Apoptotic vesicles rescue impaired mesenchymal stem cells and their therapeutic capacity for osteoporosis by restoring miR-145a-5p deficiency.

Apoptotic vesicles (apoVs) play a vital role in various pathological conditions; however, we have yet to fully understand their precise biological effects in rescuing impaired mesenchymal stem cells (MSCs) and regulating tissue homeostasis. Here, we proved that systemic infusion of bone marrow MSCs derived from wild-type (WT) mice effectively improved the osteopenia phenotype and hyperimmune state in ovariectomized (OVX) mice. Importantly, the WT MSCs rescued the impairment of OVX MSCs both in vivo and in vitro, whereas OVX MSCs did not show the same efficacy. Interestingly, treatment with apoVs derived from WT MSCs (WT apoVs) restored the impaired biological function of OVX MSCs and their ability to improve osteoporosis. This effect was not observed with OVX MSCs-derived apoVs (OVX apoVs) treatment. Mechanistically, the reduced miR-145a-5p expression hindered the osteogenic differentiation and immunomodulatory capacity of OVX MSCs by affecting the TGF-ß/Smad 2/3-Wnt/ß-catenin signaling axis, resulting in the development of osteoporosis. WT apoVs directly transferred miR-145a-5p to OVX MSCs, which were then reused to restore their impaired biological functions. Conversely, treatment with OVX apoVs did not produce significant effects due to their limited expression of miR-145a-5p. Overall, our findings unveil the remarkable potential of apoVs in rescuing the biological function and therapeutic capability of MSCs derived from individuals with diseases. This discovery offers a new avenue for exploring apoVs-based MSC engineering and expands the application scope of stem cell therapy, contributing to the maintenance of bone homeostasis through a previously unrecognized mechanism.

Mapping the cellular biogeography of human bone marrow niches using single-cell transcriptomics and proteomic imaging.

Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis.

Apoptotic Vesicular Metabolism Contributes to Organelle Assembly and Safeguards Liver Homeostasis and Regeneration.

BACKGROUND & AIMS: Apoptosis generates plenty of membrane-bound nanovesicles, the apoptotic vesicles (apoVs), which show promise for biomedical applications. The liver serves as a significant organ for apoptotic material removal. Whether and how the liver metabolizes apoptotic vesicular products and contributes to liver health and disease is unrecognized. METHODS: apoVs were labeled and traced after intravenous infusion. Apoptosis-deficient mice by Fas mutant (Fasmut) and Caspase-3 knockout (Casp3-/-) were used with apoV replenishment to evaluate the physiological apoV function. Combinations of morphologic, biochemical, cellular, and molecular assays were applied to assess the liver while hepatocyte analysis was performed. Partial hepatectomy and acetaminophen liver failure models were established to investigate liver regeneration and disease recovery. RESULTS: We discovered that the liver is a major metabolic organ of circulatory apoVs, in which apoVs undergo endocytosis by hepatocytes via a sugar recognition system. Moreover, apoVs play an indispensable role to counteract hepatocellular injury and liver impairment in apoptosis-deficient mice upon replenishment. Surprisingly, apoVs form a chimeric organelle complex with the hepatocyte Golgi apparatus through the soluble N-ethylmaleimide-sensitive factor attachment protein receptor machinery, which preserves Golgi integrity, promotes microtubule acetylation by regulating α-tubulin N-acetyltransferase 1, and consequently facilitates hepatocyte cytokinesis for liver recovery. The assembly of the apoV-Golgi complex is further revealed to contribute to liver homeostasis, regeneration, and protection against acute liver failure. CONCLUSIONS: These findings establish a previously unrecognized functional and mechanistic framework that apoptosis through vesicular metabolism safeguards liver homeostasis and regeneration, which holds promise for hepatic disease therapeutics.

Natural compounds and mesenchymal stem cells: implications for inflammatory-impaired tissue regeneration.

