Activation of fetal-like molecular programs during regeneration in the intestine and beyond.

Tissue regeneration after damage is generally thought to involve the mobilization of adult stem cells that divide and differentiate into progressively specialized progeny. However, recent studies indicate that tissue regeneration can be accompanied by reversion to a fetal-like state. During this process, cells at the injury site reactivate programs that operate during fetal development but are typically absent in adult homeostasis. Here, we summarize our current understanding of the molecular signals and epigenetic mediators that orchestrate "fetal-like reversion" during intestinal regeneration. We also explore evidence for this phenomenon in other organs and species and highlight open questions that merit future examination.

Mapping genes for human face shape: exploration of univariate phenotyping strategies.

Human facial shape, while strongly heritable, involves both genetic and structural complexity, necessitating precise phenotyping for accurate assessment. Common phenotyping strategies include simplifying 3D facial features into univariate traits such as anthropometric measurements (e.g., inter-landmark distances), unsupervised dimensionality reductions (e.g., principal component analysis (PCA) and auto-encoder (AE) approaches), and assessing resemblance to particular facial gestalts (e.g., syndromic facial archetypes). This study provides a comparative assessment of these strategies in genome-wide association studies (GWASs) of 3D facial shape. Specifically, we investigated inter-landmark distances, PCA and AE-derived latent dimensions, and facial resemblance to random, extreme, and syndromic gestalts within a GWAS of 8,426 individuals of recent European ancestry. Inter-landmark distances exhibit the highest SNP-based heritability as estimated via LD score regression, followed by AE dimensions. Conversely, resemblance scores to extreme and syndromic facial gestalts display the lowest heritability, in line with expectations. Notably, the aggregation of multiple GWASs on facial resemblance to random gestalts reveals the highest number of independent genetic loci. This novel, easy-to-implement phenotyping approach holds significant promise for capturing genetically relevant morphological traits derived from complex biomedical imaging datasets, and its applications extend beyond faces. Nevertheless, these different phenotyping strategies capture different genetic influences on craniofacial shape. Thus, it remains valuable to explore these strategies individually and in combination to gain a more comprehensive understanding of the genetic factors underlying craniofacial shape and related traits. Author Summary: Advancements linking variation in the human genome to phenotypes have rapidly evolved in recent decades and have revealed that most human traits are influenced by genetic variants to at least some degree. While many traits, such as stature, are straightforward to acquire and investigate, the multivariate and multipartite nature of facial shape makes quantification more challenging. In this study, we compared the impact of different facial phenotyping approaches on gene mapping outcomes. Our findings suggest that the choice of facial phenotyping method has an impact on apparent trait heritability and the ability to detect genetic association signals. These results offer valuable insights into the importance of phenotyping in genetic investigations, especially when dealing with highly complex morphological traits.

Myosin II mediates Shh signals to shape dental epithelia via control of cell adhesion and movement.

The development of ectodermal organs begins with the formation of a stratified epithelial placode that progressively invaginates into the underlying mesenchyme as the organ takes its shape. Signaling by secreted molecules is critical for epithelial morphogenesis, but how that information leads to cell rearrangement and tissue shape changes remains an open question. Using the mouse dentition as a model, we first establish that non-muscle myosin II is essential for dental epithelial invagination and show that it functions by promoting cell-cell adhesion and persistent convergent cell movements in the suprabasal layer. Shh signaling controls these processes by inducing myosin II activation via AKT. Pharmacological induction of AKT and myosin II can also rescue defects caused by the inhibition of Shh. Together, our results support a model in which the Shh signal is transmitted through myosin II to power effective cellular rearrangement for proper dental epithelial invagination.

Perivascular NOTCH3+ stem cells drive meningioma tumorigenesis and resistance to radiotherapy.

