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]


Introduction {#S1}
============

Keratin 16 (*KRT16* in human, *Krt16* in mouse), a type I intermediate filament protein, is constitutively expressed in a variety of epithelial appendages, including the tongue and the hair follicle, and in glabrous skin ([@R20]; [@R1]; [@R26]). Upon stressful epithelial stimuli, such as wounding or chronic inflammation, *Krt16* and its binding partner *Krt6* are selectively induced in the suprabasal layers of the epidermis ([@R21]). Mutations in *KRT16* are associated with the development of Pachyonychia Congenita (PC), a rare autosomal dominant disorder characterized by hypertrophic nail dystrophy, palmoplantar keratoderma (PPK), and oral leukokeratosis ([@R19]). PC symptoms are highly variable in penetrance, age of onset, and severity, even between individuals with the same mutation ([@R14]; [@R16]; [@R9]). In patients carrying *KRT16* mutations, palmoplantar keratoderma are the most prominent symptom and may sometimes appear as focal PPK (FPPK) with little or no nail involvement ([@R14]; [@R16]; [@R23]; [@R24]; [@R25]). *KRT16* mutation-linked PPK is typically non-epidermolytic ([@R19]; [@R16]; [@R23]), although blistering underneath and around PPK calluses has been linked to palmoplantar pain in PC patients ([@R6]). Previous mouse models harboring both dominant and recessive mutations in *Krt6* and *Krt75* recapitulate several PC-like symptoms ([@R31], [@R29]; [@R32]; [@R5]), but have failed to phenocopy palmoplantar keratoderma, which are considered the most debilitating PC symptom as they are extremely painful and significantly impact patient mobility and quality of life ([@R14]; [@R6]). In this study, we show that adult mice lacking *Krt16* develop hyperkeratotic calluses on their front and hind paws that are strikingly similar to human palmoplantar keratoderma and significantly compromise the animals' ability to walk.

Results {#S2}
=======

Failure to thrive and increased postnatal mortality in *Krt16^−/−^* mice {#S3}
------------------------------------------------------------------------

*Krt16^−/−^* mice were born alive at approximately Mendelian ratios and were initially visually indistinguishable from wild-type (WT) and *Krt16^+/−^* littermates ([Figure S1A](#SD1){ref-type="supplementary-material"}). However, 34% of *Krt16^−/−^* mice died within the first 24 h after birth, as opposed to 6% of WT and 11% of *Krt16^+/−^* mice ([Figure 1A](#F1){ref-type="fig"}). Toluidine Blue dye exclusion assays performed on newborn pups ruled out any gross early postnatal skin barrier defects in *Krt16^−/−^* ([Figure S1B](#SD1){ref-type="supplementary-material"}). Newborn *Krt16^−/−^* mice weighed less than their littermates ([Figure S1C](#SD1){ref-type="supplementary-material"}) and subsequently continued to lag behind their littermates in size and in weight ([Figure 1B--C](#F1){ref-type="fig"}). Such phenotypic changes coincided with the persistence of a markedly higher level of postnatal mortality, and over 60% of *Krt16^−/−^* mice died before weaning age. The *Krt16^−/−^* mice that did survive continued to grow and gained weight, but remained smaller and lighter than their littermate controls (data not shown).

Krt16 is essential for the structural integrity of dorsal tongue epithelium {#S4}
---------------------------------------------------------------------------

Mice lacking the Krt16 binding partners, Krt6a and Krt6b, exhibit a more severe albeit similar growth retardation and early postnatal lethality phenotype ([@R31]; [@R29]). In *Krt6a/b^−/−^* mice, the formation of large hyperplastic lesions on the dorsal posterior tongue (akin to oral leukokeratosis, a PC symptom) is thought to occlude the laryngeal space, impair feeding, and lead to early death due to starvation. Such laryngeal obstruction, although extremely rare, has been reported in pediatric PC patients carrying *KRT6a* mutations ([@R25]; [@R11]) and infants suffering from oral leukokeratosis often have trouble breastfeeding ([@R14]).

