Comprehensive ECG reference intervals in C57BL/6N substrains provide a generalizable guide for cardiac electrophysiology studies in mice.

Reference ranges provide a powerful tool for diagnostic decision-making in clinical medicine and are enormously valuable for understanding normality in pre-clinical scientific research that uses in vivo models. As yet, there are no published reference ranges for electrocardiography (ECG) in the laboratory mouse. The first mouse-specific reference ranges for the assessment of electrical conduction are reported herein generated from an ECG dataset of unprecedented scale. International Mouse Phenotyping Consortium data from over 26,000 conscious or anesthetized C57BL/6N wildtype control mice were stratified by sex and age to develop robust ECG reference ranges. Interesting findings include that heart rate and key elements from the ECG waveform (RR-, PR-, ST-, QT-interval, QT corrected, and QRS complex) demonstrate minimal sexual dimorphism. As expected, anesthesia induces a decrease in heart rate and was shown for both inhalation (isoflurane) and injectable (tribromoethanol) anesthesia. In the absence of pharmacological, environmental, or genetic challenges, we did not observe major age-related ECG changes in C57BL/6N-inbred mice as the differences in the reference ranges of 12-week-old compared to 62-week-old mice were negligible. The generalizability of the C57BL/6N substrain reference ranges was demonstrated by comparison with ECG data from a wide range of non-IMPC studies. The close overlap in data from a wide range of mouse strains suggests that the C57BL/6N-based reference ranges can be used as a robust and comprehensive indicator of normality. We report a unique ECG reference resource of fundamental importance for any experimental study of cardiac function in mice

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

<i>Mesp2</i> expression under reduced Fgf signaling.

<p>Virtual expression patterns for <i>Mesp2</i> (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 <i>Mesp2</i> expression when FGF8 signaling was reduced. The tail bud of the PSM is growing from left to right.</p

Virtual expression patterns for <i>Hes7</i> mRNA for systems of different dimensionality.

<p>Snapshots are taken at time point 680 min. At the start of the simulation 100% noise was added. The time course of our correlation function is displayed below ending at 50000 time steps equivalent to 5000 min, which shows how the different systems approach the synchronized state (Correlation function = 0). (Abscissa showing time measured in time steps.)</p

Virtual expression patterns for <i>Hes7</i> mRNA in simulation runs with different cis-inhibition values.

<p>Snapshots are taken at 500, 15000, 35000, 50000 time steps (1 time step = 0.1 min) after simulation start for a 7×7×7 cell cube for different D/N cis-inhibition strengths. In all cases 100% noise was added at the start of the simulation. On the left side the time course of the correlation function C(t) (red curve) and the synchronization measure R (blue curve) is shown.(Abscissa showing time measured in time steps.)</p

Virtual expression patterns as simulated <i>in silico</i> by the proposed gene regulatory network.

<p>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.</p

Synchronization of gene expression in somitogenesis by Delta/Notch cis-inhibition.

<p>Panel <b>A</b> shows our reaction scheme depicting the gene regulatory network. It is sketched for one cell (right) and part of a neighboring cell (left) showing those reactions that involve ligand-receptor interactions in D/N signaling and the <i>Hes7</i> oscillator. Gradient forming genes in the PSM like Fgf8, Wnt3a, and Tbx6 are not shown. Each circular area represents one gene, mRNA and protein are color coded blue and red, respectively. For fast changing gene products we simulate the transport between cellular compartments explicitly, which is indicated by subdivided circle half-areas. Activating or repressing arrows represent regulatory interactions. Degradation or decay reactions are symbolized by arrows to the empty-set symbol. For clarity, we omit in the scheme all species decays except for the D/N cis-interaction, which is assumed to lead to a fast decay of the intracellular D/N complex. Panel <b>B</b> is a sketch showing D/N interactions. D/N-transactivation (lower part of the panel): DLL1 ligand (yellow) on cell 1 binds to NOTCH1 receptor in the membrane of cell 2, whereupon the NOTCH1 intracellular domain is cleaved off, moves into the cell nucleus and activates the <i>Hes7</i> promoter. D/N cis-inhibition (upper part of the panel): We assume an excess of Notch1 in cell 2. DLL1 molecules in the membrane of cell 2 bind NOTCH1 in the same cell and are inactivated after endocytosis to a lysosome. Panel <b>C</b> shows a schematic drawing of the growing PSM: in the anterior region of the PSM (right) cells are synchronized as shown in the blow-up of a small rectangle of the PSM of this region, while in the tail bud cells are not synchronized i.e. out of phase as shown in the blow-up of the left small rectangle. Cells in the blow-ups are coupled by D/N signaling (small yellow and blue bars on the surface of the central cell). Connections are shown only for the central cell.</p

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

<p>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 <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002586#pcbi.1002586-Tiedemann1" target="_blank">[28]</a> for an extensive discussion). NICD, which originates through cleavage reactions following DLL1 ligand binding to the NOTCH1 receptor <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002586#pcbi.1002586-Fortini1" target="_blank">[3]</a>, 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.</p

Snapshots of virtual expression patterns for <i>Hes7</i> mRNA in simulations of the growing PSM.

<p>The posterior-to-anterior FGF8 gradient is coupled to the HES7 decay. One time step equals 0.1 minute. From left to right shown are the cases of 100% noise added during division of cells in the growth zone of the PSM without (shown also in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003843#pcbi.1003843.s009" target="_blank">movie S3</a>) and with (shown also in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003843#pcbi.1003843.s010" target="_blank">movie S4</a>) D/N cis-inhibition. Snapshots are also displayed for simulation runs wherein the disturbance of oscillator consonance is caused by shutting down the transcription of the core oscillator genes during mitosis. Shown are the cases of 20 min shutdown of the transcription during cell division in the growth zone of the PSM without (shown also in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003843#pcbi.1003843.s012" target="_blank">movie S6</a>) and with (shown also in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003843#pcbi.1003843.s011" target="_blank">movie S5</a>) D/N cis-inhibition. Cells are colored orange in the simulations as long as transcription of their genes is shut down.</p

<i>Mesp2</i> expression without <i>Hes7</i>.

<p>The virtual expression patterns for <i>Mesp2</i> (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 <i>Mesp2</i> expression when <i>Hes7</i> is eliminated from the GRN (virtual <i>Hes7</i> knock-out). The tail bud of the PSM is growing from left to right.</p