RESUMO
BACKGROUND & AIMS: In non-alcoholic fatty liver disease (NAFLD), monocytes infiltrate visceral adipose tissue promoting local and hepatic inflammation. However, it remains unclear what drives inflammation and how the immune landscape in adipose tissue differs across the NAFLD severity spectrum. We aimed to assess adipose tissue macrophage (ATM) heterogeneity in a NAFLD cohort. METHODS: Visceral adipose tissue macrophages from lean and obese patients, stratified by NAFLD phenotypes, underwent single-cell RNA sequencing. Adipose tissue vascular integrity and breaching was assessed on a protein level via immunohistochemistry and immunofluorescence to determine targets of interest. RESULTS: We discovered multiple ATM populations, including resident vasculature-associated macrophages (ResVAMs) and distinct metabolically active macrophages (MMacs). Using trajectory analysis, we show that ResVAMs and MMacs are replenished by a common transitional macrophage (TransMac) subtype and that, during NASH, MMacs are not effectively replenished by TransMac precursors. We postulate an accessory role for MMacs and ResVAMs in protecting the adipose tissue vascular barrier, since they both interact with endothelial cells and localize around the vasculature. However, across the NAFLD severity spectrum, alterations occur in these subsets that parallel an adipose tissue vasculature breach characterized by albumin extravasation into the perivascular tissue. CONCLUSIONS: NAFLD-related macrophage dysfunction coincides with a loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. IMPACT AND IMPLICATIONS: Our study describes for the first time the myeloid cell landscape in human visceral adipose tissue at single-cell level within a cohort of well-characterized patients with non-alcoholic fatty liver disease. We report unique non-alcoholic steatohepatitis-specific transcriptional changes within metabolically active macrophages (MMacs) and resident vasculature-associated macrophages (ResVAMs) and we demonstrate their spatial location surrounding the vasculature. These dysfunctional transcriptional macrophage states coincided with the loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. Our study provides a theoretical basis for new therapeutic strategies to be directed towards reinstating the endogenous metabolic, homeostatic and cytoprotective functions of ResVAMs and MMacs, including their role in protecting vascular integrity.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/complicações , Células Endoteliais/metabolismo , Fígado/metabolismo , Macrófagos/metabolismo , Tecido Adiposo/metabolismo , Inflamação/metabolismoRESUMO
PURPOSE: Oral-facial-digital (OFD) syndromes are genetically heterogeneous developmental disorders, caused by pathogenic variants in genes involved in primary cilia formation and function. We identified a previously undescribed type of OFD with brain anomalies, ranging from alobar holoprosencephaly to pituitary anomalies, in 6 unrelated families. METHODS: Exome sequencing of affected probands was supplemented with alternative splicing analysis in patient and control lymphoblastoid and fibroblast cell lines, and primary cilia structure analysis in patient fibroblasts. RESULTS: In 1 family with 2 affected males, we identified a germline variant in the last exon of ZRSR2, NM_005089.4:c.1211_1212del NP_005080.1:p.(Gly404GlufsTer23), whereas 7 affected males from 5 unrelated families were hemizygous for the ZRSR2 variant NM_005089.4:c.1207_1208del NP_005080.1:p.(Arg403GlyfsTer24), either occurring de novo or inherited in an X-linked recessive pattern. ZRSR2, located on chromosome Xp22.2, encodes a splicing factor of the minor spliceosome complex, which recognizes minor introns, representing 0.35% of human introns. Patient samples showed significant enrichment of minor intron retention. Among differentially spliced targets are ciliopathy-related genes, such as TMEM107 and CIBAR1. Primary fibroblasts containing the NM_005089.4:c.1207_1208del ZRSR2 variant had abnormally elongated cilia, confirming an association between defective U12-type intron splicing, OFD and abnormal primary cilia formation. CONCLUSION: We introduce a novel type of OFD associated with elongated cilia and differential splicing of minor intron-containing genes due to germline variation in ZRSR2.