Meningiomas are the most common primary intracranial tumors. Treatments for patients with meningiomas are limited to surgery and radiotherapy, and systemic therapies remain ineffective or experimental. Resistance to radiotherapy is common in high-grade meningiomas and the cell types and signaling mechanisms that drive meningioma tumorigenesis and resistance to radiotherapy are incompletely understood. Here we report NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy and find that perivascular NOTCH3+ stem cells are conserved across meningiomas from humans, dogs, and mice. Integrating single-cell transcriptomics with lineage tracing and imaging approaches in genetically engineered mouse models and xenografts, we show NOTCH3 drives tumor initiating capacity, cell proliferation, angiogenesis, and resistance to radiotherapy to increase meningioma growth and reduce survival. To translate these findings to patients, we show that an antibody stabilizing the extracellular negative regulatory region of NOTCH3 blocks meningioma tumorigenesis and sensitizes meningiomas to radiotherapy, reducing tumor growth and improving survival.

Patterning and folding of intestinal villi by active mesenchymal dewetting.

Tissue folds are structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, finger-like protrusions that enable nutrient absorption. However, the molecular and mechanical processes driving villus morphogenesis remain unclear. Here, we identify an active mechanical mechanism that simultaneously patterns and folds the intestinal epithelium to initiate villus formation. At the cellular level, we find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. This symmetry-breaking process requires altered cell and extracellular matrix interactions that are enabled by matrix metalloproteinase-mediated tissue fluidization. Computational models, together with in vitro and in vivo experiments, revealed that these cellular features manifest at the tissue level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active dewetting of a thin liquid film.

Proliferation-driven mechanical compression induces signalling centre formation during mammalian organ development.

Localized sources of morphogens, called signalling centres, play a fundamental role in coordinating tissue growth and cell fate specification during organogenesis. However, how these signalling centres are established in tissues during embryonic development is still unclear. Here we show that the main signalling centre orchestrating development of rodent incisors, the enamel knot (EK), is specified by a cell proliferation-driven buildup in compressive stresses (mechanical pressure) in the tissue. Direct mechanical measurements indicate that the stresses generated by cell proliferation are resisted by the surrounding tissue, creating a circular pattern of mechanical anisotropy with a region of high compressive stress at its centre that becomes the EK. Pharmacological inhibition of proliferation reduces stresses and suppresses EK formation, and application of external pressure in proliferation-inhibited conditions rescues the formation of the EK. Mechanical information is relayed intracellularly through YAP protein localization, which is cytoplasmic in the region of compressive stress that establishes the EK and nuclear in the stretched anisotropic cells that resist the pressure buildup around the EK. Together, our data identify a new role for proliferation-driven mechanical compression in the specification of a model signalling centre during mammalian organ development.

Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains.

A key aspect of nutrient absorption is the exquisite division of labour across the length of the small intestine, with individual nutrients taken up at different proximal:distal positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum and ileum. By examining the fine-scale longitudinal transcriptional patterns that span the mouse and human small intestine, we instead identified five domains of nutrient absorption that mount distinct responses to dietary changes, and three regional stem cell populations. Molecular domain identity can be detected with machine learning, which provides a systematic method to computationally identify intestinal domains in mice. We generated a predictive model of transcriptional control of domain identity and validated the roles of Ppar-δ and Cdx1 in patterning lipid metabolism-associated genes. These findings represent a foundational framework for the zonation of absorption across the mammalian small intestine.

Vole genomics links determinate and indeterminate growth of teeth.

Continuously growing teeth are an important innovation in mammalian evolution, yet genetic regulation of continuous growth by stem cells remains incompletely understood. Dental stem cells responsible for tooth crown growth are lost at the onset of tooth root formation. Genetic signaling that initiates this loss is difficult to study with the ever-growing incisor and rooted molars of mice, the most common mammalian dental model species, because signals for root formation overlap with signals that pattern tooth size and shape (i.e., cusp patterns). Different species of voles (Cricetidae, Rodentia, Glires) have evolved rooted and unrooted molars that have similar size and shape, providing alternative models for studying roots. We assembled a de novo genome of Myodes glareolus, a vole with high-crowned, rooted molars, and performed genomic and transcriptomic analyses in a broad phylogenetic context of Glires (rodents and lagomorphs) to assess differential selection and evolution in tooth forming genes. We identified 15 dental genes with changing synteny relationships and six dental genes undergoing positive selection across Glires, two of which were undergoing positive selection in species with unrooted molars, Dspp and Aqp1. Decreased expression of both genes in prairie voles with unrooted molars compared to bank voles supports the presence of positive selection and may underlie differences in root formation. Bulk transcriptomics analyses of embryonic molar development in bank voles also demonstrated conserved patterns of dental gene expression compared to mice, with species-specific variation likely related to developmental timing and morphological differences between mouse and vole molars. Our results support ongoing evolution of dental genes across Glires, revealing the complex evolutionary background of convergent evolution for ever-growing molars.

