Research ArticleDevelopmental Biology

Serotonergic regulation of melanocyte conversion: A bioelectrically regulated network for stochastic all-or-none hyperpigmentation

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Sci. Signal.  06 Oct 2015:
Vol. 8, Issue 397, pp. ra99
DOI: 10.1126/scisignal.aac6609
  • Fig. 1 cAMP and CREB are involved in mediating instructor cell signaling.

    (A and B) Frogs treated with pharmacological agents at stage 10 are scored for pigmentation phenotypes at stage 45. (C) Percent of the population exhibiting hyperpigmentation in embryos at stage 45 after treatment at stage 10 with the cAMP antagonist 2′5′-dideoxyadenosine (2′5′-DDA) with or without ivermectin (IVM) or the AC activator forskolin. (D) Percent of population exhibiting hyperpigmentation in embryos at stage 45 after treatment at stage 10 with the protein kinase A (PKA) antagonist H89 with or without ivermectin or injected into one cell at the four-cell stage with the mRNA for the indicated CREB with or without ivermectin. A-CREB encodes dominant-negative (DN) CREB; XlCreb1 encodes wild-type (WT) CREB; and VP16-XlCreb1 encodes constitutively active (CA) CREB. In (C) and (D), sample sizes (number of embryos) are noted for each condition. Error bars represent 1 SEM. *P < 0.0001 based on Pearson’s χ2 test.

  • Fig. 2 The pituitary gland is necessary for ivermectin-mediated hyperpigmentation.

    (A) Side view showing the main regions of Xenopus tadpole brain. (B and C) Xenopus embryos were treated with ivermectin from neurula stage (stage 10), cut at tail bud stage (stage 32), raised to tadpole stage (stage 45), and scored for hyperpigmentation (HP). Cuts were performed on tail bud stage (stage 32), removing the pineal gland, pituitary gland, or a control region below the cement gland and away from both the pituitary and pineal. (D) Effect of control cuts (n = 54 embryos), pineal cuts (n = 46 embryos), or pituitary cuts (n = 59 embryos) on the percent of ivermectin-induced hyperpigmented tadpoles. Effect of inhibition of MSH with αMSH release–inhibiting factor (MSH-RIF), an MSH agonist SHU 9119, or a 5HT receptor antagonist altanserin on the percent of hyperpigmented tadpoles. Error bars represent 1 SEM. *P < 0.0001, Pearson’s χ2 test; NS, not significant.

  • Fig. 3 Both localized depolarization and sparse, widely distributed depolarization increase Sox10 transcripts.

    (A) Expression of Sox10 in embryos injected with a mixture of XminK and β-gal mRNAs at the four-cell stage that were fixed at tail bud stages. Far left shows injection, and middle shows the section plane for the data shown at the right. (B) Effect of ivermectin treatment or XminK injection on the expression of Sox10 and Slug in tail bud stage embryos (stage 25) as assessed by RT-qPCR. XminK-injected animals were injected into one cell at the four-cell stage. Ivermectin-treated animals were exposed to the drug from neurula stage (stage 10) onward until processing for RT-qPCR at stage 25. Control animals were uninjected and untreated. Red dashed line denotes no fold change compared to control. All experimental treatments resulted in significant increase in expression of both Sox10 and Slug compared to control (P < 0.05, Student’s t test; n = 10 embryos per sample, samples run in triplicate, three biological replicates). (C) Effect of ivermectin exposure started at neurula stage on Sox10 expression as assessed by RT-qPCR in embryos collected at the indicated stages. ST, stage. NF stages 15 to 35, n = 10 embryos per sample, samples run in triplicate, three biological replicates; NF stage 45, n = 5 embryos per sample, samples run in triplicate, three biological replicates. P < 0.05, Student’s t test. (D to F) In situ hybridization for Sox10 performed on stage 15 control (CTRL) embryos (D) or embryos that had been exposed to ivermectin treatment starting at stage 10 (E and F). (F) is an enlargement of the area boxed in (E), and arrowheads indicate positive staining outside the main Sox10-positive area.