Assuntos
Processamento Alternativo , Síndromes Orofaciodigitais , Masculino , Humanos , Processamento Alternativo/genética , Síndromes Orofaciodigitais/genética , Splicing de RNA , Íntrons , Spliceossomos/genética , Ribonucleoproteínas/genéticaRESUMO
The skin epidermis is constantly renewed throughout life1,2. Disruption of the balance between renewal and differentiation can lead to uncontrolled growth and tumour initiation3. However, the ways in which oncogenic mutations affect the balance between renewal and differentiation and lead to clonal expansion, cell competition, tissue colonization and tumour development are unknown. Here, through multidisciplinary approaches that combine in vivo clonal analysis using intravital microscopy, single-cell analysis and functional analysis, we show how SmoM2-a constitutively active oncogenic mutant version of Smoothened (SMO) that induces the development of basal cell carcinoma-affects clonal competition and tumour initiation in real time. We found that expressing SmoM2 in the ear epidermis of mice induced clonal expansion together with tumour initiation and invasion. By contrast, expressing SmoM2 in the back-skin epidermis led to a clonal expansion that induced lateral cell competition without dermal invasion and tumour formation. Single-cell analysis showed that oncogene expression was associated with a cellular reprogramming of adult interfollicular cells into an embryonic hair follicle progenitor (EHFP) state in the ear but not in the back skin. Comparisons between the ear and the back skin revealed that the dermis has a very different composition in these two skin types, with increased stiffness and a denser collagen I network in the back skin. Decreasing the expression of collagen I in the back skin through treatment with collagenase, chronic UV exposure or natural ageing overcame the natural resistance of back-skin basal cells to undergoing EHFP reprogramming and tumour initiation after SmoM2 expression. Altogether, our study shows that the composition of the extracellular matrix regulates how susceptible different regions of the body are to tumour initiation and invasion.
Assuntos
Transformação Celular Neoplásica , Matriz Extracelular , Neoplasias Cutâneas , Microambiente Tumoral , Animais , Camundongos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Colágeno/metabolismo , Epiderme/patologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Neoplasias Cutâneas/patologia , Carcinoma Basocelular/patologia , Orelha/patologia , Colagenases/metabolismo , Envelhecimento , Raios Ultravioleta , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismoRESUMO
Epithelial-to-mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy1-7. Although great progress has been made in understanding the role of EMT and its regulatory mechanisms in cancer, no therapeutic strategy to pharmacologically target EMT has been identified. Here we found that netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCC) exhibiting spontaneous EMT. Pharmacological inhibition of netrin-1 by administration of NP137, a netrin-1-blocking monoclonal antibody currently used in clinical trials in human cancer (ClinicalTrials.gov identifier NCT02977195 ), decreased the proportion of EMT tumour cells in skin SCC, decreased the number of metastases and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA sequencing revealed the presence of different EMT states, including epithelial, early and late hybrid EMT, and full EMT states, in control SCC. By contrast, administration of NP137 prevented the progression of cancer cells towards a late EMT state and sustained tumour epithelial states. Short hairpin RNA knockdown of netrin-1 and its receptor UNC5B in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature that promotes tumour epithelial state and restricts EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with the A549 human cancer cell line-which undergoes EMT following TGFß1 administration8,9-with NP137. Netrin-1 inhibition decreased EMT in these transplanted A549 cells. Together, our results identify a pharmacological strategy for targeting EMT in cancer, opening up novel therapeutic interventions for anti-cancer therapy.
Assuntos
Anticorpos Monoclonais , Carcinoma de Células Escamosas , Transição Epitelial-Mesenquimal , Netrina-1 , Neoplasias Cutâneas , Animais , Humanos , Camundongos , Células A549 , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptores de Netrina/antagonistas & inibidores , Receptores de Netrina/deficiência , Receptores de Netrina/genética , Netrina-1/antagonistas & inibidores , Netrina-1/deficiência , Netrina-1/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Modelos Animais de Doenças , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Metástase Neoplásica/tratamento farmacológico , Análise da Expressão Gênica de Célula Única , RNA-Seq , Molécula de Adesão da Célula Epitelial/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Correct development and maturation of the enteric nervous system (ENS) is critical for survival1. At birth, the ENS is immature and requires considerable refinement to exert its functions in adulthood2. Here we demonstrate that resident macrophages of the muscularis externa (MMÏ) refine the ENS early in life by pruning synapses and phagocytosing enteric neurons. Depletion of MMÏ before weaning disrupts this process and results in abnormal intestinal transit. After weaning, MMÏ continue to interact closely with the ENS and acquire a neurosupportive phenotype. The latter is instructed by transforming growth factor-ß produced by the ENS; depletion of the ENS and disruption of transforming growth factor-ß signalling result in a decrease in neuron-associated MMÏ associated with loss of enteric neurons and altered intestinal transit. These findings introduce a new reciprocal cell-cell communication responsible for maintenance of the ENS and indicate that the ENS, similarly to the brain, is shaped and maintained by a dedicated population of resident macrophages that adapts its phenotype and transcriptome to the timely needs of the ENS niche.
