Mouse Hair Cycle Expression Dynamics Modeled as Coupled Mesenchymal and Epithelial Oscillators

<div><p>The hair cycle is a dynamic process where follicles repeatedly move through phases of growth, retraction, and relative quiescence. This process is an example of temporal and spatial biological complexity. Understanding of the hair cycle and its regulation would shed light on many other complex systems relevant to biological and medical research. Currently, a systematic characterization of gene expression and summarization within the context of a mathematical model is not yet available. Given the cyclic nature of the hair cycle, we felt it was important to consider a subset of genes with periodic expression. To this end, we combined several mathematical approaches with high-throughput, whole mouse skin, mRNA expression data to characterize aspects of the dynamics and the possible cell populations corresponding to potentially periodic patterns. In particular two gene clusters, demonstrating properties of out-of-phase synchronized expression, were identified. A mean field, phase coupled oscillator model was shown to quantitatively recapitulate the synchronization observed in the data. Furthermore, we found only one configuration of positive-negative coupling to be dynamically stable, which provided insight on general features of the regulation. Subsequent bifurcation analysis was able to identify and describe alternate states based on perturbation of system parameters. A 2-population mixture model and cell type enrichment was used to associate the two gene clusters to features of background mesenchymal populations and rapidly expanding follicular epithelial cells. Distinct timing and localization of expression was also shown by RNA and protein imaging for representative genes. Taken together, the evidence suggests that synchronization between expanding epithelial and background mesenchymal cells may be maintained, in part, by inhibitory regulation, and potential mediators of this regulation were identified. Furthermore, the model suggests that impairing this negative regulation will drive a bifurcation which may represent transition into a pathological state such as hair miniaturization.</p></div

Predictions on expression dynamics determined by the two population model.

<p>(<b>A</b>) Histogram of the coefficient of determination (COD) for model estimated expression, shown for all probesets (dark blue background) and probesets identified as low frequency oscillators (LOF, light pink foreground). (<b>B</b>) The magnitude of the t-statistic used to estimate differential expression between the two estimated populations, shown for all probesets (dark blue background) and probesets identified as low frequency oscillators (LOF, light pink foreground). (<b>C</b>) Normalized expression data, , for the two model populations. The left column shows actual expression data and probesets are ordered by the magnitude of the t-statistic. The right column shows expression estimated by the model ordered as in left column.</p

Simulation results from mean field coupled oscillator model.

<p>All curves are calculated by solving EQ 2 (for additional details also see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003914#pcbi.1003914.e135" target="_blank">EQ 11</a>). The magnitude of the first order parameter, shown in red, can be easily calculated from the individual order parameters, and . Here, is related to the first order parameter in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003914#pcbi-1003914-g002" target="_blank">Figure 2</a>, also shown in red (note the subscript was dropped for convenience). (<b>A</b>) Simulation results of for configuration one (config 1, solid) and configuration two (confg 2 dashed). Here config 1 relates to cluster one having negative coupling (). Note that the synchronization was stable only in config 1. We also show the incoherent result when configuration two () was set near, the steady-state value. The top plots show the values of for both cluster 1 (green) and 2 (blue) on the unit circle at time = 1, 12 and 40 days. Note that the clusters are out-of-phase. A movie of the individual oscillators corresponding to configuration 1 is available as Supplementary <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003914#pcbi.1003914.s019" target="_blank">file S2</a>. (<b>B</b>) A simulated bifurcation analysis of the model showing the stable attractors for at different values of (red dots). We note that the simulation results agree with the analytical results of , loss of the incoherent state, and , the upper bound of the wave state. The estimated period of the hair cycle is shown by the dashed line. The values corresponding to the observed hair system are highlighted, note that it is near a critical change in that corresponds to a sharp decrease in the period.</p

Predictions on population dynamics determined by the two population model.

<p>The predicted relative size of the expanding population for both the natural (green) and induced (light gray) hair cycle expression data. The time scale used was set relative to initiation, which was after morphogenesis (postnatal day 23) or after depletion for the natural and induced cycles, respectively.</p

Localization of selected candidate genes from the predicted anagen expanding population.

