ABSTRACT
Drafting gene regulatory networks (GRNs) requires embryological knowledge pertaining to the cell type families, information on the regulatory genes, causal data from gene knockdown experiments and validations of the identified interactions by cis-regulatory analysis. We use multi-omics involving next-generation sequencing to obtain the necessary information for drafting the Strongylocentrotus purpuratus (Sp) posterior gut GRN. Here, we present an update to the GRN using: (1) a single-cell RNA-sequencing-derived cell atlas highlighting the 2 day-post-fertilization (dpf) sea urchin gastrula cell type families, as well as the genes expressed at the single-cell level; (2) a set of putative cis-regulatory modules and transcription factor-binding sites obtained from chromatin accessibility ATAC-seq data; and (3) interactions directionality obtained from differential bulk RNA sequencing following knockdown of the transcription factor Sp-Pdx1, a key regulator of gut patterning in sea urchins. Combining these datasets, we draft the GRN for the hindgut Sp-Pdx1-positive cells in the 2 dpf gastrula embryo. Overall, our data suggest the complex connectivity of the posterior gut GRN and increase the resolution of gene regulatory cascades operating within it.
Footnotes
Author contributions
Conceptualization: D.V., M.I.A.; Methodology: D.V., P.P., M.S.M., C.C., M.I.A.; Software: D.V.; Validation: D.V., P.P.; Formal analysis: D.V., P.P., M.S.M.; Investigation: D.V., P.P., M.S.M., C.C.; Resources: I.M., J.L.G.-S., M.I.A.; Data curation: D.V., P.P., M.S.M.; Writing - original draft: D.V., P.P.; Writing - review & editing: M.S.M., C.C., I.M., M.I.A.; Visualization: D.V., P.P.; Supervision: I.M., J.L.G.-S., M.I.A.; Project administration: I.M., J.L.G.-S., M.I.A.; Funding acquisition: J.L.G.-S., M.I.A.
Funding
D.V. and C.C. were supported by the Stazione Zoologica Anton Dohrn PhD fellowships. This work was supported by the H2020 Marie Skłodowska-Curie Actions Innovative Training Network EvoCELL (grant number 766053 to M.I.A. and fellowship to P.P.). J.L.G.-S. was supported by the Spanish government (Ministerio de Economía y Competitividad; grant BFU2016-74961-P), the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement number 740041) and the institutional grant Unidad de Excelencia María de Maeztu (MDM-2016-0687 to the Department of Gene Regulation and Morphogenesis, Centro Andaluz de Biología del Desarrollo). M.S.M. has been granted a fellowship of the Programme for the Training of Researchers by the Ministry of Economy, Industry and Competitiveness of Spain (Ministerio de Economía y Competitividad; BES-2014-068494). I.M. acknowledges support from the Spanish Ministry of Science and Innovation (Agencia Estatal de Investigación) and the European Union (grants RYC-2016-20089, PGC2018-099392-A-I00 and PID2021-128728NB-I00).
Data availability
The raw ATAC-seq and scRNA-seq data generated for this work and described in this study can be found at NCBI Gene Expression Omnibus (accession number GSE262916). Scripts associated with the data analysis pipeline can be found at https://github.com/Danvor/spur_2dpf_hindgut_pdx1_downstream_grn. The table with various gene name synonyms can be found at the same GitHub link.