Assuntos
Sistema Nervoso Entérico , Intestinos , Macrófagos , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/crescimento & desenvolvimento , Sistema Nervoso Entérico/fisiologia , Intestinos/inervação , Linfotoxina-alfa/metabolismo , Macrófagos/metabolismo , Macrófagos/fisiologia , Neurônios/fisiologia , Desmame , Comunicação Celular , Transcriptoma , Fenótipo , Fagocitose , Sinapses , Plasticidade Neuronal , Trânsito GastrointestinalRESUMO
The joint analysis of the genome, epigenome, transcriptome, proteome and/or metabolome from single cells is transforming our understanding of cell biology in health and disease. In less than a decade, the field has seen tremendous technological revolutions that enable crucial new insights into the interplay between intracellular and intercellular molecular mechanisms that govern development, physiology and pathogenesis. In this Review, we highlight advances in the fast-developing field of single-cell and spatial multi-omics technologies (also known as multimodal omics approaches), and the computational strategies needed to integrate information across these molecular layers. We demonstrate their impact on fundamental cell biology and translational research, discuss current challenges and provide an outlook to the future.
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Genoma , Multiômica , Transcriptoma , Metaboloma , Proteoma/genética , Análise de Célula ÚnicaRESUMO
Structural variation (SV) describes a broad class of genetic variation greater than 50 bp in size. SVs can cause a wide range of genetic diseases and are prevalent in rare developmental disorders (DDs). Individuals presenting with DDs are often referred for diagnostic testing with chromosomal microarrays (CMAs) to identify large copy-number variants (CNVs) and/or with single-gene, gene-panel, or exome sequencing (ES) to identify single-nucleotide variants, small insertions/deletions, and CNVs. However, individuals with pathogenic SVs undetectable by conventional analysis often remain undiagnosed. Consequently, we have developed the tool InDelible, which interrogates short-read sequencing data for split-read clusters characteristic of SV breakpoints. We applied InDelible to 13,438 probands with severe DDs recruited as part of the Deciphering Developmental Disorders (DDD) study and discovered 63 rare, damaging variants in genes previously associated with DDs missed by standard SNV, indel, or CNV discovery approaches. Clinical review of these 63 variants determined that about half (30/63) were plausibly pathogenic. InDelible was particularly effective at ascertaining variants between 21 and 500 bp in size and increased the total number of potentially pathogenic variants identified by DDD in this size range by 42.9%. Of particular interest were seven confirmed de novo variants in MECP2, which represent 35.0% of all de novo protein-truncating variants in MECP2 among DDD study participants. InDelible provides a framework for the discovery of pathogenic SVs that are most likely missed by standard analytical workflows and has the potential to improve the diagnostic yield of ES across a broad range of genetic diseases.
Assuntos
Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/genética , Sequenciamento do Exoma/métodos , Criança , Feminino , Humanos , Masculino , Proteína 2 de Ligação a Metil-CpG/genéticaRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1009679.].
RESUMO
Numerous genetic studies have established a role for rare genomic variants in Congenital Heart Disease (CHD) at the copy number variation (CNV) and de novo variant (DNV) level. To identify novel haploinsufficient CHD disease genes, we performed an integrative analysis of CNVs and DNVs identified in probands with CHD including cases with sporadic thoracic aortic aneurysm. We assembled CNV data from 7,958 cases and 14,082 controls and performed a gene-wise analysis of the burden of rare genomic deletions in cases versus controls. In addition, we performed variation rate testing for DNVs identified in 2,489 parent-offspring trios. Our analysis revealed 21 genes which were significantly affected by rare CNVs and/or DNVs in probands. Fourteen of these genes have previously been associated with CHD while the remaining genes (FEZ1, MYO16, ARID1B, NALCN, WAC, KDM5B and WHSC1) have only been associated in small cases series or show new associations with CHD. In addition, a systems level analysis revealed affected protein-protein interaction networks involved in Notch signaling pathway, heart morphogenesis, DNA repair and cilia/centrosome function. Taken together, this approach highlights the importance of re-analyzing existing datasets to strengthen disease association and identify novel disease genes and pathways.