An interactive atlas of three-dimensional syndromic facial morphology.

Craniofacial phenotyping is critical for both syndrome delineation and diagnosis because craniofacial abnormalities occur in 30% of characterized genetic syndromes. Clinical reports, textbooks, and available software tools typically provide two-dimensional, static images and illustrations of the characteristic phenotypes of genetic syndromes. In this work, we provide an interactive web application that provides three-dimensional, dynamic visualizations for the characteristic craniofacial effects of 95 syndromes. Users can visualize syndrome facial appearance estimates quantified from data and easily compare craniofacial phenotypes of different syndromes. Our application also provides a map of morphological similarity between a target syndrome and other syndromes. Finally, users can upload 3D facial scans of individuals and compare them to our syndrome atlas estimates. In summary, we provide an interactive reference for the craniofacial phenotypes of syndromes that allows for precise, individual-specific comparisons of dysmorphology.

Adult dental epithelial stem cell-derived organoids deposit hydroxylapatite biomineral.

Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.

Syndrome-informed phenotyping identifies a polygenic background for achondroplasia-like facial variation in the general population.

Human craniofacial shape is highly variable yet highly heritable with genetic variants interacting through multiple layers of development. Here, we hypothesize that Mendelian phenotypes represent the extremes of a phenotypic spectrum and, using achondroplasia as an example, we introduce a syndrome-informed phenotyping approach to identify genomic loci associated with achondroplasia-like facial variation in the normal population. We compared three-dimensional facial scans from 43 individuals with achondroplasia and 8246 controls to calculate achondroplasia-like facial scores. Multivariate GWAS of the control scores revealed a polygenic basis for normal facial variation along an achondroplasia-specific shape axis, identifying genes primarily involved in skeletal development. Jointly modeling these genes in two independent control samples showed craniofacial effects approximating the characteristic achondroplasia phenotype. These findings suggest that both complex and Mendelian genetic variation act on the same developmentally determined axes of facial variation, providing new insights into the genetic intersection of complex traits and Mendelian disorders.

Epithelial zonation along the mouse and human small intestine defines five discrete metabolic domains.

A key aspect of nutrient absorption is the exquisite division of labor across the length of the small intestine, with individual classes of micronutrients taken up at different positions. For millennia, the small intestine was thought to comprise three segments with indefinite borders: the duodenum, jejunum, and ileum. By examining fine-scale longitudinal segmentation of the mouse and human small intestines, we identified transcriptional signatures and upstream regulatory factors that define five domains of nutrient absorption, distinct from the three traditional sections. Spatially restricted expression programs were most prominent in nutrient-absorbing enterocytes but initially arose in intestinal stem cells residing in three regional populations. While a core signature was maintained across mice and humans with different diets and environments, domain properties were influenced by dietary changes. We established the functions of Ppar-ẟ and Cdx1 in patterning lipid metabolism in distal domains and generated a predictive model of additional transcription factors that direct domain identity. Molecular domain identity can be detected with machine learning, representing the first systematic method to computationally identify specific intestinal regions in mice. These findings provide a foundational framework for the identity and control of longitudinal zonation of absorption along the proximal:distal small intestinal axis.

Bcl-6 expression by CD4+ T cells determines concomitant immunity and host resistance across distinct parasitic infections.

