The gene regulatory system for specifying germ layers in early embryos of the simple chordate

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Science Advances  09 Jun 2021:
Vol. 7, no. 24, eabf8210
DOI: 10.1126/sciadv.abf8210
  • Fig. 1 Gene expression patterns in early Ciona embryos.

    (A) Schematics of a bisymmetrical 32-cell embryo. Cells are largely specified to two ectodermal fates, vegetal marginal (largely mesodermal) fate and vegetal central (largely endodermal) fate, which are shown by different colors. (B) Possible upstream factors of genes expressed in 32-cell embryos. Cells where each upstream factor is expected to act are shown in cyan. We do not explicitly consider Ets1/2, Tcf7, Gata.a, Pem1, or the most posterior germline cells where Pem1 is localized (asterisks; see main text and Supplementary Text for details). Nine transcription factors encoded by genes expressed at the 16-cell stage are expected to act at the 32-cell stage in daughters of cells with their mRNAs. In addition, five signaling ligands act in 32-cell embryos. Fgf9/16/20 and Efna.d regulate the MAPK pathway positively and negatively, respectively. Cells in which they act have been determined by staining of doubly phosphorylated extracellular signal–regulated kinase in wild-type and Efna.d morphant embryos (18). Cells in which anti-dorsalizing morphogenetic protein (Admp) and growth differentiation factor 1/3-related (Gdf1/3-r) act have been inferred from a combination of experiments and mathematical analysis (19). Note that Efna.d is a cell membrane–bound protein transmitting the signal in a contact-dependent manner and that all cells are in direct contact with cells expressing Fgf9/16/20 and Gdf1/3-r (21). See main text and Supplementary Text for details. (C) Expression of downstream genes that initiate expression at the 32-cell stage. Cells where each gene is expressed are colored.

  • Fig. 2 Boolean representation of regulatory logics of genes that initiate expression at the 32-cell stage.

    (A) From partial truth tables Tn with missing values, disjunctive normal forms (DNFs) are inferred on the basis of experiments. We repeated experiments, in which one or more upstream factors were down-regulated, until an exhaustive search successfully identified a unique candidate DNF. (B and C) Intuitive explanation of the method to infer DNFs. In this hypothetical system, three upstream factors, A, B, and C, are expressed and possibly regulate the gene X. (B) Given a complete truth table, the corresponding regulatory function in the DNF can be determined. Conversely, given a regulatory function in the DNF, the corresponding truth table can be determined uniquely. (C) Estimation of DNFs corresponding to a partially given truth table. First, among all 27 possible conjunctions, conjunctions inconsistent with one or more cases of X = 1 are excluded (magenta). Second, conjunctions that do not explain any case of X = 1 in the partial truth table are also excluded (cyan). If any single conjunction in the remaining set does not fully explain the partial truth table, then disjunctions of multiple conjunctions are examined. In this case, three DNFs fully explain the truth table. We consider the simplest one as a primary candidate.

  • Fig. 3 Regulatory logics for gene expression in 32-cell embryo.

    (A to G) Expression of Bmp3 (A to C) and Zic-r.b (D to G) in normal embryos (A and D) and embryos injected with MOs indicated above the photographs (B, C, and E to G). Number of embryos examined and the proportion of embryos that each panel represents are shown below the panels. Arrowheads indicate loss of expression. (H) Regulatory logics that specify five different lineages at the 32-cell stage. Note that the regulatory mechanisms in B6.4 do not share the mechanisms shown here (see the text).

  • Fig. 4 Regulation of Lhx3/4 at the 16-cell stage.

    (A) While β-catenin is present in three pairs of vegetal cells (cyan boxes in the top), neither Fgf9/16/20 nor Foxd is expected to act in normal 16-cell embryos (white boxes). Therefore, FLhx3/4 predicts that Lhx3/4 is not expressed (white boxes in the center) and no expression is detected by in situ hybridization (bottom). (B) If embryos injected with Foxd mRNA (0.75 pg) are treated with FGF2, then Foxd and Fgf signaling are expected to act in all cells (cyan dots), and FLhx3/4 predicts that Lhx3/4 is expressed in the vegetal hemisphere where nuclear β-catenin is present (magenta boxes). In situ hybridization shows that this is the case. (C) If embryos are additionally treated with BIO, then Lhx3/4 is predicted to be expressed in all cells except the most posterior germline pair, and in situ hybridization revealed that this was the case.

  • Fig. 5 Evolutionary plasticity of the regulatory mechanism for Lhx3/4 expression.

    The present study revealed that FLhx3/4 is Foxd˄Fgf9/16/20˄β-catenin in an ascidian, C. intestinalis type A (or C. robusta). However, in an ascidian from a different population (C. intestinalis type B or simply called C. intestinalis), Foxa.a is necessary for Lhx3/4 expression (9); therefore, the mechanism is likely to be slightly different. However, even under the assumption that FLhx3/4 is Foxa.a˄Foxd˄Fgf9/16/20˄β-catenin, the expression pattern of Lhx3/4 is unchanged in normal embryos.

  • Table 1 Representation of regulatory logics in DNFs for expression of genes that initiate expression at the 32-cell stage.
    DNFs*Cells where gene
    expression is activated
    in normal embryos

    *Note that β-catenin acts with Tcf7, which is present in all cells, for activating targets, and that β-catenin represses Gata.a (7, 13). Because Gata.a is present in all cells, ¬β-catenin means that Gata.a becomes active.

    †This conjunction was determined by not only knockdown experiments but also overexpression experiments (fig. S2).

    These conjunctions represent expression in experimental conditions only.

    §We were not able to determine this conjunction uniquely. The expression patterns of Prdm1-r and Foxa.a indicate that either of them or both are involved. However, this conjunction represents expression in experimental conditions only but not expression in normal 32-cell embryos.

    Supplementary Materials

    • Supplementary Materials

      The gene regulatory system for specifying germ layers in early embryos of the simple chordate

      Miki Tokuoka, Kazuki Maeda, Kenji Kobayashi, Atsushi Mochizuki, Yutaka Satou

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