterns. We find that ascidian chromatin accessibility landscapes obey similar rules as in other metazoa. Open-chromatin regions are short, highly conserved within each genus and cluster around regulatory genes. The dynamics of chromatin accessibility and closest-gene expression are strongly correlated during early embryogenesis. Open-chromatin regions are highly enriched in cis-regulatory elements: 73% of 49 open chromatin regions around our test genes behaved as either distal enhancers or proximal enhancer/promoters following electroporation in Phallusia eggs. Analysis of this datasets suggests a pervasive use in ascidians of "shadow" enhancers with partially overlapping activities. Cross-species electroporations point to a deep conservation of both the trans-regulatory logic between these distantly-related ascidians and the cis-regulatory activities of individual enhancers. Finally, we found that the relative order and approximate distance to the transcription start site of open chromatin regions can be conserved between Ciona and Phallusia species despite extensive sequence divergence, a property that can be used to identify orthologous enhancers, whose regulatory activity can partially diverge.


Subject(s)
Ciona/embryology , Ciona/genetics , Embryo, Nonmammalian/metabolism , Evolution, Molecular , Genetic Variation , Regulatory Sequences, Nucleic Acid/genetics , Urochordata/embryology , Urochordata/genetics , Animals , Base Sequence , Body Patterning/genetics , Chromatin/genetics , Conserved Sequence/genetics , Embryonic Development/genetics , Enhancer Elements, Genetic , Gastrula/embryology , Gene Expression Regulation, Developmental , Species Specificity , Time Factors
4.
Front Cell Dev Biol ; 7: 372, 2019.
Article in English | MEDLINE | ID: mdl-32039199

ABSTRACT

Cis-regulatory elements (CREs) are non-coding DNA regions involved in the spatio-temporal regulation of gene expression. Gene regulatory changes drive animal development and play major roles during evolution of animal body plans. Therefore, we believe that determining CREs at different developmental stages and across animal lineages is critical to understand how evolution operates through development. The Assay for Transposase-Accessible Chromatin followed by high-throughput sequencing (ATAC-seq) is a powerful technique for the study of CREs that takes advantage of Tn5 transposase activity. Starting from fewer than 105 cells, in a 1-day procedure, it is possible to detect, at a genome-wide level, CREs located in open chromatin regions with high resolution. Here, we describe a detailed step-by-step ATAC-seq protocol for invertebrate chordate marine embryos. We have successfully applied this technique to amphioxus and two species of tunicate embryos. We also show an easy workflow to analyze data generated with this technique. Moreover, we point out that this method and our bioinformatic pipeline are efficient to detect CREs associated with Wnt signaling pathway by simply using embryos treated with a drug that perturbs this pathway. This approach can be extended to other signaling pathways and also to embryo mutants for critical genes. Our results therefore demonstrate the power of ATAC-seq for the identification of CREs that play essential functions during animal development in a wide range of invertebrate or vertebrate animals.

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