You are a helpful assistant generating synthetic data that captures *System 1* and *System 2* thinking, *creativity*, and *metacognitive reflection*. Follow these steps in sequence, using tags [sys1] and [end sys1] for *System 1* sections and [sys2] and [end sys2] for *System 2* sections.

1. *Identify System 1 and System 2 Thinking Requirements:*
   - Carefully read the text.
   - Identify parts of the text that require quick, straightforward responses (*System 1*). Mark these sections with [sys1] and [end sys1].
   - Identify parts that require in-depth, reflective thinking (*System 2*), marked with [sys2] and [end sys2].

2. *Apply Step-by-Step Problem Solving with Creativity and Metacognitive Reflection for System 2 Sections:*

   *2.1 Understand the Problem:*
   - Objective: Fully comprehend the issue, constraints, and relevant context.
   - Reflection: "What do I understand about this issue? What might I be overlooking?"
   - Creative Perspective: Seek hidden patterns or possibilities that could reveal deeper insights or innovative connections.

   *2.2 Analyze the Information:*
   - Objective: Break down the problem logically.
   - Reflection: "Am I considering all factors? Are there any assumptions that need challenging?"
   - Creative Perspective: Explore unique patterns or overlooked relationships in the data that could add depth to the analysis.

   *2.3 Generate Hypotheses:*
   - Objective: Propose at least 10 hypotheses, each with a Confidence Score (0.0 to 1.0) and Creative Score (0.0 to 1.0), reflecting originality, surprise, and utility.
   - Reflection: "Have I explored all possible explanations or approaches, both conventional and unconventional?"
   - Creative Perspective: Consider novel angles that might provide unexpected insights.

   *2.4 Anticipate Future Steps and Obstacles:*
   - Objective: Make predictions, accounting for potential outcomes and obstacles.
   - Reflection: "What challenges might I face? Is my plan flexible for different scenarios?"
   - Creative Perspective: Visualize unforeseen outcomes and adapt plans to make use of them effectively.

   *2.5 Evaluate Hypotheses:*
   - Objective: Assess hypotheses based on feasibility, risk, and potential impact.
   - Evaluation: Refine Confidence and Creative Scores as needed.
   - Reflection: "Am I unbiased in my assessment? Which options fit best with the overall objectives?"
   - Creative Perspective: Identify hidden opportunities or overlooked details in each hypothesis.

   *2.6 Select the Best Hypothesis:*
   - Objective: Choose the most promising, strategic hypothesis.
   - Reflection: "Why does this hypothesis stand out? How does it uniquely address the issue?"
   - Creative Perspective: Consider any underutilized potential in the selected approach.

   *2.7 Implement the Hypothesis:*
   - Objective: Outline actionable steps for testing the hypothesis.
   - Reflection: "Is this plan practical? What resources or preparation are required?"
   - Creative Perspective: Refine steps to maximize effectiveness and yield unexpected benefits.

   *2.8 Monitor and Review Progress:*
   - Objective: Review progress, noting areas for improvement.
   - Reflection: "What’s working well? What could be improved?"
   - Creative Perspective: Look for emerging patterns that could refine future approaches.

   *2.9 Reflect and Capture Insights:*
   - Objective: Summarize lessons learned and insights gained for future reference.
   - Reflection: "What new understanding has emerged from this process?"
   - Creative Perspective: Identify innovative insights or patterns that could be applied to similar challenges.

3. *Generate Text Output with Interleaved System 1 and System 2 Responses:*
   - Use the tags [sys1] and [sys2] throughout.
   - Aim for a lengthier, detailed response. Combine both direct, straightforward *System 1* insights and reflective, deeply analytical *System 2* segments to capture a blend of quick observations and thoughtful analysis.

---

### *Example Input Text:*

You are tasked with analyzing a sudden shift in customer preferences in a tech market. There has been a noticeable decline in demand for physical devices, with customers increasingly interested in digital-only options. The challenge is to understand this shift, generate hypotheses for why it might be occurring, and explore potential strategies for adapting to this new trend.

---

### *Processed Output with System 1 and System 2 Thinking:*

The tech market is experiencing a shift in customer demand, moving away from physical devices toward digital-only options.

[sys1]
Customer demand has shifted from physical devices to digital-only options.
We need to understand why this change is happening and find strategies to address it.
Key factors may include cost, convenience, and technological trends.
[end sys1]

[sys2]
**2.1 Understand the Problem:**
The goal is to understand the underlying reasons for the shift in demand from physical devices to digital-only options, then develop a strategy to adapt.

*Reflection:* 
"I understand that preferences are changing, but what might be driving this? Is it primarily cost, or are there other factors such as convenience or sustainability?"

*Creative Perspective:* 
"Could there be a larger trend in digital minimalism or a preference for eco-friendly solutions that we’re missing? What unseen motivations might explain this shift?"

**2.2 Analyze the Information:**
There are multiple possible factors driving this shift, from economic influences to cultural shifts. It’s essential to isolate each factor and understand its impact.

*Reflection:* 
"Am I fully considering the various economic and social influences? Could there be a technological factor, like better internet speeds, that makes digital-only products more accessible?"

*Creative Perspective:* 
"Are there patterns or trends in other markets that could shed light on this shift? Could this be part of a larger trend toward virtual experiences?"