Inflammation is a common and important pathological process occurring in any part of the body and relating to a variety of diseases. Effective tissue repair is critical for the survival of impaired organisms. Considering the side effects of the currently used anti-inflammatory medications, new therapeutic agents are urgently needed for the improvement of regenerative capacities of inflammatory-impaired tissues. Mesenchymal stromal stem/progenitor cells (MSCs) are characterized by the capabilities of self-renewal and multipotent differentiation and exhibit immunomodulatory capacity. Due to the ability to modulate inflammatory phenotypes and immune responses, MSCs have been considered as a potential alternative therapy for autoimmune and inflammatory diseases. Natural compounds (NCs) are complex small multiple-target molecules mostly derived from plants and microorganisms, exhibiting therapeutic effects in many disorders, such as osteoporosis, diabetes, cancer, and inflammatory/autoimmune diseases. Recently, increasing studies focused on the prominent effects of NCs on MSCs, including the regulation of cell survival and inflammatory response, as well as osteogenic/adipogenic differentiation capacities, which indicate the roles of NCs on MSC-based cytotherapy in several inflammatory diseases. Their therapeutic effects and fewer side effects in numerous physiological processes, compared to chemosynthetic drugs, made them to be a new therapeutic avenue combined with MSCs for impaired tissue regeneration. Here we summarize the current understanding of the influence of NCs on MSCs and related downstream signaling pathways, specifically in pathological inflammatory conditions. In addition, the emerging concepts through the combination of NCs and MSCs to expand the therapeutic perspectives are highlighted. A promising MSC source from oral/dental tissues is also discussed, with a remarkable potential for MSC-based therapy in future clinical applications.

Nanozyme-Based Robotics Approach for Targeting Fungal Infection.

Fungal pathogens have been designated by the World Health Organization as microbial threats of the highest priority for global health. It remains a major challenge to improve antifungal efficacy at the site of infection while avoiding off-target effects, fungal spreading, and drug tolerance. Here, a nanozyme-based microrobotic platform is developed that directs localized catalysis to the infection site with microscale precision to achieve targeted and rapid fungal killing. Using electromagnetic field frequency modulation and fine-scale spatiotemporal control, structured iron oxide nanozyme assemblies are formed that display tunable dynamic shape transformation and catalysis activation. The catalytic activity varies depending on the motion, velocity, and shape providing controllable reactive oxygen species (ROS) generation. Unexpectedly, nanozyme assemblies bind avidly to fungal (Candida albicans) surfaces to enable concentrated accumulation and targeted ROS-mediated killing in situ. By exploiting these tunable properties and selective binding to fungi, localized antifungal activity is achieved using in vivo-like cell spheroid and animal tissue infection models. Structured nanozyme assemblies are directed to Candida-infected sites using programmable algorithms to perform precisely guided spatial targeting and on-site catalysis resulting in fungal eradication within 10 min. This nanozyme-based microrobotics approach provides a uniquely effective and targeted therapeutic modality for pathogen elimination at the infection site.

ERK1-mediated immunomodulation of mesenchymal stem cells ameliorates inflammatory disorders.

Immune system disorders, especially T cell disorders, are important therapeutic targets of mesenchymal stem cells (MSCs) in many autoimmune diseases (ADs). Although extracellular regulated protein kinases (ERKs) play a role in MSC therapy by promoting T cell apoptosis, the mechanism remains unclear. Our findings indicate that ERK1-/- bone marrow MSCs (BMMSCs), but not ERK2-/- BMMSCs, failed to promote T cell apoptosis due to incapacity to activate the ETS2/AURKA/NF-κB/Fas/MCP-1 cascade. Moreover, ERK1-/- BMMSCs were unable to upregulate regulatory T cells and suppress T helper 17 cells. Licochalcone A (LA), which promotes ERK pathway activation, enhanced the therapeutic efficacy of MSC therapy in ulcerative colitis and collagen-induced arthritis mice. Our findings suggest that ERK1, but not ERK2, plays a crucial role in regulating T cells in MSCs. LA-treated MSCs provide a strategy to improve the efficacy of MSC-based treatments for ADs.