When we examined the tongues of *Krt16^−/−^* mice at different ages, we also detected the presence of hyperplastic lesions, which are fully penetrant and become macroscopically and microscopically visible by P3 ([Figure 1D](#F1){ref-type="fig"}). These lesions were confined to the dorsal midline area of the posterior tongue and were smaller than those observed in *Krt6a/b^−/−^* mice ([@R31]). Surviving *Krt16^−/−^* mice no longer had visible lesions, but the tongue architecture remained severely compromised, showing a thickened epithelium and loss of the characteristic filiform papillae morphology ([Figure S2A](#SD1){ref-type="supplementary-material"}). Although no epithelial fragility was detectable by routine histology in P0 *Krt16^−/−^* tongues ([Figure 1D](#F1){ref-type="fig"}), transmission electron microscopy revealed early stages of cell lysis in a subset of posterior filiform papillae ([Figure S2B](#SD1){ref-type="supplementary-material"}). Interestingly, Krt17 protein and to a lesser extent *Krt17* mRNA levels were significantly reduced in the tongue epithelium of *Krt16^−/−^* mice ([Figure 2A--B](#F2){ref-type="fig"}), and immunofluorescence staining for Krt17 was consistently absent from the anterior column of *Krt16^−/−^* filiform papillae, but not the fungiform papillae ([Figure 2A](#F2){ref-type="fig"}). The expression of Krt5, Krt6 and Krt10 was normal in *Krt16^−/−^* filiform papillae ([Figure 2A--B](#F2){ref-type="fig"}), suggesting that the loss of Krt17 was not due to an overall change in the differentiation program in anterior column of filiform papillae. The constitutive as well as inducible expression of Krt17 in other tissues, including skin, was not affected ([Figure 2A](#F2){ref-type="fig"}, [Figure 4D](#F4){ref-type="fig"}). Our findings thus extend the notion that Krt6- and Krt16-containing filaments are essential for the maintenance of dorsal tongue epithelial integrity. Loss of Krt16 and concomitant reduction of Krt17 levels deplete most, but not all, of Krt6's primary binding partners in tongue epithelium, leading to increased cell fragility and hyperplastic lesion formation, starvation, and a higher chance of postnatal death in *Krt16^−/−^* mice. Residual levels of Krt17 present in *Krt16^−/−^* tongue epithelium may explain the smaller lesions and lower number of early deaths in *Krt16^−/−^* mice as compared to *Krt6a/b^−/−^* mice ([@R31]; [@R29]). Despite their less severe nature, we cannot rule out the possibility that oral lesions in *Krt16^−/−^* mice are painful and thus impact feeding behavior.

Adult *Krt16^−/−^* develop palmoplantar keratoderma {#S5}
---------------------------------------------------

Starting at 4--6 weeks of age, surviving *Krt16^−/−^* mice developed prominent, hyperkeratotic calluses on the glabrous parts of both front and hind paws ([Figure 3A--C](#F3){ref-type="fig"}). The boundaries of these calluses did not directly correlate with the loss of endogenous *Krt16*, which is expressed in a patchy fashion throughout paw pad epithelia including the nail hyponychium, as shown by whole-mount X-gal staining ([Figure 3A](#F3){ref-type="fig"}). Instead, consistent with focal palmoplantar keratoderma (FPPK) in human PC patients, calluses in *Krt16^−/−^* mice were restricted to areas subject to physical pressure, i.e. the heel and the wrist ([Figure 3A](#F3){ref-type="fig"}). Occasionally, we observed generalized hyperkeratosis on front paw pads as early as 3 weeks of age ([Figure S3A](#SD1){ref-type="supplementary-material"}), and focal hyperkeratosis near the base of the tail. Importantly, nail morphology was not affected in *Krt16^−/−^* mice ([Figure S3B](#SD1){ref-type="supplementary-material"}).