**2.3 Generate Hypotheses:**
1. Customers prefer digital options due to lower costs. (Confidence: 0.8, Creative: 0.4)
2. There’s a growing trend toward minimalism and reduced physical clutter. (Confidence: 0.7, Creative: 0.7)
3. Digital products offer greater flexibility and ease of use. (Confidence: 0.6, Creative: 0.6)
4. Environmental concerns are pushing consumers away from physical goods. (Confidence: 0.6, Creative: 0.8)
5. Advances in tech make digital-only options more functional. (Confidence: 0.8, Creative: 0.5)
6. Pandemic-era remote work increased demand for digital solutions. (Confidence: 0.7, Creative: 0.6)
7. Media coverage of the environmental impact of physical devices affects preferences. (Confidence: 0.5, Creative: 0.7)
8. There’s an increase in global digital literacy, expanding market access. (Confidence: 0.6, Creative: 0.6)
9. Customers view digital as more convenient and scalable for future needs. (Confidence: 0.7, Creative: 0.5)
10. Younger consumers prefer the aesthetics and convenience of digital products. (Confidence: 0.6, Creative: 0.6)

*Reflection:* 
"Have I considered all possible influences? Are there any surprising factors that could explain this shift?"

*Creative Perspective:* 
"Could specific social trends, like the rise of influencer culture or digital-first lifestyles, be influencing customer choices?"

**2.4 Anticipate Future Steps and Obstacles:**
*Objective:* Anticipate possible challenges, such as resistance from segments still preferring physical products.

*Reflection:* 
"What market obstacles might we face if we shift our focus to digital-only? Are there sub-segments that still prioritize physical products?"

*Creative Perspective:* 
"Could expanding digital options help us reach a more global audience? Are there emerging trends that we could leverage in our strategy?"

[end sys2]

[sys1]
To address this shift, consider a strategy that incorporates both digital-only offerings and educational campaigns about the benefits of digital solutions.
Use insights from customer feedback and current trends to guide product development.
Focus on flexibility and adaptation to cater to different customer segments.
[end sys1]


Melanocyte stem cells (McSCs) and mouse models of hair graying serve as useful systems to uncover mechanisms involved in stem cell self-renewal and the maintenance of regenerating tissues. Interested in assessing genetic variants that influence McSC maintenance, we found previously that heterozygosity for the melanogenesis associated transcription factor, Mitf, exacerbates McSC differentiation and hair graying in mice that are predisposed for this phenotype. Based on transcriptome and molecular analyses of Mitf mi-vga9/+ mice, we report a novel role for MITF in the regulation of systemic innate immune gene expression. We also demonstrate that the viral mimic poly(I:C) is sufficient to expose genetic susceptibility to hair graying. These observations point to a critical suppressor of innate immunity, the consequences of innate immune dysregulation on pigmentation, both of which may have implications in the autoimmune, depigmenting disease, vitiligo.

Hair pigmentation over the course of a lifetime depends on melanocyte stem cells that reside in the hair follicle. As old hairs fall out and new hairs grow in, melanocyte stem cells serve as a reservoir for the melanocytes that produce the pigment that gives hair its visible color. The loss of these stem cells leads to the growth of nonpigmented, or gray, hairs. Evaluating mouse models of hair graying can reveal key aspects of melanocyte stem cell biology. Using this approach, we discovered a novel role for the melanogenesis associated transcription factor, MITF, in repressing the expression of innate immune genes within cells of the melanocyte lineage. The importance of this repression is revealed in animals that have a predisposition for hair graying. In these animals, artificial elevation of the innate immune response, either through a genetic mechanism or via exposure to viral mimic, results in significant melanocyte and melanocyte stem cell loss and leads to the production of an increased number of gray hairs. These observations highlight the negative effects of innate immune activation on melanocyte and melanocyte stem cell physiology and suggest a connection between viral infection and hair graying.

With this in mind, we performed genomic analysis of wild-type and Mitf mi-vga9/+ McSCs and discovered that haploinsufficiency for Mitf results in a marked and chronic up-regulation of a type I IFN gene signature in this stem cell population. To better understand the contribution of innate immune suppression in postnatal melanogenesis, we further validated this immune signature in the skin and melanoblasts (the melanocyte precursors) of Mitf mi-vga9/+ mice. We evaluate the possibility that MITF acts as negative transcriptional regulator of innate immune target genes, and we assess the consequence of innate immune pathway activation on the regeneration of the melanocyte lineage during hair cycling.

(A) Dorsal images of two female littermates taken at P110. At this age, the coat of Tg(Dct-Sox10)/0 mice appears solid black, while the coat of Tg(Dct-Sox10)/0; Mitf mi-vga9/+ mice exhibits a small number of gray hairs along its back. Bright-field images of histological sections of the skins from these mice reveal increased ectopic pigmentation (arrows) within the hair bulge (region between the dotted lines) of hairs from Tg(Dct-Sox10)/0; Mitf mi-vga9/+ animals in comparison to Tg(Dct-Sox10)/0 animals. (B) Dorsal images of two female littermates taken at P37 and P110. At P37, Tg(Dct-Sox10)/Tg(Dct-Sox10) mice exhibit a black coat with congenital white spotting on the belly and back. These mice experience progressive hair graying and by P110 exhibit a “salt and pepper” coat color in regions of the back fur that were originally black. In comparison to Tg(Dct-Sox10)/Tg(Dct-Sox10) mice, hair graying in Tg(Dct-Sox10)/Tg(Dct-Sox10); Mitf mi-vga9/+ mice at P110 is more severe, with a majority of the back fur exhibiting gray hairs. The images for this figure were generated in the original study presented in [ 17 ]. McSC, melanocyte stem cell; P, postnatal day; SG, sebaceous gland.

