From Dynamic Expression Patterns to Boundary Formation in the Presomitic Mesoderm

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.


Installation and Starting the Application
To run the program you need to install the packed file SIM.zip, which can be downloaded from www.helmholtz-muenchen.de/en/ieg/downloads/simu lation11 .
After unpacking the file you get a directory (SIM/) with the following configuration: • Subdirectory ICONS/ contains figures used to design the graphical user interface of the simulation program.
• Subdirectory sim_lib/ contains all supplementary jar files used by the application.
• Subdirectory sim_model/ contains the standard configuration files for the gene regulatory networks mentioned in the publication and the supplementary material: Fgf8_on_Hes1_dmC_Wnt3a_on_NICD_dpN/configfile corresponds to the model shown in Figure 2 (publication), while subdirectories Fgf8_on_Hes7_dpC/ and Fgf8_on_Hes7_dmC/ contain configuration files for the models shown in Figure S1 and Figure S2 (supplementary material). Configfile saves the parameter values needed to start the application.
• Jar file sim.jar contains the simulation program.
• Executable batch file startSIM.bat starts the application on Windows.
• Executable shell script startSim.sh starts the application on Linux and MacOS.
Execute startSim.bat to start the program on Windows and ./startSim.sh on Linux and MacOS. For more details read thefile SIM/README.

Starting the Simulation
The application starts with a graphical user interface, on which the user can see and change the parameter values needed to start the simulation. The buttons to start and stop the simulation are on the bar at the bottom of the graphical user interface (GUI): • By clicking on 'start' the simulation starts with the configuration status described on the parameter panel (see chapter Using the Graphical User Interface).
• Button 'cancel' terminates the Java application.
• It is possible to return from the simulation panel to the parameter panel by clicking 'return'.

Using the Graphical User Interface (GUI)
The picture below shows the upper half part of the GUI from which the simulation can be started. It contains informations about the genes building the gene regulatory network (GRN) and its interactions. 2. Turning it off (as below) has the consequence that all promoter parts containing NICD binding sides are also switched off.
• The value of the 'scaling factor' text field scales all parameters by a factor. This is equivalent to scaling by time. To change the scaling factor proceed as described in the following: 1. Change the text field value (e.g. to 2) and press ENTER to continue. • The 'save all model parameters' button allows the user to update the configuration file sim_model/<model_description>/configfile, i.e. all parameter modifications made on the GUI can be saved to be used by another program run. The old configfile ist moved to OLD/configfile.OLD<index>. By clicking the button a popup window opens with the option to make a notice which will be saved at the beginning of the configuration file (see picture below).
• All genes in the GRN are described on panels sharing the same space. The user can view and accordingly modify the parameter values of one specific gene by selecting the tab corresponding to it. The color of the tab indicates which gene products will be shown during the simulation run or whether gradient genes are coupled or not.
A dark gray colored tab indicates that gradient genes are coupled.

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Cyan color tags the gene which gene products are shown during the simulation.

Selected tab to view parameters of gene Mesp2
The simulation will show the mRNA in cytoplasm of Mesp2

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The Fgf8 protein is coupled on the decay rate of the Hes1 mRNA in cytoplasm.
Reset the value saved in the configuration file if it has been changed in the meantime.
Click the button to hide and accordingly show the corresponding data.
If no selection is made the gene status doesn't change by clicking the OK button.
Each gene can also be excluded from the network. This means that no computation will be done for its products. In case of Delta/Notch genes the elimination of Dll1 (Notch1) in the GRN implies also the elimination of Notch1 (Dll1) and NICD.
In the case of Fgf8 the option of a partial inhibition is also supplied. This means, that the protein production rate can be reduced by a defined amount at a defined time step during the simulation.
The gene panel contains also information about gene promoters.

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A gene knock out can be simulated by setting the mRNA production rate to 0.
To save the changes click on the OK button.
To adopt the changes click on the OK button.
The lower part of the GUI is reserved for general settings like the layout of the proliferating cells and the way they proliferate.
If the '3 dimensional growth zone' option is not selected, the growth zone consists of 1 layer, from which all new cells arise.

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Click on button to show promoter parameters.
Hide or show all promoter data. Switch promoter on or off.
Automatically switched off if corresponding gene is excluded from network.
Parameters for the Hill function describing the promoter.
Selection means that new cells arise simultaneously on multiple layers, the number of which can be set below.
Position of initial amount of cells.
Direction in which the new cells grow.
Number of intermediate states shown during cell division. One growth step corresponds <number of time steps between rendering> * <time step in minutes> minutes, in our case 3 minutes. Thus in this case new cells arise each 12 minutes.
The growth zone, which means that a new cell will randomly arise of each column in this region.

Number of cells on row.
The 'plot data for cells' field gives users the option to output the oscillators numerical data, which means that after each step of the Runge-Kutta method used to solve the system of differential equations modeling the GRN, the data will be written to a file, which can be read by an appropriate program (like gnuplot) to visualize the time course of the concentrations.

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Reset the default values saved in configfile.
The data of this cell will be saved to a file, in which the cell index corresponds the order of cell's appearance. A cell can be selected for plotting its data by typing the cell index and ENTER (important!). A multiple cell selection is also possible, ENTER inserts the cell to the list and DELETE removes the cell from the list, which corresponds the combo box items. For each selected cell a new directory CELL<index_of_cell>/ is created containing config.info with some options from the GUI and cell<index_of_cell> with the numerical data. If the numerical file already exists it will not be overwritten but moved to CELL<index_of_cell>/ OLD/ .
Clears the list of the cells selected for plotting the data.
The simulation starts by clicking the 'start' button at the bottom of the configuration panel.