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1.
Nat Genet ; 53(4): 467-476, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33731941

RESUMO

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.


Assuntos
Quimera/genética , Síndrome de Ellis-Van Creveld/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Crista Neural/metabolismo , Pan troglodytes/genética , Crânio/metabolismo , Animais , Evolução Biológica , Diferenciação Celular , Quimera/metabolismo , Síndrome de Ellis-Van Creveld/metabolismo , Síndrome de Ellis-Van Creveld/patologia , Feminino , Expressão Gênica , Genótipo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Masculino , Camundongos , Camundongos Knockout , Crista Neural/patologia , Pan troglodytes/anatomia & histologia , Pan troglodytes/metabolismo , Fenótipo , Transdução de Sinais , Crânio/anatomia & histologia , Especificidade da Espécie , Tetraploidia
2.
PLoS One ; 16(1): e0244661, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33513144

RESUMO

Amphimoschus is an extinct Eurasian ruminant genus, mostly recorded in Europe, without a close living relative and, hence, an unknown systematic position. This genus is known from around 50 localities from the late early to the middle Miocene. Two species were described during 180 years, but since their first description during the late 19th century and early 20th century, hardly any detailed taxonomic work has been done on the genus. Over the years, extensive collecting and excavating activities have enriched collections with more and more complete material of this still rare and enigmatic animal. Most interestingly, a number of skull remains have been unearthed and are promising in terms of providing phylogenetic information. In the present paper, we describe cranial material, the bony labyrinth, the dentition through 780 teeth and five skulls from different ontogenetic stages. We cannot find a clear morphometric distinction between the supposedly smaller and older species Amphimoschus artenensis and the supposedly younger and larger species A. ponteleviensis. Accordingly, we have no reason to retain the two species and propose, following the principle of priority (ICZN chapter 6 article 23), that only A. ponteleviensis Bourgeois, 1873 is valid. Our studies on the ontogenetic variation of Amphimoschus does reveal that the sagittal crest may increase in size and a supraorbital ridge may appear with age. Despite the abundant material, the family affiliation is still uncertain.


Assuntos
Artiodáctilos/anatomia & histologia , Fósseis/anatomia & histologia , Ruminantes/anatomia & histologia , Animais , Artiodáctilos/genética , Evolução Biológica , Dentição , Orelha Interna/anatomia & histologia , Orelha Interna/metabolismo , Europa (Continente) , Filogenia , Ruminantes/genética , Crânio/anatomia & histologia , Crânio/metabolismo
3.
Int J Mol Sci ; 22(3)2021 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-33498907

RESUMO

Tissue nonspecific alkaline phosphatase (TNAP/Alpl) is associated with cell stemness; however, the function of TNAP in mesenchymal progenitor cells remains largely unknown. In this study, we aimed to establish an essential role for TNAP in bone and muscle progenitor cells. We investigated the impact of TNAP deficiency on bone formation, mineralization, and differentiation of bone marrow stromal cells. We also pursued studies of proliferation, mitochondrial function and ATP levels in TNAP deficient bone and muscle progenitor cells. We find that TNAP deficiency decreases trabecular bone volume fraction and trabeculation in addition to decreased mineralization. We also find that Alpl-/- mice (global TNAP knockout mice) exhibit muscle and motor coordination deficiencies similar to those found in individuals with hypophosphatasia (TNAP deficiency). Subsequent studies demonstrate diminished proliferation, with mitochondrial hyperfunction and increased ATP levels in TNAP deficient bone and muscle progenitor cells, plus intracellular expression of TNAP in TNAP+ cranial osteoprogenitors, bone marrow stromal cells, and skeletal muscle progenitor cells. Together, our results indicate that TNAP functions inside bone and muscle progenitor cells to influence mitochondrial respiration and ATP production. Future studies are required to establish mechanisms by which TNAP influences mitochondrial function and determine if modulation of TNAP can alter mitochondrial respiration in vivo.


