Differential gene expression along the animal-vegetal axis in the ascidian embryo is maintained by a dual functional protein Foxd

Tokuhiro, Shin Ichi; Tokuoka, Miki; Kobayashi, Kenji; Kubo, Atsushi; Oda-Ishii, Izumi; Satou, Yutaka

doi:10.1371/journal.pgen.1006741

Cited by 15 publications

(21 citation statements)

References 46 publications

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“…The neural and ectodermal core logics included negative regulation by Foxd. Conversely, most genes expressed in the vegetal hemisphere were positively regulated by Foxd (Table 1), as previously indicated (23). Similarly, our results also confirmed a previously proposed model that -catenin plays a key role in discriminating medial vegetal (largely endodermal) cells from marginal vegetal (largely mesodermal) ones (5, 9, 13) (Table 1).…”

Section: Core Regulatory Logics Specifying Cell Fates Including Three Germ Layer Fatessupporting

confidence: 91%

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

et al. 2021

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In animal embryos, gene regulatory networks control the dynamics of gene expression in cells and coordinate such dynamics among cells. In ascidian embryos, gene expression dynamics have been dissected at the single-cell resolution. Here, we revealed mathematical functions that represent the regulatory logics of all regulatory genes expressed at the 32-cell stage when the germ layers are largely specified. These functions collectively explain the entire mechanism by which gene expression dynamics are controlled coordinately in early embryos. We found that regulatory functions for genes expressed in each of the specific lineages contain a common core regulatory mechanism. Last, we showed that the expression of the regulatory genes became reproducible by calculation and controllable by experimental manipulations. Thus, these regulatory functions represent an architectural design for the germ layer specification of this chordate and provide a platform for simulations and experiments to understand the operating principles of gene regulatory networks.

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Section: Core Regulatory Logics Specifying Cell Fates Including Three Germ Layer Fatessupporting

confidence: 91%

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

et al. 2021

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“…For example, Neurogenin is expressed in both A8.15 and A8.16 in our data, whereas in situ only detected expression in A8. 16. Table S1C lists all the cases where known cell types were not separated.…”

Section: Cell Type Identificationmentioning

confidence: 99%

“…RT mixture with template-switching oligos was added and proceeded using the following PCR steps: 42°C for 90 min and then 10 cycles of 50°C for 2 min, 42°C for 2 min, and last, 70°C 15 min. cDNA preamplification was performed for 12,13,14,15,16, and 17 cycles, respectively, for 1/2-cell, 4-cell, 8-cell, 16-cell, 32-cell, and 64/110-cell stage samples.…”

Section: Single-cell Rna Sequencingmentioning

confidence: 99%

A single-cell analysis of the molecular lineage of chordate embryogenesis

Zhang

Imai

et al. 2020

Sci. Adv.

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Progressive unfolding of gene expression cascades underlies diverse embryonic lineage development. Here, we report a single-cell RNA sequencing analysis of the complete and invariant embryonic cell lineage of the tunicate Ciona savignyi from fertilization to the onset of gastrulation. We reconstructed a developmental landscape of 47 cell types over eight cell cycles in the wild-type embryo and identified eight fate transformations upon fibroblast growth factor (FGF) inhibition. For most FGF-dependent asymmetric cell divisions, the bipotent mother cell displays the gene signature of the default daughter fate. In convergent differentiation of the two notochord lineages, we identified additional gene pathways parallel to the master regulator T/Brachyury. Last, we showed that the defined Ciona cell types can be matched to E6.5-E8.5 stage mouse cell types and display conserved expression of limited number of transcription factors. This study provides a high-resolution single-cell dataset to understand chordate early embryogenesis and cell lineage differentiation.

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“…As in other model organisms, however, one of the biggest challenges in GRN analysis is determining whether transcriptional regulatory effects are direct or indirect. Some ChIPchip and ChIPseq data are available in Ciona (Kubo et al, 2010;Oda-Ishii et al, 2016;Tokuhiro et al, 2017), but TF binding to particular regulatory regions does not necessarily indicate that it is functionally important (Yang et al, 2006;Hu et al, 2007;Biggin, 2011). The gold standard for determining whether transcriptional interactions are direct or indirect usually involves cis-regulatory analysis (Stolfi and Christiaen, 2012;Irvine, 2013).…”

Section: Introductionmentioning

confidence: 99%

Quantitative Dissection of the Proximal Ciona brachyury Enhancer

Shimai

Veeman

2022

Front. Cell Dev. Biol.

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A major goal in biology is to understand the rules by which cis-regulatory sequences control spatially and temporally precise expression patterns. Here we present a systematic dissection of the proximal enhancer for the notochord-specific transcription factor brachyury in the ascidian chordate Ciona. The study uses a quantitative image-based reporter assay that incorporates a dual-reporter strategy to control for variable electroporation efficiency. We identified and mutated multiple predicted transcription factor binding sites of interest based on statistical matches to the JASPAR binding motif database. Most sites (Zic, Ets, FoxA, RBPJ) were selected based on prior knowledge of cell fate specification in both the primary and secondary notochord. We also mutated predicted Brachyury sites to investigate potential autoregulation as well as Fos/Jun (AP1) sites that had very strong matches to JASPAR. Our goal was to quantitatively define the relative importance of these different sites, to explore the importance of predicted high-affinity versus low-affinity motifs, and to attempt to design mutant enhancers that were specifically expressed in only the primary or secondary notochord lineages. We found that the mutation of all predicted high-affinity sites for Zic, FoxA or Ets led to quantifiably distinct effects. The FoxA construct caused a severe loss of reporter expression whereas the Ets construct had little effect. A strong Ets phenotype was only seen when much lower-scoring binding sites were also mutated. This supports the enhancer suboptimization hypothesis proposed by Farley and Levine but suggests that it may only apply to some but not all transcription factor families. We quantified reporter expression separately in the two notochord lineages with the expectation that Ets mutations and RBPJ mutations would have distinct effects given that primary notochord is induced by Ets-mediated FGF signaling whereas secondary notochord is induced by RBPJ/Su(H)-mediated Notch/Delta signaling. We found, however, that ETS mutations affected primary and secondary notochord expression relatively equally and that RBPJ mutations were only moderately more severe in their effect on secondary versus primary notochord. Our results point to the promise of quantitative reporter assays for understanding cis-regulatory logic but also highlight the challenge of arbitrary statistical thresholds for predicting potentially important sites.

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Differential gene expression along the animal-vegetal axis in the ascidian embryo is maintained by a dual functional protein Foxd

Cited by 15 publications

References 46 publications

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

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

A single-cell analysis of the molecular lineage of chordate embryogenesis

Quantitative Dissection of the Proximal Ciona brachyury Enhancer

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