Assuntos
Variações do Número de Cópias de DNA/genética , Haploinsuficiência/genética , Cardiopatias Congênitas/genética , Bases de Dados Genéticas , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Predisposição Genética para Doença/genética , Genômica/métodos , Humanos , Canais Iônicos/genética , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único/genética , Transcriptoma/genéticaRESUMO
Over 130 X-linked genes have been robustly associated with developmental disorders, and X-linked causes have been hypothesised to underlie the higher developmental disorder rates in males. Here, we evaluate the burden of X-linked coding variation in 11,044 developmental disorder patients, and find a similar rate of X-linked causes in males and females (6.0% and 6.9%, respectively), indicating that such variants do not account for the 1.4-fold male bias. We develop an improved strategy to detect X-linked developmental disorders and identify 23 significant genes, all of which were previously known, consistent with our inference that the vast majority of the X-linked burden is in known developmental disorder-associated genes. Importantly, we estimate that, in male probands, only 13% of inherited rare missense variants in known developmental disorder-associated genes are likely to be pathogenic. Our results demonstrate that statistical analysis of large datasets can refine our understanding of modes of inheritance for individual X-linked disorders.
Assuntos
Deficiências do Desenvolvimento/genética , Genes Ligados ao Cromossomo X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Variação Genética , Cromossomos Humanos X/genética , Feminino , Genes Recessivos , Humanos , Padrões de Herança/genética , Masculino , Herança Multifatorial/genética , Mutação/genética , Fenótipo , Caracteres SexuaisRESUMO
BACKGROUND: Congenital heart disease (CHD) occurs in almost 1% of newborn children and is considered a multifactorial disorder. CHD may segregate in families due to significant contribution of genetic factors in the disease etiology. The aim of the study was to identify pathophysiological mechanisms in families segregating CHD. METHODS: We used whole exome sequencing to identify rare genetic variants in ninety consenting participants from 32 Danish families with recurrent CHD. We applied a systems biology approach to identify developmental mechanisms influenced by accumulation of rare variants. We used an independent cohort of 714 CHD cases and 4922 controls for replication and performed functional investigations using zebrafish as in vivo model. RESULTS: We identified 1785 genes, in which rare alleles were shared between affected individuals within a family. These genes were enriched for known cardiac developmental genes, and 218 of these genes were mutated in more than one family. Our analysis revealed a functional cluster, enriched for proteins with a known participation in calcium signaling. Replication in an independent cohort confirmed increased mutation burden of calcium-signaling genes in CHD patients. Functional investigation of zebrafish orthologues of ITPR1, PLCB2, and ADCY2 verified a role in cardiac development and suggests a combinatorial effect of inactivation of these genes. CONCLUSIONS: The study identifies abnormal calcium signaling as a novel pathophysiological mechanism in human CHD and confirms the complex genetic architecture underlying CHD.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Predisposição Genética para Doença , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Biologia de Sistemas/métodos , Alelos , Animais , Biologia Computacional/métodos , Bases de Dados Genéticas , Dinamarca , Feminino , Estudos de Associação Genética/métodos , Variação Genética , Humanos , Masculino , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Sistema de Registros , Sequenciamento do Exoma , Peixe-ZebraRESUMO
Glandular epithelia, including the mammary and prostate glands, are composed of basal cells (BCs) and luminal cells (LCs)1,2. Many glandular epithelia develop from multipotent basal stem cells (BSCs) that are replaced in adult life by distinct pools of unipotent stem cells1,3-8. However, adult unipotent BSCs can reactivate multipotency under regenerative conditions and upon oncogene expression3,9-13. This suggests that an active mechanism restricts BSC multipotency under normal physiological conditions, although the nature of this mechanism is unknown. Here we show that the ablation of LCs reactivates the multipotency of BSCs from multiple epithelia both in vivo in mice and in vitro in organoids. Bulk and single-cell RNA sequencing revealed that, after LC ablation, BSCs activate a hybrid basal and luminal cell differentiation program before giving rise to LCs-reminiscent of the genetic program that regulates multipotency during embryonic development7. By predicting ligand-receptor pairs from single-cell data14, we find that TNF-which is secreted by LCs-restricts BC multipotency under normal physiological conditions. By contrast, the Notch, Wnt and EGFR pathways were activated in BSCs and their progeny after LC ablation; blocking these pathways, or stimulating the TNF pathway, inhibited regeneration-induced BC multipotency. Our study demonstrates that heterotypic communication between LCs and BCs is essential to maintain lineage fidelity in glandular epithelial stem cells.