Assuntos
Trifosfato de Adenosina/biossíntese , Fosfatase Alcalina/metabolismo , Osso e Ossos/metabolismo , Respiração Celular , Células-Tronco Mesenquimais/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Fosfatase Alcalina/genética , Fosfatase Alcalina/fisiologia , Animais , Osso e Ossos/fisiologia , Calcificação Fisiológica , Diferenciação Celular , Masculino , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Knockout , Músculo Esquelético/fisiologia , Osteogênese , Crânio/metabolismo , Crânio/fisiologia
4.
Development ; 148(2)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33318149

RESUMO

Mutations in the RNA helicase DDX3 have emerged as a frequent cause of intellectual disability in humans. Because many individuals carrying DDX3 mutations have additional defects in craniofacial structures and other tissues containing neural crest (NC)-derived cells, we hypothesized that DDX3 is also important for NC development. Using Xenopus tropicalis as a model, we show that DDX3 is required for normal NC induction and craniofacial morphogenesis by regulating AKT kinase activity. Depletion of DDX3 decreases AKT activity and AKT-dependent inhibitory phosphorylation of GSK3ß, leading to reduced levels of ß-catenin and Snai1: two GSK3ß substrates that are crucial for NC induction. DDX3 function in regulating these downstream signaling events during NC induction is likely mediated by RAC1, a small GTPase whose translation depends on the RNA helicase activity of DDX3. These results suggest an evolutionarily conserved role of DDX3 in NC development by promoting AKT activity, and provide a potential mechanism for the NC-related birth defects displayed by individuals harboring mutations in DDX3 and its downstream effectors in this signaling cascade.


Assuntos
RNA Helicases DEAD-box/metabolismo , Crista Neural/embriologia , Crista Neural/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/embriologia , Xenopus/metabolismo , Animais , Cartilagem/embriologia , Cartilagem/metabolismo , Embrião não Mamífero/metabolismo , Face/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Glicogênio Sintase Quinase 3 beta/metabolismo , Morfogênese/genética , Fosforilação , Estabilidade Proteica , Crânio/embriologia , Crânio/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Via de Sinalização Wnt , Xenopus/genética , beta Catenina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(32): 19321-19327, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719137

RESUMO

Phenotypic plasticity, the ability of a single genotype to produce multiple phenotypes under different environmental conditions, is critical for the origins and maintenance of biodiversity; however, the genetic mechanisms underlying plasticity as well as how variation in those mechanisms can drive evolutionary change remain poorly understood. Here, we examine the cichlid feeding apparatus, an icon of both prodigious evolutionary divergence and adaptive phenotypic plasticity. We first provide a tissue-level mechanism for plasticity in craniofacial shape by measuring rates of bone deposition within functionally salient elements of the feeding apparatus in fishes forced to employ alternate foraging modes. We show that levels and patterns of phenotypic plasticity are distinct among closely related cichlid species, underscoring the evolutionary potential of this trait. Next, we demonstrate that hedgehog (Hh) signaling, which has been implicated in the evolutionary divergence of cichlid feeding architecture, is associated with environmentally induced rates of bone deposition. Finally, to demonstrate that Hh levels are the cause of the plastic response and not simply the consequence of producing more bone, we use transgenic zebrafish in which Hh levels could be experimentally manipulated under different foraging conditions. Notably, we find that the ability to modulate bone deposition rates in different environments is dampened when Hh levels are reduced, whereas the sensitivity of bone deposition to different mechanical demands increases with elevated Hh levels. These data advance a mechanistic understanding of phenotypic plasticity in the teleost feeding apparatus and in doing so contribute key insights into the origins of adaptive morphological radiations.


Assuntos
Ciclídeos/metabolismo , Proteínas de Peixes/metabolismo , Proteínas Hedgehog/metabolismo , Crânio/crescimento & desenvolvimento , Adaptação Fisiológica , Animais , Ciclídeos/genética , Ciclídeos/crescimento & desenvolvimento , Proteínas de Peixes/genética , Proteínas Hedgehog/genética , Transdução de Sinais , Crânio/metabolismo
6.
Sci Rep ; 10(1): 2673, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060387

RESUMO

Proximate control of the development of sexual dimorphism is still hotly debated in reptiles. In some squamates, many male-typical exaggerated traits including body size were assumed to be controlled by masculinization by male gonadal androgens. We performed a manipulative experiment to test the importance of this mechanism in the development of pronounced sexual differences in body size and size of head casque in the chameleon Chamaeleo calyptratus. Castrated males attained male-typical body size highly deviating from the body size of control females. Ontogenetic allometries of casque size on head length revealed that sexes depart considerably in casque growth later in the ontogeny; however, castrated males still follow male-typical casque growth. Paradoxically, exogenous testosterone led in females to slight increase of casque size, which might reflect interference with the feminizing effects of female gonadal hormones. The results in males strongly suggest that masculinization by male gonadal androgens during growth is not required for the development of sexual dimorphism in body size and casque size in the chameleon. The ontogeny of sexually dimorphic body size and exaggerated traits in at least some squamates is likely controlled by other proximate mechanism, possibly by feminization by ovarian hormones.