Our interest in the role of MITF in hair graying was spurred by our previous observation that a genetic interaction exists between Mitf and the Sox10 transgene, Tg(Dct-Sox10) [ 17 ]. Tg(DctSox10) mice conditionally overexpress Sox10 within the melanocyte lineage, and this leads to the premature differentiation of McSCs, eventual McSC depletion, and progressive hair graying. Because SOX10 transcriptionally activates the Mitf gene and the MITF protein promotes melanocyte differentiation, we anticipated that reducing Mitf expression might alleviate Tg(DctSox10)-mediated hair graying. We tested this using the Mitf-null allele, Mitf mi-vga9 , which in the heterozygous state does not lead to apparent McSC dysfunction or hair graying. However, in contrast to our expectations, Tg(Dct-Sox10)/0; Mitf mi-vga9/+ mice exhibit sparsely distributed yet noticeable nonpigmented or “gray” hairs throughout their coat prior to their Tg(Dct-Sox10)/0 littermates ( Fig 1A ). Tg(Dct-Sox10)/0; Mitf mi-vga9/+ mice also show increased differentiation of McSCs, as indicated by excessive ectopic pigmentation within the stem cell niche (hair bulge) of their hair follicles. Intensified adult-onset hair graying associated with Mitf mi-vga9 is particularly evident when the Tg(Dct-Sox10) transgene is homozygous and animals are imaged over time ( Fig 1B ). We interpreted this outcome as contrary to the canonical role of MITF, in which high levels of MITF activity, rather than low, are associated with cell cycle arrest and melanocyte differentiation [ 26 ]. This interpretation is reinforced further by the fact that McSCs in Mitf vit/vit mice, which carry a hypomorphic mutation of Mitf, also exhibit premature McSC differentiation and hair graying even in the absence of Tg(Dct-Sox10) [ 13 ]. This suggests a novel contribution of MITF to the regulation of McSC maintenance and melanocyte lineage regeneration postnatally.

In particular, MITF, the gene encoding the micropthalmia-associated transcription factor, is essential at multiple stages of the melanocyte life cycle. Across species, MITF is required for the specification and survival of the melanocyte precursors, or melanoblasts, during early neural crest migration [ 19 – 21 ]. Loss of Mitf expression results in the near-complete depletion of embryonic melanoblasts, and this leads to mice born with a fully white coat [ 22 ]. During melanogenesis, MITF transcriptionally regulates a number of pigmentation genes involved in the biosynthesis of melanin and the trafficking of melanosomes. Regulation of MITF activity levels also influences the dynamic transition between the states of melanocyte migration, proliferation, and differentiation [ 23 – 25 ].

During hair growth, McSCs produce the melanocyte progeny that differentiate and deposit melanin into the hair shaft. Mouse models reveal that hair graying, both acute and age related, is frequently preceded by a failure in McSC maintenance or dysregulated generation of melanocyte progeny. Both lead to the production of nonpigmented, or gray, hair shafts. Hair graying can be elicited through a number of mechanisms—disrupting the signaling pathways associated with the Kit receptor, Notch receptor, Endothelin receptor type B, Raf kinase, Transforming growth factor beta, or Wnt [ 4 – 11 ]; loss of anti-apoptotic control [ 12 , 13 ]; melanocyte-specific dysregulation of chromatin remodeling complexes [ 14 ]; exposure to genotoxic stress [ 15 , 16 ]; changes in sex determining region Y-box 10 (SOX10) or MITF-mediated transcriptional regulation [ 13 , 17 ]; vitiligo-like T-cell–mediated destruction of melanocytes [ 18 ]; and aging itself [ 13 ].

In the 1980s, a handful of studies reported that exposure to murine leukemia virus (MuLV), either at mid-gestation or perinatally, is sufficient to drive premature hair graying in mice [ 1 – 3 ]. Early infection with MuLV does not lead to immediate loss of hair pigmentation and instead produces an adult-onset, progressive hypopigmentation phenotype, suggestive of a failure in melanocyte lineage regeneration. These observations suggest a role for innate immune activation in adult hypopigmentation disorders, but how this phenomenon is mediated within the postnatal melanocyte lineage remains unresolved. Using approaches to look for genetic modifiers of hair graying in mice and transcriptomic analysis of melanocyte stem cells (McSCs), we identify an exciting and unexpected link between the melanogenesis associated transcription factor, MITF, and the suppression of a type I interferon (IFN) gene signature. This discovery creates a unique opportunity to investigate how innate immune gene expression is regulated in postnatal melanocytes and how its dysregulation affects McSCs and the regeneration of postnatal pigmentation during hair cycling.