Assuntos
Comunicação Celular , Células Epiteliais/citologia , Células-Tronco Multipotentes/citologia , Animais , Linhagem da Célula , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Feminino , Homeostase , Humanos , Masculino , Glândulas Mamárias Animais/citologia , Camundongos , Células-Tronco Multipotentes/metabolismo , Organoides/citologia , Próstata/citologia , RNA Mensageiro/genética , RNA-Seq , Receptores Notch/metabolismo , Glândulas Salivares/citologia , Análise de Célula Única , Pele/citologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Wnt/metabolismoRESUMO
The ability of the skin to grow in response to stretching has been exploited in reconstructive surgery1. Although the response of epidermal cells to stretching has been studied in vitro2,3, it remains unclear how mechanical forces affect their behaviour in vivo. Here we develop a mouse model in which the consequences of stretching on skin epidermis can be studied at single-cell resolution. Using a multidisciplinary approach that combines clonal analysis with quantitative modelling and single-cell RNA sequencing, we show that stretching induces skin expansion by creating a transient bias in the renewal activity of epidermal stem cells, while a second subpopulation of basal progenitors remains committed to differentiation. Transcriptional and chromatin profiling identifies how cell states and gene-regulatory networks are modulated by stretching. Using pharmacological inhibitors and mouse mutants, we define the step-by-step mechanisms that control stretch-mediated tissue expansion at single-cell resolution in vivo.
Assuntos
Mecanotransdução Celular/fisiologia , Análise de Célula Única , Pele/citologia , Pele/crescimento & desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Junções Aderentes/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Redes Reguladoras de Genes/efeitos dos fármacos , Hidrogéis/administração & dosagem , Hidrogéis/farmacologia , Mecanotransdução Celular/efeitos dos fármacos , Mecanotransdução Celular/genética , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação , RNA Mensageiro/genética , RNA-Seq , Pele/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Transativadores/antagonistas & inibidores , Transativadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas de Sinalização YAPRESUMO
Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.
Assuntos
Adipogenia/fisiologia , Diferenciação Celular/fisiologia , Células Intersticiais do Testículo/citologia , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/patologia , Animais , Fibrose/metabolismo , Fibrose/patologia , Humanos , Masculino , Camundongos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismoRESUMO
During embryonic and postnatal development, organs and tissues grow steadily to achieve their final size at the end of puberty. However, little is known about the cellular dynamics that mediate postnatal growth. By combining in vivo clonal lineage tracing, proliferation kinetics, single-cell transcriptomics, and in vitro micro-pattern experiments, we resolved the cellular dynamics taking place during postnatal skin epidermis expansion. Our data revealed that harmonious growth is engineered by a single population of developmental progenitors presenting a fixed fate imbalance of self-renewing divisions with an ever-decreasing proliferation rate. Single-cell RNA sequencing revealed that epidermal developmental progenitors form a more uniform population compared with adult stem and progenitor cells. Finally, we found that the spatial pattern of cell division orientation is dictated locally by the underlying collagen fiber orientation. Our results uncover a simple design principle of organ growth where progenitors and differentiated cells expand in harmony with their surrounding tissues.
Assuntos
Células Epidérmicas/metabolismo , Epiderme/crescimento & desenvolvimento , Pele/crescimento & desenvolvimento , Animais , Animais não Endogâmicos , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem da Célula/genética , Proliferação de Células/fisiologia , Células Cultivadas , Células Epidérmicas/patologia , Epiderme/metabolismo , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Células-Tronco/citologiaRESUMO
Mobile genetic Elements (MEs) are segments of DNA which can copy themselves and other transcribed sequences through the process of retrotransposition (RT). In humans several disorders have been attributed to RT, but the role of RT in severe developmental disorders (DD) has not yet been explored. Here we identify RT-derived events in 9738 exome sequenced trios with DD-affected probands. We ascertain 9 de novo MEs, 4 of which are likely causative of the patient's symptoms (0.04%), as well as 2 de novo gene retroduplications. Beyond identifying likely diagnostic RT events, we estimate genome-wide germline ME mutation rate and selective constraint and demonstrate that coding RT events have signatures of purifying selection equivalent to those of truncating mutations. Overall, our analysis represents a comprehensive interrogation of the impact of retrotransposition on protein coding genes and a framework for future evolutionary and disease studies.
