RESUMO
During development, lymph node (LN) initiation is coordinated by lymphoid tissue organizer (LTo) cells that attract lymphoid tissue inducer (LTi) cells at strategic positions within the embryo. The identity and function of LTo cells during the initial attraction of LTi cells remain poorly understood. Using lineage tracing, we demonstrated that a subset of Osr1-expressing cells was mesenchymal LTo progenitors. By investigating the heterogeneity of Osr1+ cells, we uncovered distinct mesenchymal LTo signatures at diverse anatomical locations, identifying a common progenitor of mesenchymal LTos and LN-associated adipose tissue. Osr1 was essential for LN initiation, driving the commitment of mesenchymal LTo cells independent of neural retinoic acid, and for LN-associated lymphatic vasculature assembly. The combined action of chemokines CXCL13 and CCL21 was required for LN initiation. Our results redefine the role and identity of mesenchymal organizer cells and unify current views by proposing a model of cooperative cell function in LN initiation.
Assuntos
Organogênese , Fatores de Transcrição , Diferenciação Celular , Linfonodos , Tecido LinfoideRESUMO
Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments.
Assuntos
Músculos Oculomotores/embriologia , Músculos Oculomotores/crescimento & desenvolvimento , Tretinoína/metabolismo , Animais , Tecido Conjuntivo/fisiologia , Desenvolvimento Embrionário , Olho , Imageamento Tridimensional/métodos , Camundongos/embriologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Morfogênese , Transdução de Sinais , Tendões/fisiologia , Tretinoína/fisiologiaRESUMO
PURPOSE: We aimed to identify the underlying genetic cause for a novel form of distal arthrogryposis. METHODS: Rare variant family-based genomics, exome sequencing, and disease-specific panel sequencing were used to detect ADAMTS15 variants in affected individuals. Adamts15 expression was analyzed at the single-cell level during murine embryogenesis. Expression patterns were characterized using in situ hybridization and RNAscope. RESULTS: We identified homozygous rare variant alleles of ADAMTS15 in 5 affected individuals from 4 unrelated consanguineous families presenting with congenital flexion contractures of the interphalangeal joints and hypoplastic or absent palmar creases. Radiographic investigations showed physiological interphalangeal joint morphology. Additional features included knee, Achilles tendon, and toe contractures, spinal stiffness, scoliosis, and orthodontic abnormalities. Analysis of mouse whole-embryo single-cell sequencing data revealed a tightly regulated Adamts15 expression in the limb mesenchyme between embryonic stages E11.5 and E15.0. A perimuscular and peritendinous expression was evident in in situ hybridization in the developing mouse limb. In accordance, RNAscope analysis detected a significant coexpression with Osr1, but not with markers for skeletal muscle or joint formation. CONCLUSION: In aggregate, our findings provide evidence that rare biallelic recessive trait variants in ADAMTS15 cause a novel autosomal recessive connective tissue disorder, resulting in a distal arthrogryposis syndrome.
Assuntos
Artrogripose , Contratura , Proteínas ADAMTS , Animais , Artrogripose/genética , Consanguinidade , Contratura/genética , Homozigoto , Humanos , Camundongos , Mutação , Linhagem , FenótipoRESUMO
The morphology of bones is genetically determined, but the molecular mechanisms that control shape, size and the overall gestalt of bones remain unclear. We previously showed that metacarpals in the synpolydactyly homolog (spdh) mouse, which carries a mutation in Hoxd13 similar to the human condition synpolydactyly (SPD), were transformed to carpal-like bones with cuboid shape that lack cortical bone and a perichondrium and are surrounded by a joint surface. Here we provide evidence that spdh metacarpal growth plates have a defect in cell polarization with a random instead of linear orientation. In parallel prospective perichondral cells failed to adopt the characteristic flattened cell shape. We observed a similar cell polarity defect in metacarpals of Wnt5a(-/-) mice. Wnt5a and the closely related Wnt5b were downregulated in spdh handplates, and HOXD13 induced expression of both genes in vitro. Concomitant we observed mislocalization of core planar cell polarity (PCP) components DVL2 and PRICKLE1 in spdh metacarpals indicating a defect in the WNT/PCP pathway. Conversely the WNT/ß-CATENIN pathway, a hallmark of joint cells lining carpal bones, was upregulated in the perichondral region. Finally, providing spdh limb explant cultures with cells expressing either HOXD13 or WNT5A led to a non-cell autonomous partial rescue of cell polarity the perichondral region and restored the expression of perichondral markers. This study provides a so far unrecognized link between HOX proteins and cell polarity in the perichondrium and the growth plate, a failure of which leads to transformation of metacarpals to carpal-like structures.