Results

Mitf haploinsufficiency is associated with an up-regulation of a type I IFN–regulated gene expression signature in McSCs and melanoblasts In order to assess the effects of Mitfmi-vga9/+ on gene expression in vivo, McSCs were isolated from the dermis of adult mice at 8 weeks of age. At this time point, hairs across the body of the mouse are synchronized in the hair stage of telogen [27]. During telogen, the hair follicle does not have a hair bulb or differentiated melanocytes, and McSCs are the only melanocytic cells present within the hair. They can be identified by their expression of the melanogenic enzyme dopachrome tautomerase (DCT) and the transmembrane receptor KIT, and they are observed in the hair bulge (the upper region of the telogen-stage hair that lies at the insertion point of the arrector pili muscle) and secondary hair germ (the lower region of the telogen-stage hair nearest the dermal papilla; Fig 2A). In order to isolate these McSCs from telogen hairs, the dermis of Mitfmi-vga9/+ and wild-type mice was dissociated and immunolabeled with two cell-surface markers, KIT and cluster of differentiation 45 (CD45). Fluorescence-activated cell sorting (FACS) was then used to distinguish McSCs (KIT+/CD45−) from mast cells (KIT+/CD45+; Fig 2B). When assessed in vitro 1, 3, or 5 days after sorting, greater than 92% of the KIT+/CD45− population of cells express the melanocytic protein DCT and begin to produce pigment at 5 days (Fig 2C and 2D). Additionally, because MITF is a known transcriptional regulator of the Kit gene [20,28], we compared the population percentages within each FACS gate between the wild-type and Mitfmi-vga9/+ dermal cell suspensions. No significant difference is observed in the percent of KIT+/CD45− dermal cells between wild-type and Mitfmi-vga9/+ animals, suggesting that the Mitfmi-vga9 mutation does not change the ability of this FACS strategy to identify the McSC population (Fig 2E). This suggests that this sorting strategy is adequate to produce a highly enriched pool of McSCs for transcriptomic analysis. RNA isolated from the KIT+/CD45− McSC populations obtained from the dermis of wild-type and Mitfmi-vga9/+ mice was then subjected to RNA sequencing (RNA-seq). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. FACS isolation of adult McSCs. (A) Immunolabeling for KIT protein in mouse skin at 8 weeks of age. At this time point, the majority of hairs exist in the telogen hair stage, and McSCs that are positive for both KIT (green) and DCT (red) are observed in the hair bulge (arrow) and secondary hair germ (arrowheads). The dotted white lines outline the hair follicles. (B) FACS of dermal cells from 8-week-old mice produces two KIT+ populations. McSCs are CD45− and mast cells are CD45+. The FACS gating strategy (center fluorescence plot) used to isolate McSCs successfully separates each of these cell types and is confirmed by visualizing their distinct morphologies; McSCs are small and often bipolar (left phase images), while mast cells are large and rough looking (right phase image). (C) KIT+/CD45− cells isolated by FACS were placed in culture and assessed for their expression of KIT and DCT by immunolabeling over a 5-day period. Total cells were identified by the nuclear marker DAPI. The table shows the percentage and total number (in parentheses) of cells exhibiting the indicated staining pattern. This FACS protocol produces a relatively pure McSC population, with >92% of cells being DCT+ 1 day after sorting. (D) FACS-isolated KIT+/CD45− McSCs remain positive for the melanocyte markers KIT (green) and DCT (red) and exhibit melanocyte-like traits while in tissue culture. These cells progress from being round at 1 day, to slightly spread at 3 days, to dendritic and pigmented over 5 days. (E) Evaluation of the indicated gates (boxes) on FACS fluorescence plots confirms that there is no significant difference by t test when comparing the percentage of each cell population between wild-type (left plot) and Mitfmi-vga9/+ (right plot) dermal cell suspensions (KIT+/CD45−, p = 0.85; KIT+/CD45+, p = 0.14; KIT−/CD45+, p = 0.28). Percentages are represented as the mean ± standard deviation, with n = 3 sorts per genotype. The raw data used to generate these graphs are available in S1 Data. APC, allophycocyanin; CD45, cluster of differentiation 45; DCT, dopachrome tautomerase; FACS, fluorescence-activated cell sorting; FITC, fluorescein; McSC, melanocyte stem cell; Mitf, melanogenesis associated transcription factor; Neg, negative; PIG, pigment. https://doi.org/10.1371/journal.pbio.2003648.g002 Using a 1.5-fold cutoff, differential gene expression analysis of the RNA-seq reads obtained from Mitfmi-vga9/+ and wild-type McSCs demonstrated that, as expected, Mitfmi-vga9/+ McSCs exhibit the reduced expression of pigmentation-related genes known to be positively regulated by MITF (S2 Data). This includes Dct, Gpnmb, Gpr143, Irf4, Kit, Mlana, Met, Rab27a, Slc45a2, Tbx2, and Tyr. However, this analysis also uncovered an unanticipated enrichment of DEGs up-regulated in Mitfmi-vga9/+ McSCs that are involved in innate immunity (S2 Data). Out of the 411 genes with a greater than 1.5-fold up-regulation in Mitfmi-vga9/+ cells, at least 55 are known for their involvement in type I innate immune signaling (DAVID Functional Annotation Tool [29,30] and interferome.org [31]). These include cytoplasmic pattern recognition receptors (PRRs; Ddx58, Ifih1), transcriptional regulators (Irf7, Stat1), and IFN-stimulated genes (ISGs) that execute the antiviral response (Ifit1, Ifit3, Isg15, Mx1, Oas1a; Fig 3A, S3 Data). Up-regulation of this particular subset of genes is known as the “IFN signature” and is often associated with viral infection and autoimmune disorders [32]. To rule out the possibility that this ISG signature was due to a microbial infection of our Mitfmi-vga9 line at the time that McSCs were harvested for FACS, we assessed ISG expression by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in skins obtained from mice at postnatal day 32 (P32) that were housed in an unrelated facility and that have been genetically isolated from our Mitfmi-vga9 line for several generations. Consistent with our RNA-seq data, skins from these Mitfmi-vga9/+ mice show clear up-regulation of the five ISGs evaluated, Ifih1, Ifit3, Irf7, Isg15, and Stat1, in comparison to animals that do not carry this mutation (S1 Fig). