Craig Johnson

The stress-induced keratin 16 intermediate filament gene, KRT16 , and K16 protein are spatially restricted to the suprabasal layers of the epidermis and widely used as biomarkers in psoriasis, hidradenitis suppurativa, atopic dermatitis, and other inflammatory disorders. Pathogenic variants in KRT16 cause pachyonychia congenita (PC), a rare genetic condition in which tissue homeostasis is disrupted in palmoplantar epidermis, nail, oral mucosa, and other KRT16 -expressing epithelial appendages. How K16 may regulate the molecular mechanisms underlying these conditions is poorly understood. Here, we show that K16 negatively regulates type I interferon (IFN) signaling and innate immune responses in skin. Clinically, the signature palmoplantar keratoderma lesions in individuals with PC showed enhanced type I IFN signaling. In mouse skin in vivo, loss of Krt16 led to exacerbation of imiquimod-induced psoriasiform disease and heightened recruitment of neutrophils in a phorbol ester–induced model of acute sterile inflammation. In KRT16 -null human keratinocytes, loss of K16 amplified IFN signaling including phosphorylated interferon regulatory factor 7 (IRF7) and interferon-stimulated gene 15 (ISG15) after treatment with synthetic dsRNA poly(I:C). Mechanistically, K16 interacted with effectors of the RIG-I–like receptor (RLR) pathway, including 14-3-3ɛ, and inhibited the 14-3-3ɛ:RIG-I interaction upstream of IFN activation in mouse and immortalized human keratinocytes ex vivo. Topical application of the Janus kinase inhibitor cream ruxolitinib to palmoplantar keratoderma lesions of Krt16 -null mice reduced lesion severity. These findings uncover a previously unrecognized paradigm for keratin-dependent regulation of innate immunity, with implications for our understanding of inflammatory skin diseases and potential treatments for PC.

The type I intermediate filament proteins keratin 14 (K14) and keratin 15 (K15) are common to all complex epithelia. K14 is highly expressed by progenitor keratinocytes, in which it provides essential mechanical integrity and gates keratinocyte entry into differentiation by sequestering YAP1, a transcriptional effector of Hippo signaling, to the cytoplasm. K15 has long been used as a marker of hair bulge stem cells, though its specific role in skin epithelia is unknown. Here, we show that the lack of two biochemical determinants, a cysteine residue within the stutter motif of the central rod domain and a 14-3-3 binding site in the N-terminal head domain, renders K15 unable to effectively sequester YAP1 in the cytoplasm like K14 does. We combine insight obtained from cell culture and transgenic mouse models with computational analyses of transcriptomics data and propose a model in which a higher K15:K14 ratio promotes a progenitor state and antagonizes differentiation in keratinocytes of the epidermis.

Formation of skin epithelial appendages like hair follicles requires hedgehog (Hh) signal reception, its conduction through the primary cilium and activation of Gli transcription factors. How Hh signalling induces cell-type-specific responses through Gli transcription factors in hair follicle stem cells and their cilia-dependence remains unclear. Here, we use conditional mouse mutants to genetically dissect the roles of Gli2 and Gli3 transcription factors and cilia in the skin epithelium. Upon keratinocyte-specific depletion of Gli2, hair follicle morphogenesis is delayed whereas sebaceous gland formation is enhanced, suggesting a dual role for Gli2 during appendage development. Gli2 promotes proliferation of sebaceous gland stem cells, impacting the number and size of individual sebaceous gland lobes. While ablation of Gli3 shows no detectable phenotypes, hair follicle cell fate is blocked in Gli2/Gli3 double knockout (dKO) mice, suggesting functional compensation. Finally, loss of cilia phenocopies the depletion of Gli2 but not the Gli2/3 dKO mutants. Our study reveals compartment-specific regulation of murine skin morpohogenesis by Gli2 and cilia-independent activator functions of Gli3 in the absence of Gli2.

Micropatterned human pluripotent stem cells treated with BMP4 (two-dimensional (2D) gastruloids) are among the most widely used stem cell models for human gastrulation. Due to its simplicity and reproducibility, this system is ideal for high-throughput quantitative studies of tissue patterning and has led to many insights into the mechanisms of mammalian gastrulation. However, 2D gastruloids have been studied only up to about 2 days owing to a loss of organization beyond this time with earlier protocols. Here we report an extended 2D gastruloid model to up to 10 days. We discovered a phase of highly reproducible morphogenesis between 2 and 4 days during which directed migration from the primitive streak-like region gives rise to a mesodermal layer beneath an epiblast-like layer. Multiple types of mesoderm arise with striking spatial organization including lateral plate mesoderm-like cells on the colony border and paraxial mesoderm-like cells further inside the colony. Single-cell transcriptomics showed strong similarity of these cells to mesoderm in human and nonhuman primate embryos. Our results illustrate that extended culture of 2D gastruloids provides a powerful model for human mesoderm differentiation and morphogenesis.