Apoptotic vesicles ameliorate lupus and arthritis via phosphatidylserine-mediated modulation of T cell receptor signaling.

Mesenchymal stem cells (MSCs) influence T cells in health, disease and therapy through messengers of intercellular communication including extracellular vesicles (EVs). Apoptosis is a mode of cell death that tends to promote immune tolerance, and a large number of apoptotic vesicles (apoVs) are generated from MSCs during apoptosis. In an effort to characterize these apoVs and explore their immunomodulatory potential, here we show that after replenishing them systemically, the apoV deficiency in Fas mutant mice and pathological lymphoproliferation were rescued, leading to the amelioration of inflammation and lupus activity. ApoVs directly interacted with CD4+ T cells and inhibited CD25 expression and IL-2 production in a dose-dependent manner. A broad range of Th1/2/17 subsets and cytokines including IFNγ, IL17A and IL-10 were suppressed while Foxp3+ cells were maintained. Mechanistically, exposed phosphatidylserine (PtdSer/PS) on apoVs mediated the interaction with T cells to disrupt proximal T cell receptor signaling transduction. Remarkably, administration of apoVs prevented Th17 differentiation and memory formation, and ameliorated inflammation and joint erosion in murine arthritis. Collectively, our findings unveil a previously unrecognized crosstalk between MSC apoVs and CD4+ T cells and suggest a promising therapeutic use of apoVs for autoimmune diseases.

Accurate gingival segmentation from 3D images with artificial intelligence: an animal pilot study.

BACKGROUND: Gingival phenotype plays an important role in dental diagnosis and treatment planning. Traditionally, determining the gingival phenotype is done by manual probing of the gingival soft tissues, an invasive and time-consuming procedure. This study aims to evaluate the feasibility and accuracy of an alternatively novel, non-invasive technology based on the precise 3-dimension (3D) soft tissue reconstruction from intraoral scanning and cone beam computed tomography (CBCT) to predict the gingival biotype. METHODS: As a proof-of-concept, Yorkshire pig mandibles were scanned, and the CBCT data were fed into a deep-learning model to reconstruct the teeth and surrounding bone structure in 3D. By overlaying the CBCT scan with the intraoral scans, an accurate superposition was created and used for virtual measurements of the soft tissue thickness. Meanwhile, gingival thicknesses were also measured by a periodontal probe and digital caliper on the buccal and lingual sides at 3 mm apical to the gingival margin of the posterior teeth and compared with the virtual assessment at the same location. The data obtained from virtual and clinical measurements were compared by Wilcoxon matched-pairs signed-rank analysis, while their correlation was determined by Pearson's r value. The Mann-Whitney U test was used for intergroup comparisons of the amount of difference. RESULTS: Among 108 investigated locations, the clinical and virtual measurements are strongly positively correlated (r = 0.9656, P < 0.0001), and only clinically insignificant differences (0.066 ± 0.223 mm) were observed between the two assessments. There is no difference in the agreement between the virtual and clinical measurements on sexually matured samples (0.087 ± 0.240 mm) and pre-pubertal samples (0.033 ± 0.195 mm). Noticeably, there is a greater agreement between the virtual and clinical measurements at the buccal sites (0.019 ± 0.233 mm) than at the lingual sites (0.116 ± 0.215 mm). CONCLUSION: In summary, the artificial intelligence-based virtual measurement proposed in this work provides an innovative technique potentially for accurately measuring soft tissue thickness using clinical routine 3D imaging systems, which will aid clinicians in generating a more comprehensive diagnosis with less invasive procedures and, in turn, optimize the treatment plans with more predictable outcomes.

Csf1 from marrow adipogenic precursors is required for osteoclast formation and hematopoiesis in bone.