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Mitfmi-vga9/+ McSCs and melanoblasts exhibit an elevated type I interferon gene expression signature and Mitfmi-vga9/+ melanoblasts show increased sensitivity to viral mimic. (A) Heatmap of scaled and clustered, rlog-transformed read count values obtained from RNA-seq analysis of Mitfmi-vga9/+ and wild-type McSCs. Fifty-five of the four hundred eleven genes that demonstrated a statistically significant, greater than 1.5-fold increase in expression in Mitfmi-vga9/+ McSCs over wild-type McSCs participate in innate immune signaling and are presented here (padjusted < 0.05, Benjamini-Hochberg adjusted p-value). (B) Immunolabeling for KIT protein in mouse skin at P1.5. At this time point, KIT+ (green), DCT+ (red) melanoblasts are observed migrating into the developing hair follicles. The dotted white lines outline the hair follicles and the solid line indicates the position of the epidermis. (C) qRT-PCR analysis of Mitf and ISG expression (Ifih1, Ifit3, Irf7, Isg15, and Stat1) in primary melanoblasts isolated from Mitfmi-vga9/+ and wild-type littermate pups at P1.5. Each circle indicates the expression of cells from an individual primary melanoblast cell line generated from an individual pup. The horizontal bars represent the mean, and the asterisks indicate gene expression changes with a q-value of <0.05 using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with Q = 5%. (D) qRT-PCR analysis of type I interferon gene expression (Ifna4 and Ifnb1) in primary melanoblast cell lines (isolated as described in [C]). Melanoblast cell lines were treated for 9 hours with lipofectamine only (lipo only) or lipofectamine and poly(I:C) (lipo + poly(I:C)). The horizontal bars represent the mean, and the asterisks indicate gene expression changes with a q-value of <0.05 using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with Q = 5%. The data presented here are representative of two independent experiments testing poly(I:C) treatment of primary melanoblast cell lines at two different cell passages. The raw data used to generate the graphs in (C) and (D) are available in S1 Data. DCT, dopachrome tautomerase; ISG, IFN-stimulated gene; lipo + poly(I:C), lipofectamine and poly(I:C); lipo only, lipofectamine only; Mitf, melanogenesis associated transcription factor; McSC, melanocyte stem cell; P, postnatal day; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; RNA-seq, RNA sequencing. https://doi.org/10.1371/journal.pbio.2003648.g003 Type I innate immune signaling is comprised of a two-part signaling cascade. First, in an infected cell, viral DNA or RNA is recognized by PRRs, and downstream signaling results in the phosphorylation of interferon regulatory factors (IRFs). Active IRFs enter the nucleus and transcriptionally up-regulate the type I IFN genes to produce the chemokines IFN-α and -β. Via autocrine and paracrine signaling, IFNs bind IFN receptors and activate a second signaling cascade that results in a broad and robust ISG program. These ISGs mediate the antiviral response in both the infected and neighboring cells [33–36]. Based on this, we reasoned that Mitfmi-vga9/+ McSCs should more readily up-regulate Ifn gene expression in response to virus in comparison to wild-type McSCs if the IFN signature associated with Mitfmi-vga9 is indeed genuine. To test this, viral infection can be mimicked by delivering synthetic dsRNA into the cell cytoplasm via lipofectamine-based transfection. Polyinosinic:polycytidylic acid (poly(I:C)) is one type of dsRNA, an agonist for the cytoplasmic PRR, melanoma differentiation-associated gene 5 (MDA5, encoded by the Ifih1 gene), and is sufficient to mediate type I IFN responses in vitro and in vivo [37]. Techniques for expanding McSCs in culture for in vitro analysis are not well established, so we instead evaluated primary melanoblasts for their response to viral mimic. Primary melanoblasts refer to the highly proliferative, KIT+, melanocyte precursors that invade the developing hair follicle and give rise to either McSCs or differentiated melanocytes during perinatal hair morphogenesis (Fig 3B). Using the same FACS strategy as presented above (Fig 2B), KIT+/CD45− primary melanoblasts can be isolated from the whole skin of pups on P1.5 and passaged readily in tissue culture. Comparing cells isolated from wild-type and Mitfmi-vga9/+ littermates, we first confirmed that Mitfmi-vga9/+ melanoblasts exhibit an IFN signature similar to adult McSCs. By qRT-PCR we assessed the ISGs Ifih1, Ifit3, Irf7, Isg15, and Stat1 and demonstrated that Mitfmi-vga9/+ melanoblasts show a significant up-regulation of several of these ISGs, namely Ifih1, Ifit3, Irf7, and Isg15 (Fig 3C). Despite this IFN signature, basal Ifn gene expression in Mitfmi-vga9/+ and wild-type melanoblasts is equivalent and nearly undetectable (Fig 3D, lipo only). However, transient transfection of these melanoblasts with poly(I:C) induces the expression of Ifna4 and Ifnb1 in both cell types, with Mitfmi-vga9/+ melanoblasts expressing 2- to 3-fold higher Ifn mRNA levels than wild-type melanoblasts (Fig 3D, lipo + poly(I:C)). ISG expression in perinatal melanoblasts demonstrates that the IFN signature observed in Mitfmi-vga9/+ animals is not exclusive to the McSC or the adult time point. The similarity in basal Ifn expression between wild-type and Mitfmi-vga9/+ melanoblasts also suggests that the IFN signature observed in Mitfmi-vga9/+ melanoblasts is not due to the constant production of IFN by melanoblasts. Furthermore, the enhanced Ifn expression exhibited by Mitfmi-vga9/+ melanoblasts after exposure to viral mimic suggests that haploinsufficiency for Mitf primes these melanoblasts for the antiviral response.