The age of onset for callus formation on both front and hind paws was variable, and calluses differed in severity between animals ([Figure 3A](#F3){ref-type="fig"} shows two representative examples of callus severity in *Krt16^−/−^*). 36% of Krt16^−/−^ mice showed no alteration of their hind paw pads at all. Hind paw calluses also consistently formed after front paw pad calluses were already established. Visual changes in *Krt16^−/−^* front paw pads correlated with an expanded epidermal compartment ([Figure 3B](#F3){ref-type="fig"}) that correlated with a 2-fold increase in proliferation ([Figure 4C](#F4){ref-type="fig"}) in the absence of apoptotic cell death ([Figure S3C](#SD1){ref-type="supplementary-material"}). Areas adjacent to the callus showed a thickened epithelium, but no significant increase in proliferation ([Figure 3B](#F3){ref-type="fig"}, [Figure 4C](#F4){ref-type="fig"}). Paw pad epithelia in younger mice with no obvious signs of callus formation were indistinguishable from controls (data not shown). Since palmoplantar keratoderma in humans greatly impair mobility, we asked whether callus formation in *Krt16^−/−^* mice negatively impacts the animals' activity level in a behavioral assay. We found that adult *Krt16^−/−^* mice are significantly less active than control animals and spent more time resting than walking ([Figure 4A](#F4){ref-type="fig"}, [Figure S3D](#SD1){ref-type="supplementary-material"}). Several anomalies, including the patchy expression of Krt17 (which is absent from control mouse paw pad epidermis) and focal toluidine blue dye penetration ([Figure 4B and 4D](#F4){ref-type="fig"}) suggested a disruption of the outside-in epidermal barrier in *Krt16^−/−^* front paw calluses, which may lead to secondary infections as implied by the presence of prominent hyper-pigmentation in those areas ([Figure 3A](#F3){ref-type="fig"}). *Krt17* expression is induced in response to barrier breach ([@R8]; [@R17]) and a mutation in the barrier protein filaggrin has been shown to intensify PC-related symptoms in a human patient with a coincident *Krt16* mutation ([@R16]; [@R10]). Filaggrin expression is normal in *Krt16^−/−^* paw pad epithelia, as reported for PC plantar lesions ([@R30]), but is reduced in established front paw calluses ([Figure 4D](#F4){ref-type="fig"}), confirming that *Krt16^−/−^* glabrous skin, while initially intact, is eventually unable to fend off the continuous physical pressure generated by walking and/or cleaning behavior, resulting in hyperproliferation and a focal loss of barrier protection. In humans, PPK are very painful. The cause of the pain is unknown, but clinical observations have noted the formation of blisters underneath or adjacent to calluses ([@R6]), as well as secondary infections following fissuring of the hyperkeratotic tissue ([@R14]). Alternatively, it is possible that PPK calluses exert an increased pressure on nerve endings in plantar skin, especially while walking. While an objective assessment of the level and type of pain or its source in *Krt16^−/−^* mice is beyond the scope of this study, we hypothesize that *Krt16^−/−^* mice experience substantial discomfort as a direct result of palmoplantar lesions and thus exhibit a significant decrease in their overall mobility.

Discussion {#S6}
==========

Here, we show that the loss of *Krt16* function in mice causes the development of prominent calluses on the plantar side of front and hind paws, which significantly compromise mobility and eventually lead to overt loss of barrier properties. While the molecular mechanism of PPK pathogenesis in PC is still unclear, this symptom is currently thought to develop as a consequence of intermediate filament network disruption by dominantly-acting mutations in relevant keratins ([@R9]). To our surprise, deletion of *Krt16* produced spontaneously arising PPK-like lesions in mice, suggesting that PPK pathogenesis in PC is more complex than previously appreciated and may represent, at least in part, a loss of function phenotype. Almost all human PC patients harboring *KRT16* mutations report PPK ([@R14], [@R16], [@R9]). However, many mutations in *KRT16* only elicit milder overall PC phenotypes, often diagnosed as FPPK because of the limited and at times absent nail involvement ([@R16]; [@R23]; [@R24]; [@R25]). In particular, this is seen with small deletions in *KRT16*, which are thought to cause exclusion of the mutant form of KRT16 from filament assembly ([@R24]; [@R28]; [@R4]). These findings together with our data suggest that both the dominant negative disruption of intermediate filaments as well as the exclusion of keratins from the network can contribute to PPK pathogenesis. Nail dystrophy, on the other hand, was not observed in *Krt16^−/−^* mice, and may depend more heavily on the dominant negative interference of keratin filaments. Accordingly, the deletion of individual or even multiple keratins leads to no or very minor nail defects in mice ([@R31]; [@R29]; [@R32]; this study), whereas a dominant mutation in *Krt75* elicits the characteristic nail overgrowth in mouse ([@R5]). In humans, where all known PC-causative keratin mutations are dominant, nail involvement is highly penetrant. These observations in humans, mouse models, and now our findings in *Krt16^−/−^* mice showcase how a combination of loss and gain of function phenotypes can contribute to the complex overall clinical presentation of PC. Furthermore, our data substantiate the hypothesis that FPPK and PC can share a common pathogenesis ([@R28]; [@R2]), since *Krt16^−/−^* mice developed FPPK along with oral lesions, another PC-like symptom. We also noticed differences in disease onset, phenotype severity, and change in barrier permeability in *Krt16^−/−^* mice, supporting the idea of keratin, non-keratin and/or environmental modifiers in PPK pathogenesis ([@R24]). It will be interesting to examine whether the mechanism(s) by which loss of *Krt16* results in the development of PPK overlap(s) with the pathogenesis of other keratoderma, e.g. striate PPK (*Desmoplakin*) or diffuse PPK (*KRT1*, *Desmoglein*).