Colony-stimulating factor 1 (Csf1) is an essential growth factor for osteoclast progenitors and an important regulator for bone resorption. It remains elusive which mesenchymal cells synthesize Csf1 to stimulate osteoclastogenesis. We recently identified a novel mesenchymal cell population, marrow adipogenic lineage precursors (MALPs), in bone. Compared to other mesenchymal subpopulations, MALPs expressed Csf1 at a much higher level and this expression was further increased during aging. To investigate its role, we constructed MALP-deficient Csf1 CKO mice using AdipoqCre. These mice had increased femoral trabecular bone mass, but their cortical bone appeared normal. In comparison, depletion of Csf1 in the entire mesenchymal lineage using Prrx1Cre led to a more striking high bone mass phenotype, suggesting that additional mesenchymal subpopulations secrete Csf1. TRAP staining revealed diminished osteoclasts in the femoral secondary spongiosa region of Csf1 CKOAdipoq mice, but not at the chondral-osseous junction nor at the endosteal surface of cortical bone. Moreover, Csf1 CKOAdipoq mice were resistant to LPS-induced calvarial osteolysis. Bone marrow cellularity, hematopoietic progenitors, and macrophages were also reduced in these mice. Taken together, our studies demonstrate that MALPs synthesize Csf1 to control bone remodeling and hematopoiesis.

Apoptotic extracellular vesicles are metabolized regulators nurturing the skin and hair.

Over 300 billion of cells die every day in the human body, producing a large number of endogenous apoptotic extracellular vesicles (apoEVs). Also, allogenic stem cell transplantation, a commonly used therapeutic approach in current clinical practice, generates exogenous apoEVs. It is well known that phagocytic cells engulf and digest apoEVs to maintain the body's homeostasis. In this study, we show that a fraction of exogenous apoEVs is metabolized in the integumentary skin and hair follicles. Mechanistically, apoEVs activate the Wnt/ß-catenin pathway to facilitate their metabolism in a wave-like pattern. The migration of apoEVs is enhanced by treadmill exercise and inhibited by tail suspension, which is associated with the mechanical force-regulated expression of DKK1 in circulation. Furthermore, we show that exogenous apoEVs promote wound healing and hair growth via activation of Wnt/ß-catenin pathway in skin and hair follicle mesenchymal stem cells. This study reveals a previously unrecognized metabolic pathway of apoEVs and opens a new avenue for exploring apoEV-based therapy for skin and hair disorders.

Exocrine pancreas regeneration modifies original pancreas to alleviate diabetes in mouse models.

Diabetes is a major public health issue because of its widely epidemic nature and lack of cure. Here, we show that pancreas-derived mesenchymal stem cells (PMSCs) are capable of regenerating exocrine pancreas when implanted into the kidney capsule of mice with streptozotocin (STZ)-induced diabetes. Mechanistically, we found that the regenerated exocrine pancreas elevated interleukin-6 (IL-6) in PMSC implants, which transiently activated tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) to inhibit IL-17, thereby rescuing damaged exocrine pancreas and islet ß cells. In addition, we used knockout mouse models to show that global lack of IL-6, TNF-α, or IFN-γ resulted in increased severity of STZ-induced diabetes and resistance to PMSC implantation therapy, confirming the roles of these factors in safeguarding pancreatic ß cells. Furthermore, removal of the kidney capsule PMSC implants at 28 days after implantation did not affect the PMSC-initiated therapeutic effect on diabetic mice. This study reveals a previously unknown role of exocrine pancreas regeneration in safeguarding ß cells and demonstrates a "soil-rescues-seed" strategy for type 1 diabetes therapy.

The Vaccine Efficacy Against the SARS-CoV-2 Omicron: A Systemic Review and Meta-Analysis.

