From dynamic expression patterns to boundary formation in the presomitic mesoderm

Abstract

The segmentation of the vertebrate body is laid down during early embryogenesis. The formation of signaling gradients, the periodic expression of genes of the Notch-, Fgf- and Wnt-pathways and their interplay in the unsegmented presomitic mesoderm (PSM) precedes the rhythmic budding of nascent somites at its anterior end, which later develops into epithelialized structures, the somites. Although many in silico models describing partial aspects of somitogenesis already exist, simulations of a complete causal chain from gene expression in the growth zone via the interaction of multiple cells to segmentation are rare. Here, we present an enhanced gene regulatory network (GRN) for mice in a simulation program that models the growing PSM by many virtual cells and integrates WNT3A and FGF8 gradient formation, periodic gene expression and Delta/Notch signaling. Assuming Hes7 as core of the somitogenesis clock and LFNG as modulator, we postulate a negative feedback of HES7 on Dll1 leading to an oscillating Dll1 expression as seen in vivo. Furthermore, we are able to simulate the experimentally observed wave of activated NOTCH (NICD) as a result of the interactions in the GRN. We esteem our model as robust for a wide range of parameter values with the Hes7 mRNA and protein decays exerting a strong influence on the core oscillator. Moreover, our model predicts interference between Hes1 and HES7 oscillators when their intrinsic frequencies differ. In conclusion, we have built a comprehensive model of somitogenesis with HES7 as core oscillator that is able to reproduce many experimentally observed data in mice.

Figure 1. Relation between temporal evolution of gene expression and spatial pattern in somitogenesis.

Panels A, C, and D show the time evolution of gene expressions in our model for one cell (not all drawn to scale). Panel B depicts a sketch of the PSM (anterior to the right) with formed somites, the growth zone, and a forming somitic cleft, in which the anterior boundary of Mesp2 expression marks the upcoming somite boundary. (A) Mesp2 is induced by dynamic NICD expression in concert with TBX6. The Mesp2 expression boundary moves to the right together with the WNT3A and FGF8 gradients that are generated by decay of their gene products after their expression has stopped outside the growth zone. At the same time, Mesp2 is repressed by FGF8 signaling and by RIPPLY2, which is induced by MESP2. By the growing PSM the temporal expression is mapped into a spatial pattern: in a moving ‘window of opportunity’ between activating TBX6 and repressive FGF8 expression, Mesp2 is induced when NICD is highly expressed, i.e. the NICD wave ‘moves’ into this window. (C) NICD expression oscillates as a result of the reaction of NOTCH1 (static expression) with DLL1, which is dynamic and controlled by the negative feedback oscillator HES7. (D) NICD forms a ‘wave’ because its oscillations are slowed down by interaction with the WNT3A gradient.

Figure 2. Reaction scheme of the proposed gene regulatory network (GRN).

The scheme details the full GRN for one cell and part of a neighboring cell for those reactions that involve ligand-receptor interactions like in Delta-Notch signaling or input from the Fgf8 or Wnt3a signal transduction pathways. Color-coded circular areas for each gene symbolize mRNA and protein. For fast changing gene products the transport of mRNA or protein between cytoplasm and nucleus or between cytoplasm and membrane is explicitly simulated, which is indicated by dividing each half-area of the circle again. Regulatory interactions are shown as activating or repressing arrows. Broken lines indicate that the interaction is simulated only in an even more course-grained manner than the other gene regulatory reactions (see for an extensive discussion). NICD, which originates through cleavage reactions following DLL1 ligand binding to the NOTCH1 receptor , was assigned a separate symbol to clarify that only the intracellular domain of the Notch receptor acts in the nucleus as a transcription (co)-factor. The (weak) modulating action of LFNG on D/N signaling is shown as dashed lines - (red for the case of inhibiting action, green for the case of a positive effect on the D/N reaction rate.) Arrows pointing to the symbol for the empty set designate decay reactions of a species. We suppressed them for all species' decays except for those decay rates that we assume as controlled by signal transduction pathways. This applies also to the removal of DLL1 and NOTCH1 from the membrane after their binding, resulting in NOTCH1 cleavage and NICD split-off.

Figure 3. Virtual expression patterns as simulated in silico by the proposed gene regulatory network.

Expression patterns are shown at three different time points in one oscillation cycle for one half of the PSM. Cytoplasmic mRNAs are colored in blue, proteins in red. The tail bud is growing from left to right. When EPHA4 concentration has reached a certain threshold, the virtual cells change their shape to symbolize epithelialization at the forming somite border.

Figure 4. Mesp2 expression without Hes7.

The virtual expression patterns for Mesp2 (cytoplasmic mRNA) are shown at five different time points in one complete and part of the following oscillation cycle. Panels on the left show the wild-type situation, panels on the right show Mesp2 expression when Hes7 is eliminated from the GRN (virtual Hes7 knock-out). The tail bud of the PSM is growing from left to right.

Figure 5. Mesp2 expression without Ripply2.

The virtual expression pattern for Mesp2 (cytoplasmic mRNA) is shown at three different time points in one oscillation cycle when Ripply2 is eliminated from the GRN. The tail bud of the PSM is growing from left to right.

Figure 6. Mesp2 expression under reduced Fgf signaling.

Virtual expression patterns for Mesp2 (cytoplasmic protein) at five different time points in one complete and part of the following oscillation cycle, when FGF8 protein production rate is reduced by 50%, 600 minutes after the simulation has been started. Panels on the left show the wild-type situation, panels on the right show Mesp2 expression when FGF8 signaling was reduced. The tail bud of the PSM is growing from left to right.

Figure 7. Changing the HES7 nuclear decay rate leads to a disturbance of the Hes1 oscillator.

The top panel shows the virtual expression pattern for Hes1 (cytoplasmic mRNA) with default parameters values, the panel below shows Hes1 expression when the nuclear decay rate of HES7 was changed resulting in the occurrence of beats, which are visualized in the uppermost concentration plot over time. The concentration plot in the middle shows the time course without the influence of the gradient decay. The plot at the bottom shows the time course of Hes7 mRNA for comparison.