We only observed occasional and locally restricted areas of cell lysis in established *Krt16^−/−^* front paw calluses ([Figure 3B](#F3){ref-type="fig"}, arrowhead and inset), but never in hind paw calluses or uninvolved skin, despite the global expression pattern of *Krt16* in glabrous tissue. Expression of other structurally important keratins in the suprabasal layer, such as *Krt1* and *Krt10*, was also generally intact ([Figure 4D](#F4){ref-type="fig"}) and likely contributes to the maintenance of cellular integrity in the absence of *Krt16*. Thus, we speculate that *Krt16* has an additional function in glabrous skin, separate from its role of structural support. Several studies have already implicated *Krt16* and other type 1 keratins, such as *Krt10* and *Krt17*, as significant players in epidermal homeostasis ([@R21]; [@R8]; [@R27]; [@R22]; [@R13]). A key feature of *Krt16* expression in the skin is its selective induction following injury, UV exposure, or in chronic disease states (e.g. psoriasis) ([@R21]; [@R7]; [@R15]). Glabrous skin is a specialized tissue designed to withstand and adapt to significant mechanical trauma ([@R26]; [@R3]). It will be worth investigating the function of constitutive *Krt16* expression in this unique environment, and whether the underlying mechanism bears any relationship to the role(s) fulfilled by Krt16 when induced by injury and other relevant stressors.

Materials and Methods {#S7}
=====================

Generation of Krt16*^−/−^* mice {#S8}
-------------------------------

*C57 Bl/6* ES cells in which the *Krt16* coding sequence has been replaced with a lacZ-loxP-Neo^R^-loxP cassette were created by Velocigene using funds provided by the trans-NIH Knock-Out Mouse Project (KOMP), and obtained from the KOMP repository (supported by the NCRR-NIH). ES cells were injected into *C57 Bl/6^cBrd/cBrd^* albino blastocysts (distributed by the NCI, Frederick, MD). A high coat color male chimera was bred to *C57 Bl/6^cBrd/cBrd^* albino females and 100% germline transmission was observed. *Krt16^+/−^* F1 offspring were born at the expected 50:50 ratio and inter-crossed to generate *Krt16^−/−^* mice. All experiments involving mice were reviewed and approved by the Johns Hopkins Institutional Animal Care and Use Committee. Mouse lines were maintained under specific pathogen-free conditions (SPF), and fed chow and water *ad libitum*. All experiments were performed using littermate controls (*wild-type* or *Krt16^+/−^*).

Histopathology and immunofluorescence {#S9}
-------------------------------------

Whole tongues and paws were fixed overnight in Bouin's (Sigma) or 4% para-formaldehyde/PBS. Tissue was then either rinsed and embedded in Sakura Tissue-Tek O.C.T. (VWR, Radnor, PA), or dehydrated and processed for routine paraffin embedding. Sections were cut at 5μm and stained with hematoxylin/eosin (H&E) according to standard protocols. For immunofluorescence, frozen sections or rehydrated paraffin-embedded sections were washed in PBS, blocked in 5% NGS, 0.1% Triton-X100 for 1 h at room temperature, incubated in primary antibody solution (2.5% NGS, 0.1% Triton-X100) for 1 h at room temperature, washed in PBS, incubated in secondary antibody (2.5% NGS) for 1 h, counterstained with DAPI, mounted, and imaged using an inverted Zeiss fluorescence microscope with ApoTome attachment. Antibodies used were directed against: Krt17 (1:1000; [@R17]), Krt6 (1:250; [@R17]), Krt10 (1:500; Covance, Princeton, NJ), filaggrin (1:500; Covance, Princeton, NJ), Ki67 (Sp6 clone,1:200; ThermoFisher, Pittsburgh, PA), and AlexaFluor488 (1:1000; Invitrogen, Carlsbad, CA). TUNEL staining was performed as described ([@R18]).

Whole-mount Xgal and barrier function assays {#S10}
--------------------------------------------

Whole paws were fixed for 1 h in 4% paraformaldehyde/PBS at 4°C, washed in PBS, and permeabilized via 3×15 min washes in PBS containing 2mM MgCl~2~, 0.01% sodium deoxycholate, and 0.02% NP-40. The tissue was then transferred to scintillation vials containing a 1mg/ml X-gal solution (30mM K~4~Fe(CN)~6~, 30mM K~3~Fe(CN)~6~, 2mM MgCl~2~, 0.01% sodium deoxycholate, 0.02% NP-40, PBS) for 30 min at 30°C. Samples were photographed immediately. Toluidine Blue dye staining, to assess outside-in barrier function, was carried out on newborn pups and adult paws as described ([@R12]).