Background: COVID-19 is a respiratory illness caused by SARS-CoV-2. The most recent variant is Omicron (line B.1.1.529), which was first identified in South Africa in November 2021. The concern with this variant is the ineffectiveness of vaccines currently available. We aim to systematically evaluate the effectiveness of the currently available COVID-19 vaccines and boosters for the Omicron variant. Methods: We searched the PubMed, Embase, the Cochrane Library and Web of Science databases from inception to June 5th, 2022. Studies that examined the effectiveness of SARS-CoV-2 vaccines against the Omicron variant infection were included. Random-effects model was used to estimate the pooled vaccine effectiveness against the Omicron variant. Results: A total of 13 studies were included to evaluate the effectiveness of the vaccine against the Omicron variant, and 11 studies were included to compare the effectiveness between the two-dose and three-dose (booster) vaccinations. Full vaccination (two-dose with or without booster) showed a protective effect against the Omicron variant compared to no vaccination (OR = 0.62, 95% CI: 0.56-0.69), while the effectiveness decreased significantly over 6 months after the last dose. The two-dose vaccination plus booster provided better protection against the Omicron variant compared to the two-dose vaccination without booster (OR = 0.60, 95% CI: 0.52-0.68). Additional analysis was performed for the most commonly used vaccines in the United Staes: BNT162b2(Pfizer) (OR = 0.65, 95% CI: 0.52-0.82) and mRNA-1273(Moderna) (OR = 0.67, 95% CI: 0.58-0.88) vaccines in the US, which showed similar effectiveness compared to no vaccination. Conclusions: The full dose of SARS-CoV-2 vaccination effectively reduces infection from the SARS-CoV-2 Omicron variant; however, the effectiveness wanes over time. The booster vaccine provides additional protection against the Omicron variant.

Metabolic Reconfiguration Activates Stemness and Immunomodulation of PDLSCs.

Periodontal ligament derived stem cells (PDLSC) are adult multipotent mesenchymal-like stem cells (MSCs) that can induce a promising immunomodulation to interact with immune cells for disease treatment. Metabolic reconfiguration has been shown to be involved in the immunomodulatory activity of MSCs. However, the underlying mechanisms are largely unknown, and it remains a challenging to establish a therapeutic avenue to enhance immunomodulation of endogenous stem cells for disease management. In the present study, RNA-sequencing (RNA-seq) analysis explores that curcumin significantly promotes PDLSC function through activation of MSC-related markers and metabolic pathways. In vitro stem cell characterization further confirms that self-renewal and multipotent differentiation capabilities are largely elevated in curcumin treated PDLSCs. Mechanistically, RNA-seq reveals that curcumin activates ERK and mTOR cascades through upregulating growth factor pathways for metabolic reconfiguration toward glycolysis. Interestingly, PDLSCs immunomodulation is significantly increased after curcumin treatment through activation of prostaglandin E2-Indoleamine 2,3 dioxygenase (PGE2-IDO) signaling, whereas inhibition of glycolysis activity by 2-deoxyglucose (2-DG) largely blocked immunomodulatory capacity of PDLSCs. Taken together, this study provides a novel pharmacological approach to activate endogenous stem cells through metabolic reprogramming for immunomodulation and tissue regeneration.

mTOR Signaling in the Regulation of CD4+ T Cell Subsets in Periodontal Diseases.

Periodontal disease results from the inflammatory infiltration by the microbial community which is marked through tooth mobility and alveolar bone resorption. The inflammation in periodontal disease is mediated by CD4+ T cells through cytokine secretion and osteoclastogenetic activity. Historically, the inflammatory model in periodontal disease is described through disruption of the balance between two subsets of T helper cells which are T-helper type 1 (Th1) and T-helper type 2 (Th2). However, more and more studies have found that apart from subsets of helper T cells, regulatory T-cells and Th17 cells are also involved in the pathogenesis of periodontal diseases. Growing evidence proves that helper T cells differentiation, activation, and subset determination are under the strong impact of mTOR signaling. mTOR signaling could promote Th1 and Th17 cell differentiation and inhibit Treg commitment through different mTOR complexes, therefore we anticipate a regulation effect of mTOR signaling on periodontal diseases by regulating CD4+ T cell subsets. This review aims to integrate the topical researches about the role of different types of Th cells in the pathogenesis of periodontal diseases, as well as the regulation of mTOR signaling in the specification and selection of Th cell commitment.

Proteomic analysis of MSC-derived apoptotic vesicles identifies Fas inheritance to ameliorate haemophilia a via activating platelet functions.