Assuntos
Cartilagem/embriologia , Lâmina de Crescimento/embriologia , Proteínas de Homeodomínio/metabolismo , Ossos Metacarpais/embriologia , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cartilagem/metabolismo , Polaridade Celular , Células Cultivadas , Proteínas Desgrenhadas , Lâmina de Crescimento/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Proteínas com Domínio LIM/metabolismo , Ossos Metacarpais/metabolismo , Camundongos , Camundongos Knockout , Morfogênese/genética , Fosfoproteínas/metabolismo , Receptores da Fenciclidina/metabolismo , Sindactilia/genética , Fatores de Transcrição/genética , Proteínas Wnt/genética , Proteína Wnt-5a , beta Catenina/metabolismoRESUMO
Skeletal muscle regeneration requires the coordinated interplay of diverse tissue-resident- and infiltrating cells. Fibro-adipogenic progenitors (FAPs) are an interstitial cell population that provides a beneficial microenvironment for muscle stem cells (MuSCs) during muscle regeneration. Here we show that the transcription factor Osr1 is essential for FAPs to communicate with MuSCs and infiltrating macrophages, thus coordinating muscle regeneration. Conditional inactivation of Osr1 impaired muscle regeneration with reduced myofiber growth and formation of excessive fibrotic tissue with reduced stiffness. Osr1-deficient FAPs acquired a fibrogenic identity with altered matrix secretion and cytokine expression resulting in impaired MuSC viability, expansion and differentiation. Immune cell profiling suggested a novel role for Osr1-FAPs in macrophage polarization. In vitro analysis suggested that increased TGFß signaling and altered matrix deposition by Osr1-deficient FAPs actively suppressed regenerative myogenesis. In conclusion, we show that Osr1 is central to FAP function orchestrating key regenerative events such as inflammation, matrix secretion and myogenesis.
RESUMO
Heterotopic ossification is a disorder caused by abnormal mineralization of soft tissues in which signaling pathways such as BMP, TGFß and WNT are known key players in driving ectopic bone formation. Identifying novel genes and pathways related to the mineralization process are important steps for future gene therapy in bone disorders. In this study, we detect an inter-chromosomal insertional duplication in a female proband disrupting a topologically associating domain and causing an ultra-rare progressive form of heterotopic ossification. This structural variant lead to enhancer hijacking and misexpression of ARHGAP36 in fibroblasts, validated here by orthogonal in vitro studies. In addition, ARHGAP36 overexpression inhibits TGFß, and activates hedgehog signaling and genes/proteins related to extracellular matrix production. Our work on the genetic cause of this heterotopic ossification case has revealed that ARHGAP36 plays a role in bone formation and metabolism, outlining first details of this gene contributing to bone-formation and -disease.
Assuntos
Proteínas Hedgehog , Ossificação Heterotópica , Feminino , Humanos , Tecido Conjuntivo/metabolismo , Proteínas Hedgehog/metabolismo , Ossificação Heterotópica/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador betaRESUMO
Fibro-adipogenic progenitors (FAPs) are resident mesenchymal progenitors in adult skeletal muscle that support muscle repair, but also give rise to fibrous and adipose infiltration in response to disease and chronic injury. FAPs are identified using cell surface markers that do not distinguish between quiescent FAPs and FAPs actively engaged in the regenerative process. We have shown previously that FAPs are derived from cells that express the transcription factor Osr1 during development. Here we show that adult FAPs express Osr1 at low levels and frequency, however upon acute injury FAPs reactivate Osr1 expression in the injured tissue. Osr1+ FAPs are enriched in proliferating and apoptotic cells demonstrating that Osr1 identifies activated FAPs. In vivo genetic lineage tracing shows that Osr1+ activated FAPs return to the resident FAP pool after regeneration as well as contribute to adipocytes after glycerol-induced fatty degeneration. In conclusion, reporter LacZ or eGFP-CreERt2 expression from the endogenous Osr1 locus serves as marker for FACS isolation and tamoxifen-induced manipulation of activated FAPs.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Músculo Esquelético/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Proteínas de Ligação ao Cálcio , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Cistos , Citometria de Fluxo , Edição de Genes , Regulação da Expressão Gênica , Glucosidases/genética , Glucosidases/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Hepatopatias , Músculo Esquelético/citologia , Fatores de TranscriçãoRESUMO
Fibro-adipogenic progenitors (FAPs) are an interstitial cell population in adult skeletal muscle that support muscle regeneration. During development, interstitial muscle connective tissue (MCT) cells support proper muscle patterning, however the underlying molecular mechanisms are not well understood and it remains unclear whether adult FAPs and embryonic MCT cells share a common lineage. We show here that mouse embryonic limb MCT cells expressing the transcription factor Osr1, differentiate into fibrogenic and adipogenic cells in vivo and in vitro defining an embryonic FAP-like population. Genetic lineage tracing shows that developmental Osr1+ cells give rise to a subset of adult FAPs. Loss of Osr1 function leads to a reduction of myogenic progenitor proliferation and survival resulting in limb muscle patterning defects. Transcriptome and functional analyses reveal that Osr1+ cells provide a critical pro-myogenic niche via the production of MCT specific extracellular matrix components and secreted signaling factors.