<p>In Situ Hybridization (ISH; RNA) and immunofluorescence (protein) were performed on mouse skin sections taken from telogen (day0) or anagen (Day 16) phases of the hair cycle, determined by Supplementary <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003914#pcbi.1003914.s008" target="_blank">Figure S8A</a>. DAPI was used as a counterstain for cell nuclei (blue). The expression of candidate genes for ISH is seen as bright foci (red and green) in specific cell types. Note comparisons to technical negative control and positive controls in Supplementary <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003914#pcbi.1003914.s009" target="_blank">Figure S9</a>. Foxn1 (red) was identified as a candidate matrix derived cell marker and was used here as a positive control for localization to matrix cells <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003914#pcbi.1003914-Mecklenburg1" target="_blank">[50]</a>. (<b>A</b>) ISH: R1 and 2 shows RNA expression for Ovol1 and Smad6 (green), respectively, which were predicted to be expressed in follicle cell populations that expand during the anagen phase. No expression was observed during the telogen phase (Day 0). (<b>B</b>) Protein staining by immunofluorescence: R1 and 2 shows RNA expression for Ovol1 and Smad6 (green), respectively. Again, no expression was observed during the telogen phase (Day 0).</p

Localization of selected candidate genes from the predicted static background population markers.

<p>In Situ Hybridization (ISH; RNA) and immunofluorescence (protein) were performed on mouse skin sections taken from telogen (day0) or anagen (Day 16) phases of the hair cycle, determined by Supplementary <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003914#pcbi.1003914.s008" target="_blank">Figure S8</a> A. DAPI was used as a counterstain for cell nuclei (blue). The expression of candidate genes for ISH is seen as bright foci (red and green) in specific cell types. Note comparisons to technical negative control and positive controls in Supplementary <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003914#pcbi.1003914.s009" target="_blank">Figure S9</a>. (<b>A</b>) ISH: Fgf7 (red) was used as a positive control marker, which has been reported to be expressed in DP cells <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003914#pcbi.1003914-Rosenquist1" target="_blank">[42]</a>. R1 and 2 shows RNA expression for Stat5a and Fermt2 (green), respectively, which were predicted to be expressed in dermal papilla, or other background population during the telogen and anagen phase. (<b>B</b>) Protein staining by immunofluorescence: Due to technical issues with Fgf7 and Fermt2 antibody selection, morphology was used to determine localization and Vim was chosen as an alternate candidate marker. Vim was predicted to be expressed in dermal papilla or other background population during the telogen and anagen phases. R1 and 2 shows protein expression for Stat5a and Vim (green), respectively.</p

Periodic gene expression in mouse hair cycle.

<p>An approximation of the hair cycle phase corresponding to the time scale is indicated via a color bar, see legend in lower right. (<b>A</b>) Heat map of actual expression data for probesets identified as periodic. For visualization, the data is normalized relative to the corresponding maximum and minimum values (this normalization was not used in statistical analysis), and sorted first by principal frequency and second by phase. (<b>B</b>) Heat map of expression estimated by the principal periodic component corresponding to A. Sorted and normalized as in A. (<b>C</b>) Histogram of the phases for probeset expression patterns corresponding to the longest period, 31 days, estimated by the principal periodic component.</p

48 h Western blot data for control and RA-treated WT, R38+ and R38− HL60 cells.

<p>A representative blot is displayed above its respective bar graph, and each bar graph (error bars represent standard error) presents the fold change respective to each control. The fold change was calculated after performing densitometry across three or more repeated blots. Note that the scale of the y-axis for each bar graph differs. A: There was no change in total ERK or MEK levels for any cell line. RA induced MEK and ERK phosphorylation in all three cell lines. Only RA-treated WT HL60 cells showed upregulation of c-Raf expression. Also, only RA-treated WT HL60 cells exhibited increased c-Raf phosphorylation at S259, S621 and S289/296/301. Neither R38+ nor R38− displayed increased c-Raf expression or phosphorylation after RA treatment. B: RA-treated WT HL60 cells showed upregulation of Lyn, Fgr, Vav1, and c-Cbl expression. RA-inducible Slp76 expression was evident in R38+ and R38−. Immunoprecipitation of c-Cbl followed by blotting of CD38 reveals that there is little CD38 and c-Cbl interaction in RA-treated R38+ compared to RA-induced WT HL60. GAPDH (not shown) served as loading control; c-Cbl (not shown) served as control for c-Cbl immunoprecipitation.</p

Diagrams of the signaling proteins investigated in this study for each treatment case in R38+ and R38− compared to the RA-treated WT HL60 cells.