  • Fig. 4 Human homologs of the genes with increased expression resulting from ivermectin-induced depolarization are associated with neoplasms and cancer.

    (A) Venn diagram of the transcripts that were differentially regulated (increased or decreased) in transcriptional microarrays of Xenopus tadpoles treated with ivermectin starting at stage 10 and collected at early (stage 15) and late stage (stage 45). See fig. S1 for the differentially expressed transcripts clustered by stage and condition. See tables S1 and S2 for a list of the transcripts and genes. (B and C) Pathway analysis of the differentially expressed transcripts in stage 15 embryos (B) and stage 45 embryos (C). Proteins are red shapes, diseases are purple boxes, stimulatory regulatory events are indicated by an arrow and a plus sign on the relationship line, inhibitory regulatory events are indicated by a blunt line, and arrows without any sign indicate direct binding of proteins.

  • Fig. 5 Inferred dynamic stochastic model recapitulating the observed phenotype frequencies.

    (A) Reverse-engineered network that produces hyperpigmentation phenotype at frequencies matching those observed experimentally. Starting network elements were the drugs (text without a shape) and the named nodes and blue interaction lines, representing known elements and relationships in the hyperpigmentation (HP node) pathway. Elements added algorithmically included three unknown required components (nodes a, b, and c) and green interaction lines. The model also described the kinetic parameters (see text S3 for details). Multiple signaling interactions are combined together in a necessary (dashed lines) or sufficient (solid lines) fashion (see Materials and Methods for details). Activating regulatory interactions are indicated with arrows, and inhibiting interactions are indicated with blunt lines. (B) Simulations of the model showing the dynamic changes in concentration of each colored node after running the model to steady state and the proportion of normally pigmented (pigmentation value of 0) and hyperpigmented (pigmentation value of 1) tadpoles at the end of the simulation. Simulations of control (no treatment), the presence of cyanopindolol, an inhibitor of 5HT-R1, and ivermectin and cyanopindolol are shown.

  • Fig. 6 Inferred model performance with the experiments in the training data set and the experiments in the validation data set.

    (A) Percentage of correct outcomes of the model for each of the experiments used to reverse-engineer the model (training data set). Dashed line indicates 85% performance accuracy. (B) Percentage of correct predictions of the model for a set of experiments not used in model generation (validation data set). See data S2 for details. MTP, methiothepin.

  • Fig. 7 Phase space of the inferred model showing stochastic developmental trajectories and pharmacological treatment bifurcations.

    (A to C) Trajectories of the state of two serotonin receptors (5HT-R1 and 5HT-R2) and degree of hyperpigmentation in the inferred model during 100 simulations for each of three representative treatments. “0” represents inactive on the 5HT-R axes; “2” or “2.5” represents full activity. “0” on the hyperpigmentation axis represents normally pigmented outcome; “1” represents a fully hyperpigmented outcome. The initial state of the embryo (yellow dot) is the same for all the simulations for a given treatment. The attractors represent hyperpigmented phenotypes (red dots) and nonhyperpigmented phenotypes (blue dots) and are the stable states to which the system converges. The attractor dot size is proportional to the number of trajectories that converge to that phenotype. Note that in the ivermectin condition, there are two attractors for the hyperpigmented state, a representation of how different trajectories can produce the same phenotypic outcome. (D) Qualitative phase-space trajectories summarizing the dynamics of the inferred model during three different treatments. In the model, each treatment represents a change to the initial concentration of the indicated pharmacological drug, resulting in a bifurcation (a global change in the dynamics of the system) in the signaling network, a shift in the attractors, and different frequencies for the resultant phenotypes. The trajectory linewidths are proportional to the frequency of simulations producing the trajectory.

  • Fig. 8 Schematic pathways for melanocyte control downstream of instructor cell signaling.