Cluster of differentiation (CD4+) T cells consist of multiple subtypes, defined by expression of lineage-specific transcription factors, that contribute to the control of infectious diseases by providing help to immune and nonimmune target cells. In the current study, we examined the role of B cell lymphoma (Bcl)-6, a transcriptional repressor and master regulator of T follicular helper cell differentiation, in T cell-mediated host defense against intestinal and systemic parasitic infections. We demonstrate that while Bcl-6 expression by CD4+ T cells is critical for antibody-mediated protective immunity against secondary infection with the nematode Heligmosoides polygyrus bakeri, it paradoxically compromises worm expulsion during primary infection by limiting the generation of interleukin-10 (IL-10)-producing Gata3+ T helper 2 cells. Enhanced worm expulsion in the absence of Bcl-6 expressing T cells was associated with amplified intestinal goblet cell differentiation and increased generation of alternatively activated macrophages, effects that were reversed by neutralization of IL-10 signals. An increase in IL-10 production by Bcl-6-deficient CD4+ T cells was also evident in the context of systemic Leishmania donovani infection, but in contrast to Heligmosoides polygyrus bakeri infection, compromised T helper 1-mediated liver macrophage activation and increased susceptibility to this distinct parasitic challenge. Collectively, our studies suggest that host defense pathways that protect against parasite superinfection and lethal systemic protozoal infections can be engaged at the cost of compromised primary resistance to well-tolerated helminths.

Epithelial TNF controls cell differentiation and CFTR activity to maintain intestinal mucin homeostasis.

The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and noninflamed samples from patients with inflammatory bowel disease (IBD) undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF/CFTR signaling axis in the adult intestine and identify epithelial cell-derived TNF as an upstream regulator of mucin homeostasis.

Direct reprogramming of oral epithelial progenitor cells to cancer stem cells at single cell resolution in vivo.

Tumor initiation represents the initial step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Most studies investigating cancer-driving mechanisms in solid tumors rely on analyses of established malignant lesions, and thus cannot directly capture processes underlying the reprogramming of normal progenitor cells into cancer cells. Here, using spatiotemporally controlled oncogene expression in a genetically engineered system we demonstrate that concomitant YAP activation and HPV E6-E7 -mediated inhibition of tumor suppressive pathways is sufficient to rapidly reprogram oral epithelial progenitor cells (OEPCs) into cancer stem cells (CSCs). Single cell analyses of these nascent CSCs revealed hallmark transcriptional programs driving tumor initiation. Importantly, these CSC-enriched expression signatures distinguish normal tissue from malignant head and neck tumors and are associated with poor patient survival. Elucidating mechanisms underlying OEPC to CSC reprogramming may offer new insights to halt the conversion of premalignant cells into invasive carcinoma. HIGHLIGHTS: YAP and HPV E6-E7 reprogram oral epithelial progenitor cells into cancer stem cells. Single cell analyses reveal the transcriptional architecture of tumor initiation.CSC transcriptional programs distinguish normal tissue from carcinoma.CSC signatures are associated with poor head and neck cancer survival.

SPRY4-dependent ERK negative feedback demarcates functional adult stem cells in the male mouse germline†.

Niche-derived growth factors support self-renewal of mouse spermatogonial stem and progenitor cells through ERK MAPK signaling and other pathways. At the same time, dysregulated growth factor-dependent signaling has been associated with loss of stem cell activity and aberrant differentiation. We hypothesized that growth factor signaling through the ERK MAPK pathway in spermatogonial stem cells is tightly regulated within a narrow range through distinct intracellular negative feedback regulators. Evaluation of candidate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)-responsive genes known to dampen downstream signaling revealed robust induction of specific negative feedback regulators, including Spry4, in cultured mouse spermatogonial stem cells in response to glial cell line-derived neurotrophic factor or fibroblast growth factor 2. Undifferentiated spermatogonia in vivo exhibited high levels of Spry4 mRNA. Quantitative single-cell analysis of ERK MAPK signaling in spermatogonial stem cell cultures revealed both dynamic signaling patterns in response to growth factors and disruption of such effects when Spry4 was ablated, due to dysregulation of ERK MAPK downstream of RAS. Whereas negative feedback regulator expression decreased during differentiation, loss of Spry4 shifted cell fate toward early differentiation with concomitant loss of stem cell activity. Finally, a mouse Spry4 reporter line revealed that the adult spermatogonial stem cell population in vivo is demarcated by strong Spry4 promoter activity. Collectively, our data suggest that negative feedback-dependent regulation of ERK MAPK is critical for preservation of spermatogonial stem cell fate within the mammalian testis.

