Figure 8. Delayed onset of transcription restricts expression range.

(A) Simulation of efnb2b expression using the kinetic model without (left) or with (right) a co-transcriptional activator Y. The dependence on Y delays the onset of efnb2b expression and reduces its range. (B) Top: Experimental design. Bottom: Time-course induction of efnb2b after injecting recombinant Nodal protein at 3.5 hpf (green) and 4.5 hpf (orange). The induction kinetics of this gene are very slow, but the later Nodal is injected, the faster its induction. Note that counts for the expression of late target genes are higher after early injection compared to later injections. This effect might be due to the fact that after early injections phospho-Smad2 levels are high for a longer period before a gene becomes competent to respond as compared to late injections, when there is a shorter time window of high phospho-Smad2 levels. There might be a priming mechanism in which longer exposure to activated Smad2 increases gene expression when competence is reached. (C) RNA fluorescent in situ hybridization for efnb2b at 3, 4.5 and 6 hpf. Expression of efnb2b is only detected at 6 hpf, although Nodal signaling and the expression of most other Nodal targets commences much earlier.

DOI: http://dx.doi.org/10.7554/eLife.05042.027

Figure 8—figure supplement 1. Characterization of co-regulated Nodal target genes.

Left panel: binding peaks of Smad2 and the associated transcription factor FoxH1 at dome stage after injection of Squint mRNA (red) or after treatment with the Nodal signaling inhibitor SB505124 (blue). Peaks called by the MACS algorithm are indicated (gray blocks). Middle panel: NanoString count levels after mNodal injection (red) or after SB treatment (blue) in the absence (−cycloH) or the presence (+cycloH) of the translation inhibitor cycloheximide. Right panel: time course induction of target genes after Nodal injection at 3.5 hpf (green) or 4.5 hpf (orange). Although these genes have Smad2/FoxH1 binding sites, their induction is abolished in the presence of cycloheximide, suggesting that an additional transcriptional co-regulator controls their transcription. Moreover, these genes are delayed in their onset of expression. The length of the delay depends on the stage at which Nodal is applied.

DOI: http://dx.doi.org/10.7554/eLife.05042.028

Figure 8—figure supplement 2. Transcriptional competence regulates the onset and range of bra expression.

(A) Concentration-dependent induction of flh (left), bra (middle) and gsc (right). bra and flh can be induced at low Nodal concentrations. (B) Induction dynamics of bra after injecting Nodal at 3.25, 4.25 and 5.25 hpf as a function of time after injection (left) or as a function of absolute embryonic time (right). Arrowheads in the right panel indicate the time of Nodal injection. bra can only be induced when the embryo has reached a specific embryonic stage. bra expression is detected by RT-qPCR. (C) Fluorescent RNA in situ hybridization with bra probe at 3, 4.5, 6 hpf. bra is only detected at 6 hpf in 5 cell tiers.

DOI: http://dx.doi.org/10.7554/eLife.05042.029