    Under normal conditions the polarized instructor cells produce a relatively small serotonergic signal (stars) due to reuptake and retention of 5HT by the SERT into the instructor cells. At this relatively low concentration of serotonin, only a high-affinity serotonin receptor 5HT-R5 on the pituitary melanotrope cells may become activated, thereby producing normally pigmented animals. Right: When the instructor cell population is depolarized, SERT exports serotonin, resulting in increased serotonin concentrations in the microenvironment of the melanocytes and the pituitary. At this higher concentration, serotonin may activate 5HT-R5 and 5HT-R2 on both the pituitary melanotrope cells and on melanocyte cells. In the melanotrope cells, activation of 5HT receptors stimulates AC activity (not shown), thereby enhancing MSH release. Serotonin signaling increases the abundance of pro-opiomelanocortin (POMC), a precursor of MSH. MSH binding to melanocortin 1 receptors (MC1R) on melanocytes stimulates AC activity (not shown), increasing cAMP production, PKA activity (not shown), and CREB phosphorylation and activity (not shown), thereby increasing the expression of Sox10 and Slug. By increasing cAMP in melanocytes, serotonin may also contribute proliferation, invasive migration, and the altered morphology of the melanocytes. Although melanocytes can also respond to serotonin directly, this pathway is not shown here because the most parsimonious network that explains all of the results, including the long-range signaling by instructor cells, does not require this link.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/397/ra99/DC1

    Text S1. Model implementation, simulation, and evaluation.

    Text S2. Method for reverse-engineering a stochastic model of hyperpigmentation.

    Text S3. System of equations and kinetic parameters for the reverse-engineered model.

    Fig. S1. Hierarchical clustering of the differentially regulated transcripts.

    Table S1. A list of the 45 transcripts differentially expressed by stage 15 in response to ivermectin.

    Table S2. A list of the 517 transcripts differentially expressed by stage 45 in response to ivermectin.

    Table S3. Enriched GO affected in stage 45 embryos.

    Table S4. Cancer-related genes in humans of the homologs of the genes differentially expressed in stage 45 Xenopus tadpole embryos after depolarizing ivermectin treatment.

    Table S5. Reference genes and primers for RT-qPCR.

    Data S1. Differentially expressed transcripts in early and late embryos and their association with disease or cellular process.

    Data S2. Results of the training set and validation set experiments and the performance of the model for each.

  • Supplementary Materials for:

    Serotonergic regulation of melanocyte conversion: A bioelectrically regulated network for stochastic all-or-none hyperpigmentation

    Maria Lobikin, Daniel Lobo, Douglas J. Blackiston, Christopher J. Martyniuk, Elizabeth Tkachenko, Michael Levin*

    *Corresponding author. E-mail: michael.levin{at}tufts.edu

    This PDF file includes:

    • Text S1. Model implementation, simulation, and evaluation.
    • Text S2. Method for reverse-engineering a stochastic model of hyperpigmentation.
    • Text S3. System of equations and kinetic parameters for the reverse-engineered model.
    • Fig. S1. Hierarchical clustering of the differentially regulated transcripts.
    • Legends for tables S1 and S2
    • Table S3. Enriched GO affected in stage 45 embryos.
    • Table S4. Cancer-related genes in humans of the homologs of the genes differentially expressed in stage 45 Xenopus tadpole embryos after depolarizing ivermectin treatment.
    • Table S5. Reference genes and primers for RT-qPCR.
    • Legends for data S1 and S2

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 495 KB

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). A list of the 45 transcripts differentially expressed by stage 15 in response to ivermectin.
    • Table S2 (Microsoft Excel format). A list of the 517 transcripts differentially expressed by stage 45 in response to ivermectin.
    • Data S1 (Microsoft Excel format). Differentially expressed transcripts in early and late embryos and their association with disease or cellular process.
    • Data S2 (Microsoft Excel format). Results of the training set and validation set experiments and the performance of the model for each.

    Citation: M. Lobikin, D. Lobo, D. J. Blackiston, C. J. Martyniuk, E. Tkachenko, M. Levin, Serotonergic regulation of melanocyte conversion: A bioelectrically regulated network for stochastic all-or-none hyperpigmentation. Sci. Signal. 8, ra99 (2015).

    © 2015 American Association for the Advancement of Science

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