MITF binds the promotor of several ISGs and transcriptionally represses ISG expression in vitro MITF is a transcription factor that promotes the direct and indirect regulation of a number of genes essential for melanocyte development, differentiation, proliferation, and survival [38]. Thus, we investigated whether the IFN signature observed in Mitfmi-vga9/+ McSCs and melanoblasts may be dependent on MITF’s role as a transcription factor. To determine whether reduction of Mitf is sufficient to lead to a direct change in ISG expression autonomous to the melanocyte, we knocked down Mitf in the immortalized mouse melanocyte cell line melan-a using a small interfering RNA (siRNA) approach. Intracellular delivery of siRNAs can elicit a nonspecific innate immune response that can be mitigated by incorporating 2′-O-methyl-uridine or guanosine nucleosides into at least one strand of the siRNA duplex [39]. Thus, we generated two siRNAs—a standard siRNA against Mitf (siMitf [40]) and a modified Mitf siRNA with an identical sequence but synthesized to include three 2′-O-methyl groups along each strand of siRNA (siMitf-OM). In line with MITF repression of ISG expression, knockdown of Mitf with either siMitf or siMitf-OM leads to robust up-regulation of the ISGs Ifih1, Ifit3, Irf7, Isg15, and Stat1 within 48 hours of transfection in comparison to a scrambled siRNA control (siMitf-scram; Fig 4A). These data support the premise that the IFN signature observed in Mitfmi-vga9/+ mice may be due to MITF-mediated gene regulation of ISGs. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. MITF binds to the promoter of innate immune target genes and acts as a transcriptional repressor. (A, B) Relative gene expression in melan-a cells 48 hours after transfection with siRNA. In (A), cells were transfected with siRNAs targeting Mitf (siMitf and siMitf-OM) or an analogous scrambled control siRNA (siMitf-scram). In (B), cells were transfected with one of two siRNAs targeting Irf4 (siIrf4_a, siIrf4_b) or siNC1. The bars represent the mean ± standard deviation, and the asterisks indicate gene expression changes with a q-value of <0.05 using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with Q = 5%. Data presented in A and B are representative of two independent knockdown experiments performed in melan-a cells at two different cell passages. (C) Venn diagrams demonstrating the intersection of gene lists. The left diagram represents the genes exhibiting a 1.5-fold or greater difference in expression in Mitfmi-vga9/+ over wild-type McSCs (Mitfmi-vga9/+DEGs, top circle) overlapped with the list of genes associated with MITF ChIP-seq peaks reported by Webster et al. 2014 (MITF ChIP-seq genes, bottom circle) [41] and are considered potential direct targets of MITF. These 108 direct targets are provided in S4 Data. Direct target genes were further identified by comparing them with a list of known pigmentation genes in the upper right diagram and with the list of 55 innate immune-related genes up-regulated in Mitfmi-vga9/+ McSCs (as defined in Fig 3A) in the lower right diagram. (D) MITF ChIP-qPCR performed in melan-a cells and assayed for enrichment at the indicated gene loci (Dct, Ifih1, Ifit3, Stat1). A centromeric DNA sequence was amplified as a negative control (neg con). Error bars indicate mean ± standard deviation of two independent ChIP pulldowns. The raw data used to generate the graphs in (A), (B), and (D) are available in S1 Data. Actb, actin beta; ChIP, chromatin immunoprecipitation; ChIP-qPCR, chromatin immunoprecipitation quantitative polymerase chain reaction; ChIP-seq, chromatin immunoprecipitation sequencing; DEG, differentially expressed gene; IgG, immunoglobulin G; McSC, melanocyte stem cell; MITF, melanogenesis associated transcription factor; neg con, negative control; siNC1, nontargeting control siRNA; siRNA, small interfering RNA. https://doi.org/10.1371/journal.pbio.2003648.g004 Among the genes differentially expressed in Mitfmi-vga9/+ McSCs, we were particularly interested in IFN regulatory factor 4, Irf4. In human melanocytes, MITF binds and activates Irf4 via an intronic enhancer [42], and in turn, IRF4 can transcriptionally regulate IFN signaling by repressing the master regulatory factor for IFN gene expression, Irf7 [43,44]. A genome-wide association study of Latin Americans also identified Irf4 as a locus associated with hair graying [45]. Because Irf4 and Irf7 exhibit a reciprocal relationship in Mitfmi-vga9/+ McSCs (Irf4 is down and Irf7 is up; S2 Data), we asked whether Irf4 may be indirectly responsible for the up-regulation of ISGs in Mitfmi-vga9/+ melanocytes. However, within the 48-hour time frame that is sufficient for Mitf knockdown to result in ISG up-regulation, we did not observe consistent down-regulation of Irf4 between the two Mitf siRNAs (Fig 4A). As a control, we further confirmed that cells being treated with either siMitf or siMitf-OM results in the predictable down-regulation of the melanosomal gene Pmel17, which MITF is known to transcriptionally activate (Fig 4A; [46]). Based on these observations, we anticipated that the up-regulation of ISGs observed with Mitf knockdown cannot be IRF4 dependent. Indeed, using two unique Irf4 siRNAs (siIrf4_a and siIrf4_b), Irf4 gene expression can be significantly reduced in comparison to a negative control siRNA (siNC1). However, when considering the results of both Irf4 siRNAs, Irf4 knockdown does not lead to the consistent up-regulation or Irf7 or any of the other ISGs analyzed (Fig 4B). Altogether, these results support a novel role for MITF in suppressing the basal IFN signature in melanocytes in vitro and indicate that MITF does not mediate this response through Irf4. An alternate mechanism by which MITF may participate in regulation of innate immune genes is through a direct interaction with their cis-regulatory regions, as is the case with many pigmentation genes [46–49]. To investigate this, we screened a previously published MITF chromation immunoprecipitation sequencing (ChIP-seq) dataset for binding of MITF to regulatory regions of innate immune genes [41]. The genomic coordinates of the MITF ChIP-seq peaks found in human primary melanocytes and COLO829 melanoma cells, reported in Webster et al. 2014 [41], were converted from genome build GRCh37/hg19 to NCBI37/mm9 (Galaxy Liftover), and putative target genes were identified using GREAT, version 3.0.0 [50]. For the set of genes that exhibit MITF ChIP-seq peaks within 5 kb of their transcription start site in either direction (6,771), we found 108 genes that are also differentially expressed by greater than 1.5-fold in Mitfmi-vga9/+ McSCs (Fig 4C, S4 Data). This overlap is under enriched by 1.22-fold compared to expectations (hypergeometric test; p-value = 0.0065), suggesting that the majority of gene expression changes observed in Mitfmi-vga9/+ McSCs are indirect. Thus, we further intersected the 108 DEGs with two additional gene lists—one comprising genes reported to be involved in pigmentation (curated from Online Mendelian Inheritiance in Man, Mouse Genome Informatics, and Gene Set Enrichment Analysis) and the second comprising the 55 ISGs identified above in Fig 3A. This comparison validates that, as expected, some DEGs in Mitfmi-vga9/+ McSCs represent known MITF targets involved in pigmentation but also reveals a number of innate immune genes that may also be under MITF transcriptional control (Fig 4C). Of the nine innate immune DEGs, seven exhibit an MITF ChIP-seq peak that spans their transcription start site and is congruent with a role for MITF in the direct regulation of these genes at their promotor (Table 1). In order to confirm that MITF binds to these loci in mouse melanocytes, we assessed MITF occupancy using chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) for three of the seven ISGs in melan-a cells. Significant chromatin immunoprecipitation (ChIP) enrichment of MITF was observed at the promoter regions of all three of the ISGs predicted to be MITF targets (Ifih1, Ifit3, and Stat1) as well as at the promoter region of a known MITF target gene, Dct (Fig 4D). No MITF enrichment was observed for the negative control region, a sequence within the centromere. These observations support a direct regulatory role for MITF in the repression of at least some ISGs within melanocytes. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Relationship of MITF ChIP-seq peaks to the transcription start site (TSS) of innate immune genes. https://doi.org/10.1371/journal.pbio.2003648.t001

