Brachyury controls Ciona notochord fate as part of a feed-forward network

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 is unaffected by CRISPR Bra disruption. We identify Foxa.a and Mnx as potential co-regulators, and find that combinatorial cocktails are more effective at reprogramming 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 classically defined master regulator.

Evolution of Developmental Programs for the Midline Structures in Chordates: Insights From Gene Regulation in the Floor Plate and Hypochord Homologues of Ciona Embryos

“…The present findings, along with a number of previous studies, illustrate common features and the difference of midline development between vertebrates and tunicates ( Figure 6 ). The gene regulatory network for notochord development in ascidian embryos has been extensively studied ( Imai et al, 2006 ; Hotta et al, 2008 ; Passamaneck et al, 2009 ; Kubo et al, 2010 ; José-Edwards et al, 2015 ; Reeves et al, 2021 ). Brachyury is a key specifier gene for the notochord formation.…”

“…Brachyury is a key specifier gene for the notochord formation. FoxA.a is an upstream activator of Brachyury ( Imai et al, 2006 ; Hotta et al, 2008 ; Kubo et al, 2010 ), but it also directly activates notochord-specific genes ( Passamaneck et al, 2009 ; José-Edwards et al, 2015 ; Reeves et al, 2021 ).…”

Quantitative Dissection of the Proximal Ciona brachyury Enhancer

“…We mutated predicted binding sites for 6 different TFs of interest. Zic, Ets and FoxA are all known upstream regulators of Bra (Yagi et al, 2004;Imai et al, 2006;Matsumoto et al, 2007;Farley et al, 2016;Reeves et al, 2021). Primary notochord cell fate specification depends on FGF signaling that culminates in the activation of Ets family TFs.…”

“…Several previous studies have mapped out cis-regulatory modules controlling brachyury expression in ascidian models (Corbo et al, 1998;Fujiwara et al, 1998;Takahashi et al, 1999;Yagi et al, 2004;Matsumoto et al, 2007;Farley et al, 2016;Harder et al, 2021;Reeves et al, 2021). Various transcription factor binding motifs of interest have been mutated, but these efforts have been split between Halocynthia and Ciona and also between the proximal bra enhancer and the more distal "shadow" bra enhancer in Ciona.…”

“…That is our goal here, using the proximal enhancer for the notochord transcription factor Brachyury (Bra) as a test case. Brachyury induction in both the primary A-line and secondary B-line notochord lineages has been extensively investigated and numerous upstream regulators are known (Miya and Nishida, 2003;Yagi et al, 2004;Hudson and Yasuo, 2006;Imai et al, 2006;Matsumoto et al, 2007;Farley et al, 2016;Veeman, 2018;Harder et al, 2021;Reeves et al, 2021). Several of these interactions are likely direct based on mutating predicted TFBSs in reporter assays (Corbo et al, 1998;Fujiwara et al, 1998;Takahashi et al, 1999;Yagi et al, 2004;Matsumoto et al, 2007;Farley et al, 2016;Reeves et al, 2021), but there has been no systematic attempt to predict and disrupt sites for all of these putative upstream regulators in the same quantitative framework.…”