An FGF-driven feed-forward circuit for spatiotemporal patterning of the cardiopharyngeal mesoderm in a simple chordate

In embryos, pluripotent stem cells and multipotent progenitors must divide and produce distinct progeny to express their full developmental potential. In vertebrates, mounting evidence point to the existence of multipotent cardiopharyngeal progenitors that produce second-heart-field-derived cardiomyocytes, and branchiomeric skeletal head muscles. However, the cellular and molecular mechanisms underlying these early fate choices remain largely elusive. The tunicate Ciona has emerged as an attractive model to study early cardiopharyngeal development at high spatial and temporal resolution: through two asymmetric and oriented cell divisions, defined multipotent 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 MAPK-negative heart precursors, and MAPK-positive multipotent progenitors and ASM precursors. We characterize an FGF/MAPK-driven feed-forward circuit that promotes the successive activations of essential cardiopharyngeal determinants, Tbx1/10 and Ebf. Finally, we show that coupling FGF/MAPK restriction and cardiopharyngeal network deployment with cell divisions permits the emergence of diverse cell types from common multipotent progenitors.


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In the past few years, studies guided by developmental genetics knowledge    7 born TVCs, marked by the B7.5-lineage-specific Mesp>H2B::mCherry transgene, as 126 previously observed (Davidson et al, 2006). We also detected weaker but persistent 127 dpERK staining in the TVCs during migration (Figs. 1 and S1). Following the first and 128 second asymmetric divisions of the TVCs and STVCs, dpERK staining was successively 129 restricted to the more lateral STVCs and ASMFs, respectively ( Figures 1A, B; S1).

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The canonical FGF/Ras/MEK/ERK pathway is necessary and sufficient to 132 promote pharyngeal muscle specification in the cardiopharyngeal lineage.

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This exclusion of MAPK activity from the medial first and second heart precursors 134 opened the possibility that differential ERK activity is required for proper STVC and

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Since Ebf expression is maintained for several days in the ASMF derivatives as they 226 differentiate into body wall and siphon muscles (Razy-Krajka et al., 2014), we tested 227 whether continued MEK activity is also required for the maintenance of Ebf expression 228 11 past its initial onset and cells' commitment to an ASM fate. Using both regular and 229 intron-specific antisense probes, which specifically detect nascent transcripts (Wang et

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

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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)).

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Treatments starting before TVC divisions (12 hpf) did not block Tbx1/10 or Ebf 359 expression in embryos fixed after their normal onset at either 16 or 19hpf, respectively 360 ( Figure 5B). Similarly, treatment starting between the first and second asymmetric 361 divisions (15hpf) did not block localized Ebf expression at 19hpf ( Figure 5B). This     expressed Tbx1/10 to variable extents. This suggests that the cardiopharyngeal 382 regulatory network can qualitatively unfold independently of cell cycle progression, but 383 the latter is necessary for Tbx1/10 expression to its wild-type levels.

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Noto16 and Wee1, and assay Ebf expression at later stages. Inhibitors of the G1/S 386 transition failed to block STVC divisions (data not shown), most likely because T12-387 driven products did not accumulate quickly enough to interfere with the G1/S transition 388 in STVCs (this cell cycle lasts only ~2 hours compared to ~6 hours for the TVC 389 interphase), suggesting that the G1 phase is too short for T12-driven gene products to 390 accumulate before the G1/S transition. Therefore, we focused the analyses of Ebf

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Focusing on ASMFs, we found that the proportion of Ebf+ cells in control embryos 407 progressively increased from ~20% showing "weak" expression at 15.5hpf to >90%

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showing "strong" expression by 18hpf ( Figure 5F; see Figure 5D for examples of "weak" 409 and "strong" expression). This semi-quantitative analysis revealed an under-appreciated 410 dynamic at the onset of Ebf expression, which appears to take at least one hour to be 411 "strongly" expressed in >75% of newborn ASMFs ( Figure 4F).

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To evaluate the impact of Wee1-induced mitosis inhibition on Ebf accumulation, we 413 focused on undivided STVCs at each time point (hence the lower numbers in Figure 4F 414 compare to Figure 4E). By 17hpf, wee1-expressing delayed STVCs showed "strong" Ebf

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Larvae were also treated with U0126 (+) or DMSO (as negative control, (-)), starting at 17hpf, which 990 corresponds to the transition from a MAPK-dependent to a MAPK-independent autoregulative mode of Ebf 991 expression (see Figure 6A). Wee1-induced delays in cell cycle progression increased the sensitivity of late Ebf 992 expression to MAPK inhibition, further supporting the notion that cell divisions accelerate the transition 993 from MAPK-dependent to MAPK-independent self-activating regulation of Ebf transcription.