Behavioral assays to assess mobility {#S11}
------------------------------------

Individual mice were allowed to acclimate for 30 min and then placed into a rectangular plexiglas chamber with a stainless steel floor. To eliminate outside positional cues, the chamber was uniformly illuminated and its sides masked with cardboard. Mobility was monitored over a time frame of 30 min for infrared beam-breaking activity and the data was recorded using the Optimax software (Columbus Instruments, Columbus, OH). Results represent an average of three separate experiments.

Supplementary Material {#S15}
======================

The authors thank Dr. Michael Caterina for help with the behavioral assays, Janet Folmer for assistance with electron microscopy, and members of the Coulombe lab for discussion and critical reading of the manuscript. These studies were supported by grant AR44232 to P.A.C. from the National Institute of Arthritis, Musculoskeletal, and Skin Diseases (NIAMS) at the National Institutes of Health.

**Conflict of Interest**

The authors declare no conflict of interest.

![Failure to thrive, high postnatal mortality, and posterior tongue lesions in *Krt16^−/−^* mice\
(**a**) High postnatal mortality in *Krt16^−/−^* mice. 34% of *Krt16^−/−^* mice die within 24 h after birth; only a third survive past weaning. (**b**) *Krt16^−/−^* mice weigh significantly less than control littermates. Error bars = SEM. (**c**) P17 *Krt16^−/−^* mice are smaller than littermates and a white-yellow plaque covers the posterior dorsal area of their tongues (arrowhead). (**d**) H&E-stained sections of posterior tongues at P0 and P7. *Krt16^−/−^* mice show no obvious defects at birth. By P7, the architecture of filiform papillae (fp) is markedly disrupted, and massive hyperkeratosis leads to macroscopically visible plaques. Note the paucity of cell lysis in the suprabasal layer. Dotted line = epithelial/muscle junction. Scale bar, 50μm.](nihms346163f1){#F1}

![Krt17 levels are selectively reduced in *Krt16^−/−^* filiform papillae\
(**a**) Immunofluorescent stainings in P0 and P7 tongue cross-sections show a reduction of Krt17 in *Krt16^−/−^* filiform papillae (white arrowheads), but not in fungiform papillae (yellow arrowhead). Constitutive expression of Krt17 in hair follicles at P0 is not affected. Krt6 and Krt10 are unchanged in both filiform papillae in *Krt16^−/−^* tongues and skin (white arrowheads). Scale bars, 50μm. (**b**) Whole tongue lysates show a universal reduction in Krt17 expression in *Krt16^−/−^* tongue epithelium. Krt5 expression is unaltered. To a lesser extent, *Krt17* mRNA levels are also reduced (data not shown).](nihms346163f2){#F2}

![*Krt16^−/−^* mice develop palmoplantar keratoderma\
(**a**) Adult *Krt16^−/−^* mice develop PPK-like lesions in their front and hind paws. Callus formation is common in areas of high physical impact (arrowheads), despite the global *Krt16* expression pattern in glabrous skin (see Xgal stain). Note the presence of focal hyperpigmentation in front paw calluses. (**b**) H&E-stained cross-sections of front paws, showing expansion of the epidermis and massive hyperkeratosis in *Krt16^−/−^* mice. Occasionally, focal suprabasal lysis is observed in *Krt16^−/−^* calluses (arrowheads). sc = stratum corneum, epi = epidermis. Scale bars, 100μm (calluses), 50μm (insets). (**c**) Hyperkeratosis without lysis in *Krt16^−/−^* hind paw epidermis. Note the change in shape and arrangement of suprabasal nuclei. sc = stratum corneum, sb = suprabasal layer, b = basal layer. Scale bar, 50μm.](nihms346163f3){#F3}

![*Krt16^−/−^* calluses - impaired mobility, hyperproliferation, and compromised epidermal barrier function\
(**a**) *Krt16^−/−^* mice are less active than controls in a behavioral assay (experimental time frame of 30 min). Asterisks = p-value \< 0.002, Student's T-test. (**b**) Toluidine Blue dye penetration in adult front paws highlights focal loss of epidermal barrier function in weight bearing areas (arrowheads). (**c**) *Krt16^−/−^* front paw calluses show a 2-fold increase in proliferation and a more modest increase in uninvolved areas adjacent to established calluses. Asterisk = p-value \< 0.03, Student's T-test. (**d**) Basal/suprabasal induction of Krt17 and marked reduction of barrier protein filaggrin in *Krt16^−/−^* front paw calluses. Krt1 expression is patchy in areas subject to barrier breach, but otherwise normal. Dotted line = epidermal/dermal junction. Scale bar, 50μm.](nihms346163f4){#F4}