Bipolar disorder (BP) is a recurring psychiatric condition characterized by alternating episodes of low energy (depressions) followed by manias (high energy). Cortical network activity produced by GABAergic interneurons may be critical in maintaining the balance in excitatory/inhibitory activity in the brain during development. Initially, GABAergic signaling is excitatory; with maturation, these cells undergo a functional switch that converts GABA_A channels from depolarizing (excitatory) to hyperpolarizing (inhibitory), which is controlled by the intracellular concentration of two chloride transporters. The earliest, NKCC1, promotes chloride entry into the cell and depolarization, while the second (KCC2) stimulates movement of chloride from the neuron, hyperpolarizing it. Perturbations in the timing or expression of NKCC1/KCC2 may affect essential morphogenetic events including cell proliferation, migration, synaptogenesis and plasticity, and thereby the structure and function of the cortex. We derived induced pluripotent stem cells (iPSC) from BP patients and undiagnosed control (C) individuals, then modified a differentiation protocol to form GABAergic interneurons, harvesting cells at sequential stages of differentiation. qRT-PCR and RNA sequencing indicated that after six weeks of differentiation, controls transiently expressed high levels of NKCC1. Using multi-electrode array (MEA) analysis, we observed that BP neurons exhibit increased firing, network bursting and decreased synchrony compared to C. Understanding GABA signaling in differentiation may identify novel approaches and new targets for treatment of neuropsychiatric disorders such as BP.

Micropatterned human pluripotent stem cells (hPSCs) treated with BMP4 (2D gastruloids) are among the most widely used stem cell models for human gastrulation. Due to its simplicity and reproducibility, this system is ideal for high throughput quantitative studies of tissue patterning and has led to many insights into the mechanisms of mammalian gastrulation. However, 2D gastruloids have only been studied up to 48h. Here we extended this system to 96h. We discovered a phase of highly reproducible morphogenesis during which directed migration from the primitive streak-like region gives rise to a mesodermal layer beneath an epiblast-like layer. Multiple types of mesoderm arise with striking spatial organization including lateral mesoderm-like cells on the colony border and paraxial mesoderm-like further inside the colony. Single cell transcriptomics showed strong similarity of these cells to mesoderm in human and non-human primate embryos. However, our data suggest that the annotation of the reference human embryo may need to be revised. This illustrates that extended culture of 2D gastruloids provides a powerful model for human mesoderm differentiation and morphogenesis.

Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta (TGF-β) in the tumor microenvironment reportedly contributes to the skewing of neutrophils to a more pro-tumor phenotype. The effects of TGF-β on neutrophil signaling and migration are, however, unclear. We sought to characterize TGF-β signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether it directly induces neutrophil migration. We found that TGF-β1 does not induce neutrophil chemotaxis in transwell or underagarose migration assays. TGF-β1 does activate canonical signaling through SMAD3 and noncanonical signaling through ERK1/2 in neutrophils in a time- and dose-dependent manner. Additionally, TGF-β1 present in the tumor-conditioned media (TCM) of invasive breast cancer cells results in SMAD3 activation. We discovered that TCM induces neutrophils to secrete leukotriene B₄ (LTB₄), which is a lipid mediator important for amplifying the range of neutrophil recruitment. However, TGF-β1 alone does not induce secretion of LTB₄. RNA-sequencing revealed that TGF-β1 and TCM alter gene expression in HL-60 cells, including the mRNA levels of the pro-tumor oncostatin M (OSM) and vascular endothelial growth factor A (VEGFA). These new insights into the role and impact of TGF-β1 on neutrophil signaling, migration, and gene expression have significant implications in the understanding of the changes in neutrophils that occur in the tumor microenvironment.

How paracrine signals are interpreted to yield multiple cell fate decisions in a dynamic context during human development in vivo and in vitro remains poorly understood. Here we report an automated tracking method to follow signaling histories linked to cell fate in large numbers of human pluripotent stem cells (hPSCs). Using an unbiased statistical approach, we discovered that measured BMP signaling history correlates strongly with fate in individual cells. We found that BMP response in hPSCs varies more strongly in the duration of signaling than the level. However, we discovered that both the level and duration of signaling activity control cell fate choices only by changing the time integral of signaling and that duration and level are therefore interchangeable in this context. In a stem cell model for patterning of the human embryo, we showed that signaling histories predict the fate pattern and that the integral model correctly predicts changes in cell fate domains when signaling is perturbed. Using an RNA-seq screen we then found that mechanistically, BMP signaling is integrated by SOX2.

Purpose:Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of Hedgehog signaling in PDAC have been contradictory, with Hedgehog signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how Hedgehog pathway inhibition reprograms the PDAC microenvironment.Experimental Design:We used a combination of pharmacologic inhibition, gain- and loss-of-function genetic experiments, cytometry by time-of-flight, and single-cell RNA sequencing to study the roles of Hedgehog signaling in PDAC.Results:We found that Hedgehog signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAF) compared with inflammatory CAFs (iCAF). Sonic Hedgehog overexpression promotes tumor growth, while Hedgehog pathway inhibition with the smoothened antagonist, LDE225, impairs tumor growth. Furthermore, Hedgehog pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in regulatory T cells, consistent with increased immunosuppression.Conclusions:Hedgehog pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment.