Brachyury controlsCionanotochord fate as part of a feedforward network and not as a unitary master regulator

Abstract

The notochord is a defining feature of the chordates. The transcription factor Brachyury (Bra) is a key regulator of notochord fate but here we show that it is not a unitary master regulator in the model chordate Ciona. Ectopic Bra expression only partially reprograms other cell types to a notochord-like transcriptional profile and a subset of notochord-enriched genes are unaffected by CRISPR Bra disruption. We identify Foxa.a and Mnx as potential co-regulators and find that combinatorial cocktails are more effective at reprograming other cell types than Bra alone. We reassess the network relationships between Bra, Foxa.a and other components of the notochord gene regulatory network and find that Foxa.a expression in the notochord is regulated by vegetal FGF signaling. It is a direct activator of Bra expression and has a binding motif that is significantly enriched in the regulatory regions of notochord-enriched genes. These and other results indicate that Bra and Foxa.a act together in a regulatory network dominated by positive feed-forward interactions, with neither being a classicallydefined master regulator.

Single-cell analysis of cell fate bifurcation in the chordateCiona

“…There is very little overlap between the genes differentially expressed in the notochord at the 64-cell stage and those induced by ectopic expression of Brachyury in other studies (Figure 6 B) [73,74], suggesting that these earliest differentially expressed genes may be expressed in parallel to, and not downstream of Brachyury We found that the most common pattern was for a gene to be upregulated with respect to the parental state in one cell type while not changing in the sibling. All possible combinations were observed, however, and there were a surprising number of differentially expressed genes that were upregulated in both sibling cell types as compared to the parental cell type, but to varying extents.…”

“…The hourglass metaphor is an apt way to think about the establishment of new transcriptional states, but note that we are not making reference to the concept of the phylotypic stage where an hourglass metaphor has also been widely used [84]. [71,73]. We also found that Zic becomes enriched in primary notochord compared to its A-line neural sibling cells.…”

“…TFBS analysis was performed similar to [73]. Control and target sequence sites were determined using the FindMarkers function implemented in Seurat to find statically significantly up and downregulated genes as desired.…”

“…Much like in the endoderm, muscle, and mesenchyme, we find that the DE TFs are either expressed strongly in a short time frame around fate induction or are stably expressed and increase their expression over time (Figure 6 C).The notochord exhibits distinct waves of transcriptionTo further characterize the temporal dynamics of the Ciona notochord transcriptome, we plotted the notochord expression levels over time of all the genes that are enriched in the notochord compared to other non-notochord tissues at each stage.Hierarchical clustering of these notochord enriched genes revealed 5 relatively distinct temporal waves of expression (Figure 6D-E). Characteristic temporal expression profiles have previously been identified by in situ hybridization for select notochord genes,[65][66][67]70,71,73,76,77] but the genome-wide transcriptional profiling performed here allows the comprehensive identification of distinct suites of genes that co-vary in expression in the notochord over time. A similar set of distinct temporal waves was seen when clustering the temporal expression profiles of the notochord-enriched transcription factors alone, suggesting that these waves might be controlled by distinct TFs or combinations of TFs acting in a differentiation cascade.…”

Ciona Brachyuryproximal and distal enhancers have different FGF dose-response relationships

“…Bra expression requires both the transcription factor Zic-r.b as well as active FGF signaling mediated by Ets family transcription factors (Yagi et al, 2004, Imai et al, 2006. These interactions are likely direct because mutation of predicted Zic and Ets binding sites in both the Proximal (Reeves and Shimai et al, 2020) and Distal (Farley et al, 2016) enhancers abrogates Bra expression. Essential Zic and Ets sites have also been identified in the Bra enhancer in the stolidobranch ascidian Halocynthia (Matsumoto et al, 2007).…”