Apoptotic vesicles (apoVs) are apoptotic cell-derived nanosized vesicles that play a crucial role in multiple pathophysiological settings. However, their detailed characteristics, specific surface markers, and biological properties are not fully elucidated. In this study, we compared mesenchymal stem cell (MSC)-derived apoVs and exosomes from three different types of MSCs including human bone marrow MSCs (hBMSCs), human adipose MSCs (hASCs), and mouse bone marrow MSCs (mBMSCs). We established a unique protein map of MSC-derived apoVs and identified the differences between apoVs and exosomes in terms of functional protein cargo and surface markers. Furthermore, we identified 13 proteins specifically enriched in apoVs compared to exosomes, which can be used as apoV-specific biomarkers. In addition, we showed that apoVs inherited apoptotic imprints such as Fas to ameliorate haemophilia A in factor VIII knockout mice via binding to the platelets' FasL to activate platelet functions, and therefore rescuing the blood clotting disorder. In summary, we systemically characterized MSC-derived apoVs and identified their therapeutic role in haemophilia A treatment through a previously unknown Fas/FasL linkage mechanism.

Whitlockite-Enabled Hydrogel for Craniofacial Bone Regeneration.

Growth-factor-free bone regeneration remains a challenge in craniofacial engineering. Here, we engineered an osteogenic niche composed of a commercially modified alginate hydrogel and whitlockite microparticles (WHMPs), which impart tunable physicochemical properties that can direct osteogenesis of human gingival mesenchymal stem cells (GMSCs). Our in vitro studies demonstrate that WHMPs induce osteogenesis of GMSCs more effectively than previously demonstrated hydroxyapatite microparticles (HApMPs). Alginate-WHMP hydrogels showed higher elasticity without any adverse effects on the viability of the encapsulated GMSCs. Moreover, the alginate-WHMP hydrogels upregulate the mitogen-activated protein kinase (MAPK) pathway, which in turn orchestrates several osteogenic markers, such as RUNX2 and OCN, in the encapsulated GMSCs. Concurrent coculture studies with human osteoclasts demonstrate that GMSCs encapsulated in alginate-WHMP hydrogels downregulate osteoclastic activity, potentially due to release of Mg2+ ions from the WHMPs along with secretion of osteoprotegerin from the GMSCs. In vivo studies demonstrated that the GMSCs encapsulated in our osteogenic niche were able to promote bone repair in calvarial defects in murine models. Altogether, our results confirmed the development of a promising treatment modality for craniofacial bone regeneration based on an injectable growth-factor-free hydrogel delivery system.

RGD-Modified Alginate-GelMA Hydrogel Sheet Containing Gingival Mesenchymal Stem Cells: A Unique Platform for Wound Healing and Soft Tissue Regeneration.

Soft tissue reconstruction has remained a major clinical challenge in dentistry and regenerative medicine. Although current methods have shown partial success, there are several disadvantages associated with these approaches. Gingival mesenchymal stem cells (GMSCs) can be simply obtained in the oral cavity for soft tissue augmentation. Regenerative potential of mesenchymal stem cells (MSCs) encapsulated in hydrogels is well documented. Here, an alginate-gelatin methacrylate (GelMA) hydrogel formulation is developed encapsulating GMSCs within the developed hydrogel. The results confirm that the encapsulated MSCs remain viable within the hydrogel with enhanced collagen deposition. An excisional wound model in mice is utilized to evaluate the in vivo functionality of the GMSC-hydrogel construct for wound healing and soft tissue regeneration. The histology and immunofluorescence analyses confirm the effectiveness of the GMSC-hydrogel in expediting wound healing via enhancing angiogenesis and suppressing local proinflammatory cytokines. Altogether, the findings demonstrate that GMSCs encapsulated in an engineered hydrogel sheet based on alginate and GelMA can be used to expedite wound healing and soft tissue regeneration, with potential applications in plastic and reconstructive surgery as well as dentistry.

Human-chimpanzee fused cells reveal cis-regulatory divergence underlying skeletal evolution.

Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human-chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human-chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.