An FGF-driven feed-forward circuit patterns the cardiopharyngeal mesoderm in space and time

In embryos, multipotent progenitors divide to produce distinct progeny and express their full potential. In vertebrates, multipotent cardiopharyngeal progenitors produce second-heart-field-derived cardiomyocytes, and branchiomeric skeletal head muscles. However, the mechanisms underlying these early fate choices remain largely elusive. The tunicate Ciona emerged as an attractive model to study early cardiopharyngeal development at high resolution: through two asymmetric and oriented divisions, defined cardiopharyngeal progenitors produce distinct first and second heart precursors, and pharyngeal muscle (aka atrial siphon muscle, ASM) precursors. Here, we demonstrate that differential FGF-MAPK signaling distinguishes between heart and ASM precursors. We characterize a feed-forward circuit that promotes the successive activations of essential ASM determinants, Hand-related, Tbx1/10 and Ebf. Finally, we show that coupling FGF-MAPK restriction and cardiopharyngeal network deployment with cell divisions defines the timing of gene expression and permits the emergence of diverse cell types from multipotent progenitors.


53
Taken together, this growing body of evidence points to the existence of a mesodermal 54 All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/138701 doi: bioRxiv preprint first posted online May. 17, 2017; 8 Tbx1/10 in the STVCs and Ebf in the ASMFs ( Figure 1C). This data indicate that FGF-147 MAPK signaling is required in the cardiopharyngeal progenitors and/or their progeny 148 for ASM fate specification, beyond the initial TVC induction. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

192
To further probe the signal transduction pathway, we engineered a constitutively 193 active version of the Ciona Mek1/2 protein by introducing phosphomimetic mutations 194 of two conserved serine residues in the catalytic domain, as previously shown for the 195 mammalian homolog (Cowley et al., 1994;Mansour et al., 1994). Early misexpression of

196
Mek S220E,S216D in the B7.5 lineage using the Mesp enhancer caused ectopic TVC 197 induction, mimicking the effects of gain of Ets1/2 function ( Figure S4; (Davidson et al., 198 All rights reserved. No reuse allowed without permission. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.   also did not alter STVC divisions ( Figure S3G), but it blocked the ASMF-specific 223 expression of Ebf ( Figure 3H). These results indicate that continuous MEK activity is 224 All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

248
MAPK activity is necessary and sufficient for ASMF-specific expression of Ebf.

250
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.    (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.    4E). To test whether Tbx1/10 was also required for ectopic Ebf expression in response to 301 All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.    (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

329
CRISPR/Cas9-mediated Hand-r mutagenesis strongly inhibited Ebf expression, and 330 this effect could be rescued by a CRISPR-resistant Hand-r cDNA ( Figure 4F). To test 331 whether this effect was mediated by a loss of Tbx1/10 expression, we attempted to 332 rescue the Hand-r loss-of-function by over-expressing Tbx1/10 using the Foxf enhancer.  Hand-r is also required in parallel to Tbx1/10 for Ebf activation in the ASMFs.

338
Taken together, these analyses suggest that coherent feed-forward circuits govern 339 the sequential activation of Hand-r, Tbx1/10 and Ebf in response to continuous but

346
In principle, the feed-forward circuit described above is sufficient to explain the

351
This sequence of events -divisions followed by gene activation-is paramount as it 352 permits the birth of first and second heart precursors, whose fates are antagonized by 353 All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

354
Therefore, we sought to investigate the role(s) of the cell cycle in controlling the timing 355 of Tbx1/10 and Ebf activation.

356
We first evaluated the effects of cytochalasin B, a classic inhibitor of cytokinesis 357 widely used to study cell fate specification in ascidians ( Figure 5A; (Whittaker, 1973)).

358
Treatments starting before TVC divisions (12 hpf) did not block Tbx1/10 or Ebf   Figure 5D). In these delayed TVCs, Tbx1/10 expression was strongly reduced 378 compared to control STVCs ( Figure 5D). However, 20 to 40% of the delayed TVCs 379 All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/138701 doi: bioRxiv preprint first posted online May. 17, 2017; expressed Tbx1/10 to variable extents. This suggests that the cardiopharyngeal 380 regulatory network can qualitatively unfold independently of cell cycle progression, but 381 the latter is necessary for Tbx1/10 expression to its wild-type levels.

401
The proportion of Ebf+ ASMFs in control embryos progressively increased from 402 ~20% at 15.5hpf to >90% by 18hpf, revealing an under-appreciated dynamic at the 403 onset of Ebf expression, which appears to take at least one hour to be "strongly" 404 expressed in >75% of newborn ASMFs ( Figure S6).

405
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

18
To evaluate the impact of Wee1-induced mitosis inhibition on Ebf accumulation, we 406 focused on undivided STVCs at each time point (hence the lower numbers in Figure S6A 407 compare to Figure S6B). By 17hpf, wee1-expressing delayed STVCs showed "strong" Ebf 408 expression in comparably high proportions of embryos. However, these proportions

425
We sought to further probe the mechanisms that regulate the initiation of Ebf 426 expression in early ASMFs, and their biological significance for fate specification. Since (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.    (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.    (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/138701 doi: bioRxiv preprint first posted online May. 17, 2017; signaling and cell-cycle-regulated transcriptional input(s) govern the onset and initial 485 accumulation of Ebf gene products during the first hour of the ASMF cycle, whereas the 486 maintenance of Ebf expression relies primarily on MAPK-independent autoactivation, 487 following initial accumulation (Figure 7).

488
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

489
Here, we demonstrated that the progressive restriction of FGF-MAPK signaling   (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. prospective TVC membrane to the ventral epidermis. The latter differential integrin-

536
Our preliminary analyses indicate that perturbations of the FGF-Ras-MEK pathway 537 can alter cardiopharyngeal cell division patterns ( Figure S3). While these effects did not 538 account for the observed changes in gene expression, future studies will unravel FGF-539 All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.   (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.     (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.   (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/138701 doi: bioRxiv preprint first posted online May. 17, 2017; These observations imply that the intrinsic dynamic of the network is slower than 618 observed. This allows first and second heart precursors to be born prior to the onset of 619 Tbx1/10 and Ebf, respectively. The latter sequence is essentially for the heart    (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 29 and a fate-restricted non-cardiac muscle identity, while MAPK inhibition is required for 670 myocardial specification and differentiation in the first and second heart field, 671 successively.

672
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.    (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

767
We cloned a full-length cis-regulatory DNA construct (~7kbp) that was analyzed by 768 introducing large deletions to map the functional elements. We found a fragment of 769 1264 bp, that we called T6, located at -4682 bp upstream from the initiation codon that (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

793
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

819
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

837
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

865
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.    (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

920
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

938
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

966
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

983
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

1001
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.