<p>For a given protein, solid black arrows indicate increased expression while white arrows indicate increased phosphorylation. Dashed black lines indicate an interaction that has previously been demonstrated in this lab by immunoprecipitation and/or FRET, with the exception of Fgr:Slp76 and Fgr:c-Raf. These two interactions are implied by the known binding of Slp76 and c-Raf to Src-family kinase members. Dashed black lines with arrowheads indicate phosphorylation (kinase) events known in the literature. Solid-filled factors vs. white-filled factors serve to clarify the expression indicated by the black arrows. Gradient-filled factors indicate expression but to a lower level than the RA-treated WT case. Downstream effects (CD11b expression, p47^phox expression, etc) are written in black if they occur, grey if they do not occur, or in gradient if they occur but to a lesser extent than the RA-treated WT HL60 case. A: In RA-treated WT HL60, CD38 is upregulated, along with its intracellular binding partners Slp76, Vav1, c-Cbl, and Lyn. Fgr is also upregulated. MEK and ERK show increased phosphorylation, while c-Raf is upregulated and shows increased phosphorylation at S259, S621 and S289/296/301. Differentiation markers that occur include CD11b expression, cell cycle arrest, p47^phox expression and inducible ROS production. B: In RA-treated R38+ HL60, CD38 is upregulated, but not Vav1, c-Cbl, or Lyn. Fgr is not upregulated. MEK and ERK show increased phosphorylation; however c-Raf is not upregulated, nor shows increased phosphorylation. Increased CD11b expression, cell cycle arrest, p47^phox expression and inducible ROS production do not occur. C: In RA-treated R38− HL60, CD38 is not upregulated, nor Vav1, c-Cbl, or Lyn. Fgr is not upregulated. MEK and ERK show increased phosphorylation; however c-Raf is not upregulated, nor shows increased phosphorylation. Increased CD11b expression, cell cycle arrest, p47^phox expression and inducible ROS production do not occur. D: In PP2-treated R38+ HL60, CD38 is partially upregulated (indicated by the gradient CD38), and Slp76, Vav1, c-Cbl, and Lyn are upregulated. Fgr is not upregulated. MEK and ERK phosphorylation is decreased; however c-Raf is upregulated and shows increased phosphorylation. Increased cell cycle arrest occurs, but not increased CD11b expression, p47^phox expression or inducible ROS production. E: In PP2-treated R38− HL60, CD38 is not upregulated, but Slp76, Vav1, c-Cbl, and Lyn are upregulated. Fgr is not upregulated. MEK and ERK phosphorylation is decreased; however c-Raf is upregulated and shows increased phosphorylation. Increased cell cycle arrest occurs, but not increased CD11b expression, p47^phox expression or inducible ROS production. F: In PP2+RA-treated R38+ HL60, CD38 is upregulated, along with Slp76, Vav1, c-Cbl, and Lyn. Fgr is also upregulated. MEK and ERK phosphorylation is decreased; however c-Raf is upregulated and shows increased phosphorylation. Differentiation markers that occur include CD11b expression, cell cycle arrest, and p47^phox expression, but not inducible ROS production. G: In PP2+RA-treated R38− HL60, CD38 is partially upregulated, along with Slp76, Vav1, c-Cbl, and Lyn. Fgr is also upregulated. MEK and ERK phosphorylation is decreased; however c-Raf is upregulated and shows increased phosphorylation. Differentiation markers that occur include partial CD11b expression, cell cycle arrest, and p47^phox expression, but not inducible ROS production.</p

The Src-Family Kinase Inhibitor PP2 Rescues Inducible Differentiation Events in Emergent Retinoic Acid-Resistant Myeloblastic Leukemia Cells

<div><p>Retinoic acid is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing maturation in leukemia cells. Using HL60 model human myeloid leukemia cells, where all-<i>trans</i> retinoic acid (RA) induces granulocytic differentiation, we developed two emergent RA-resistant HL60 cell lines which are characterized by loss of RA-inducible G1/G0 arrest, CD11b expression, inducible oxidative metabolism and p47^phox expression. However, RA-treated RA-resistant HL60 continue to exhibit sustained MEK/ERK activation, and one of the two sequentially emergent resistant lines retains RA-inducible CD38 expression. Other signaling events that define the wild-type (WT) response are compromised, including c-Raf phosphorylation and increased expression of c-Cbl, Vav1, and the Src-family kinases (SFKs) Lyn and Fgr. As shown previously in WT HL60 cells, we found that the SFK inhibitor PP2 significantly increases G1/G0 cell cycle arrest, CD38 and CD11b expression, c-Raf phosphorylation and expression of the aforementioned regulators in RA-resistant HL60. The resistant cells were potentially incapable of developing inducible oxidative metabolism. These results motivate the concept that RA resistance can occur in steps, wherein growth arrest and other differentiation events may be recovered in both emergent lines. Investigating the mechanistic anomalies in resistant cell lines is of therapeutic significance and helps to mechanistically understand the response to retinoic acid’s biological effects in WT HL60 cells.</p> </div