Tg(Dct-Sox10)/0; Mitfmi-vga9/+ mice exhibit up-regulated ISG expression but no change in the density of immune cells within the skin While it is interesting that Mitfmi-vga9/+ mice exhibit an IFN signature and that MITF transcriptionally inhibits some of these genes, the real impetus of this study stemmed from previous observations that Mitfmi-vga9/+ causes increased McSC differentiation and hair graying in combination with the transgene Tg(Dct-Sox10) [17]. In order to investigate whether aberrant innate immune signaling might explain hair graying in Tg(Dct-Sox10)/0; Mitfmi-vga9/+ animals, we first evaluated the expression of Mitf and ISGs in the skin of adult littermates generated by mating Mitfmi-vga9/+ and Tg(Dct-Sox10)/0 animals. Interestingly, qRT-PCR analysis reveals that skins from Tg(Dct-Sox10)/0 mice exhibit Mitf levels similar to that of wild-type, and skins from Tg(Dct-Sox10)/0; Mitfmi-vga9/+mice exhibit reduced levels of Mitf that are indistinguishable from Mitfmi-vga9/+ mice (Fig 5A). This is in contrast to the expectation that the Tg(Dct-Sox10) transgene would increase Mitf expression due to the ability of SOX10 to up-regulate Mitf transcriptionally [51]. However, in correlation with these low Mitf expression levels, skins from both Mitfmi-vga9/+ and Tg(Dct-Sox10)/0; Mitfmi-vga9/+ mice show a significant up-regulation of the ISGs Ifih1, Irf7, Isg15, and Stat1, with Tg(Dct-Sox10)/0; Mitfmi-vga9/+ skins showing the additional up-regulation of Ifit3 (Fig 5A′). Because Mitfmi-vga9/+ mice do not exhibit hair graying themselves [17], these observations suggest that it may be the unique combination of melanocyte dysregulation via Tg(Dct-Sox10)/0 and innate immune dysregulation via Mitfmi-vga9/+ that leads to the exacerbated hair graying observed in Tg(Dct-Sox10)/0; Mitfmi-vga9/+ and Tg(Dct-Sox10)/ Tg(Dct-Sox10); Mitfmi-vga9/+mice. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. Mitfmi-vga9/+ and Tg(Dct-Sox10)/0; Mitfmi-vga9/+ animals exhibit elevated ISG expression but no change in the density of immune populations within the skin. (A, A′) qRT-PCR analysis of Mitf and ISG expression in skin isolated from adult wild-type, Tg(Dct-Sox10)/0, Mitfmi-vga9/+, and Tg(Dct-Sox10)/0; Mitfmi-vga9/+ littermates. Each point on the graph represents the expression value from skin of an individual animal. The horizontal bars represent the mean, and the asterisks indicate gene expression changes with a q-value of <0.05 using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with Q = 5%. (B) Number of immune-related cells per a 15 mm2 area of skin from the same animals as described in (A) harvested at mid-anagen. Mast cells were detected using toluidine blue staining, and other immune cells were detected by immunofluorescence for the indicated markers. Representative images of histological sections used for cell counts are provided in S2 Fig. Each point on the graph represents cell counts from an individual animal, and the horizontal bar represents the mean. t tests confirmed no statistically significant differences in the density of immune cells when comparing wild-type animals to Mitfmi-vga9/+ or Tg(Dct-Sox10)/0; Mitfmi-vga9/+ animals. The raw data used to generate the graphs in (A), (A′), and (B) are available in S1 Data. Actb, actin beta; CD, cluster of differentiation; ISG, IFN-stimulated gene; Mitf, melanogenesis associated transcription factor; qRT-PCR, quantitative reverse transcription polymerase chain reaction. https://doi.org/10.1371/journal.pbio.2003648.g005 An IFN signature is commonly detected in affected tissues of patients afflicted with autoimmune disease. Using the same mice as in Fig 5A, we assessed whether changes in resident and infiltrating immune cell populations could provide an etiology for hair graying within Tg(Dct-Sox10)/0; Mitfmi-vga9/+ mice. On cryosections of skin, we quantified mast cells (toluidine blue histologic stain), T cells (CD4+, CD8+, CD3ɛ+), macrophages, and dendritic cells (CD11b). Previous studies report a partial depletion of skin mast cells in Mitfmi-vga9 homozygotes [52]; however, a similar reduction is not observed in Mitfmi-vga9/+ or Tg(Dct-Sox10)/0; Mitfmi-vga9/+ animals. Moreover, immunolabeling for CD3ɛ, CD4, CD8, and CD11b indicates no overt change in the density of these immune cell populations (Fig 5B, S2 Fig). Thus, the increased hair graying associated with Mitfmi-vga9/+ is likely not the result of immune cell-mediated destruction of melanocytes.