Patterning and folding of intestinal villi by active mesenchymal dewetting.

Tissue folding generates structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, the numerous finger-like protrusions that are essential for nutrient absorption. However, the molecular and mechanical mechanisms driving the initiation and morphogenesis of villi remain a matter of debate. Here, we identify an active mechanical mechanism that simultaneously patterns and folds intestinal villi. We find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. At the cell-level, this occurs through a process dependent upon matrix metalloproteinase-mediated tissue fluidization and altered cell-ECM adhesion. By combining computational models with in vivo experiments, we reveal these cellular features manifest at the tissue-level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active de-wetting of a thin liquid film.

NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy.

Meningiomas are the most common primary intracranial tumors1-3. Treatments for patients with meningiomas are limited to surgery and radiotherapy, and systemic therapies remain ineffective or experimental4,5. Resistance to radiotherapy is common in high-grade meningiomas6, and the cell types and signaling mechanisms driving meningioma tumorigenesis or resistance to radiotherapy are incompletely understood. Here we report NOTCH3 drives meningioma tumorigenesis and resistance to radiotherapy and find NOTCH3+ meningioma mural cells are conserved across meningiomas from humans, dogs, and mice. NOTCH3+ cells are restricted to the perivascular niche during meningeal development and homeostasis and in low-grade meningiomas but are expressed throughout high-grade meningiomas that are resistant to radiotherapy. Integrating single-cell transcriptomics with lineage tracing and imaging approaches across mouse genetic and xenograft models, we show NOTCH3 drives tumor initiating capacity, cell proliferation, angiogenesis, and resistance to radiotherapy to increase meningioma growth and reduce survival. An antibody stabilizing the extracellular negative regulatory region of NOTCH37,8 blocks meningioma tumorigenesis and sensitizes meningiomas to radiotherapy, reducing tumor growth and improving survival in preclinical models. In summary, our results identify a conserved cell type and signaling mechanism that underlie meningioma tumorigenesis and resistance to radiotherapy, revealing a new therapeutic vulnerability to treat meningiomas that are resistant to standard interventions.

The developmental basis for scaling of mammalian tooth size.

When evolution leads to differences in body size, organs generally scale along. A well-known example of the tight relationship between organ and body size is the scaling of mammalian molar teeth. To investigate how teeth scale during development and evolution, we compared molar development from initiation through final size in the mouse and the rat. Whereas the linear dimensions of the rat molars are twice that of the mouse molars, their shapes are largely the same. Here, we focus on the first lower molars that are considered the most reliable dental proxy for size-related patterns due to their low within-species variability. We found that scaling of the molars starts early, and that the rat molar is patterned equally as fast but in a larger size than the mouse molar. Using transcriptomics, we discovered that a known regulator of body size, insulin-like growth factor 1 (Igf1), is more highly expressed in the rat molars compared to the mouse molars. Ex vivo and in vivo mouse models demonstrated that modulation of the IGF pathway reproduces several aspects of the observed scaling process. Furthermore, analysis of IGF1-treated mouse molars and computational modeling indicate that IGF signaling scales teeth by simultaneously enhancing growth and by inhibiting the cusp-patterning program, thereby providing a relatively simple mechanism for scaling teeth during development and evolution. Finally, comparative data from shrews to elephants suggest that this scaling mechanism regulates the minimum tooth size possible, as well as the patterning potential of large teeth.