Assuntos
Deficiências do Desenvolvimento/genética , Variação Genética , Retroelementos/fisiologia , Humanos , Taxa de Mutação , Retroelementos/genéticaRESUMO
In the version of this Article originally published, ref. 52 was incorrectly only attributed to its corresponding author, Fre, S., and an older title was used. The correct citation should have been: Lilja, A. M. et al. Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland. Nat. Cell Biol. https://doi.org/10.1038/s41556-018-0108-1 (2018)'. This has now been amended in all online versions of the Article.
RESUMO
The mammary gland is composed of basal cells and luminal cells. It is generally believed that the mammary gland arises from embryonic multipotent progenitors, but it remains unclear when lineage restriction occurs and what mechanisms are responsible for the switch from multipotency to unipotency during its morphogenesis. Here, we perform multicolour lineage tracing and assess the fate of single progenitors, and demonstrate the existence of a developmental switch from multipotency to unipotency during embryonic mammary gland development. Molecular profiling and single cell RNA-seq revealed that embryonic multipotent progenitors express a unique hybrid basal and luminal signature and the factors associated with the different lineages. Sustained p63 expression in embryonic multipotent progenitors promotes unipotent basal cell fate and was sufficient to reprogram adult luminal cells into basal cells by promoting an intermediate hybrid multipotent-like state. Altogether, this study identifies the timing and the mechanisms mediating early lineage segregation of multipotent progenitors during mammary gland development.
Assuntos
Linhagem da Célula , Células Epiteliais/fisiologia , Glândulas Mamárias Animais/fisiologia , Células-Tronco Embrionárias Murinas/fisiologia , Células-Tronco Multipotentes/fisiologia , Animais , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Glândulas Mamárias Animais/embriologia , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Morfogênese , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Multipotentes/metabolismo , Fenótipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Análise de Sequência de RNA/métodos , Transdução de Sinais , Análise de Célula Única/métodos , Fatores de Tempo , Transativadores/genética , Transativadores/metabolismo , TranscriptomaRESUMO
In cancer, the epithelial-to-mesenchymal transition (EMT) is associated with tumour stemness, metastasis and resistance to therapy. It has recently been proposed that, rather than being a binary process, EMT occurs through distinct intermediate states. However, there is no direct in vivo evidence for this idea. Here we screen a large panel of cell surface markers in skin and mammary primary tumours, and identify the existence of multiple tumour subpopulations associated with different EMT stages: from epithelial to completely mesenchymal states, passing through intermediate hybrid states. Although all EMT subpopulations presented similar tumour-propagating cell capacity, they displayed differences in cellular plasticity, invasiveness and metastatic potential. Their transcriptional and epigenetic landscapes identify the underlying gene regulatory networks, transcription factors and signalling pathways that control these different EMT transition states. Finally, these tumour subpopulations are localized in different niches that differentially regulate EMT transition states.
Assuntos
Transição Epitelial-Mesenquimal , Neoplasias/patologia , Animais , Cromatina/genética , Epigênese Genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Masculino , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Neoplasias/genética , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Transcrição GênicaRESUMO
We previously estimated that 42% of patients with severe developmental disorders carry pathogenic de novo mutations in coding sequences. The role of de novo mutations in regulatory elements affecting genes associated with developmental disorders, or other genes, has been essentially unexplored. We identified de novo mutations in three classes of putative regulatory elements in almost 8,000 patients with developmental disorders. Here we show that de novo mutations in highly evolutionarily conserved fetal brain-active elements are significantly and specifically enriched in neurodevelopmental disorders. We identified a significant twofold enrichment of recurrently mutated elements. We estimate that, genome-wide, 1-3% of patients without a diagnostic coding variant carry pathogenic de novo mutations in fetal brain-active regulatory elements and that only 0.15% of all possible mutations within highly conserved fetal brain-active elements cause neurodevelopmental disorders with a dominant mechanism. Our findings represent a robust estimate of the contribution of de novo mutations in regulatory elements to this genetically heterogeneous set of disorders, and emphasize the importance of combining functional and evolutionary evidence to identify regulatory causes of genetic disorders.