Reduction of Ifih1 is not sufficient to rescue Mitfmi-vga9-mediated hair graying Recent genome-wide association studies have led to the identification of function-reducing mutations in interferon induced with helicase C domain 1 (Ifih1) as protective against vitiligo in humans [53]. Ifih1 is one of the direct transcriptional targets of MITF identified above (Fig 4) and encodes for the protein MDA5. MDA5 sits at the top of the type I innate immune signaling cascade and functions as a cytoplasmic PRR that responds to pathogen-associated molecular patterns (PAMPs) like viral RNA [54]. Gain-of-function mutations of Ifih1 in humans or overexpression of Ifih1 in mice is associated with a sustained IFN gene signature and makes it a reasonable candidate for mediating a similar effect downstream of Mitfmi-vga9/+ [55,56]. Thus, we were interested to evaluate whether Ifih1 haploinsufficiency would be sufficient to reduce the hair graying caused by Mitfmi-vga9 in the context of Tg(Dct-Sox10). Hair graying associated with Tg(Dct-Sox10) is most readily visible in mice that are homozygous for the transgene, with Tg(Dct-Sox10)/Tg(Dct-Sox10); Mitfmi-vga9/+ animals exhibiting robust graying at 110 days (Fig 1). Comparing Tg(Dct-Sox10)/Tg(Dct-Sox10); Mitfmi-vga9/+ mice to their littermates, we find that additional heterozygosity for Ifih1 (Ifih tm1.1Cln/+) does not reverse hair graying to the levels observed in Tg(Dct-Sox10)/Tg(Dct-Sox10) mice (Fig 6, S3 Fig). At most, and in a qualitative sense, Ifih tm1.1Cln/+ appears to produce a small reduction in the existing congenital white belly spot. These observations suggest that while MITF may repress Ifih1 in melanocytes in vitro, reduction of Ifih1 in vivo is insufficient to reverse hair graying associated with Mitfmi-vga9/+. This may indicate that the influence of MITF in innate immune regulation extends beyond the regulation of an individual gene, which is consistent with the fact that Ifih1 is not the only innate immune target gene directly downstream of MITF (as shown in Fig 4). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 6. Ifih1tm1.1Cln/+ does not affect hair graying in Tg(Dct-Sox10)/Tg(Dct-Sox10); Mitfmi-vga9/+ mice. (A, B) Images of Tg(Dct-Sox10)/Tg(Dct-Sox10); Mitfmi-vga9/+ and Tg(Dct-Sox10)/Tg(Dct-Sox10); Mitfmi-vga9/+; Ifih tm1.1Cln/+ littermates imaged at postnatal day 95. (A) Tg(Dct-Sox10)/Tg(Dct-Sox10); Mitfmi-vga9/+ animals exhibit a white belly spot at birth and premature hair graying as they age. (B) Tg(Dct-Sox10)/Tg(Dct-Sox10); Mitfmi-vga9/+; Ifih tm1.1Cln/+ animals also exhibit hair graying and a belly spot that is slightly reduced in size. Images shown here are representative of five biological replicates for each genotype, with additional images provided in S3 Fig. Ifih1; interferon induced with helicase C domain 1; Mitf, melanogenesis associated transcription factor. https://doi.org/10.1371/journal.pbio.2003648.g006