Assuntos
Androgênios/metabolismo , Lagartos/crescimento & desenvolvimento , Caracteres Sexuais , Testosterona/metabolismo , Androgênios/genética , Animais , Tamanho Corporal/genética , Castração , Feminino , Gônadas/metabolismo , Lagartos/anatomia & histologia , Lagartos/genética , Lagartos/metabolismo , Masculino , Crânio/anatomia & histologia , Crânio/metabolismo , Testosterona/genética
7.
Nat Rev Endocrinol ; 16(3): 147-164, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31974498

RESUMO

The development of the craniofacial skeleton relies on complex temporospatial organization of diverse cell types by key signalling molecules. Even minor disruptions to these processes can result in deleterious consequences for the structure and function of the skull. Thyroid hormone deficiency causes delayed craniofacial and tooth development, dysplastic facial features and delayed development of the ossicles in the middle ear. Thyroid hormone excess, by contrast, accelerates development of the skull and, in severe cases, might lead to craniosynostosis with neurological sequelae and facial hypoplasia. The pathogenesis of these important abnormalities remains poorly understood and underinvestigated. The orchestration of craniofacial development and regulation of suture and synchondrosis growth is dependent on several critical signalling pathways. The underlying mechanisms by which these key pathways regulate craniofacial growth and maturation are largely unclear, but studies of single-gene disorders resulting in craniofacial malformations have identified a number of critical signalling molecules and receptors. The craniofacial consequences resulting from gain-of-function and loss-of-function mutations affecting insulin-like growth factor 1, fibroblast growth factor receptor and WNT signalling are similar to the effects of altered thyroid status and mutations affecting thyroid hormone action, suggesting that these critical pathways interact in the regulation of craniofacial development.


Assuntos
Anormalidades Craniofaciais/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Craniossinostoses/metabolismo , Humanos , Transdução de Sinais/fisiologia , Crânio/metabolismo
8.
Cell Mol Life Sci ; 77(10): 1933-1945, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31722070

RESUMO

Vertebrate cranial mesoderm is a discrete developmental unit compared to the mesoderm below the developing neck. An extraordinary feature of the cranial mesoderm is that it includes a common progenitor pool contributing to the chambered heart and the craniofacial skeletal muscles. This striking developmental potential and the excitement it generated led to advances in our understanding of cranial mesoderm developmental mechanism. Remarkably, recent findings have begun to unravel the origin of its distinct developmental characteristics. Here, we take a detailed view of the ontogenetic trajectory of cranial mesoderm and its regulatory network. Based on the emerging evidence, we propose that cranial and posterior mesoderm diverge at the earliest step of the process that patterns the mesoderm germ layer along the anterior-posterior body axis. Further, we discuss the latest evidence and their impact on our current understanding of the evolutionary origin of cranial mesoderm. Overall, the review highlights the findings from contemporary research, which lays the foundation to probe the molecular basis of unique developmental potential and evolutionary origin of cranial mesoderm.


Assuntos
Evolução Biológica , Mesoderma/crescimento & desenvolvimento , Desenvolvimento Muscular/genética , Crânio/crescimento & desenvolvimento , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Mesoderma/embriologia , Músculo Esquelético/embriologia , Músculo Esquelético/crescimento & desenvolvimento , Crista Neural/crescimento & desenvolvimento , Crista Neural/metabolismo , Crânio/metabolismo , Vertebrados/embriologia , Vertebrados/genética
9.
Dev Biol ; 458(2): 189-199, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31733190

RESUMO

The N-ethyl-N-nitrosourea (ENU) ←forward genetic screen is a useful tool for the unbiased discovery of novel mechanisms regulating developmental processes. We recovered the dorothy mutation in such a screen designed to recover recessive mutations affecting craniofacial development in the mouse. Dorothy embryos die prenatally and exhibit many striking phenotypes commonly associated with ciliopathies, including a severe midfacial clefting phenotype. We used exome sequencing to discover a missense mutation in nucleotide binding protein 2 (Nubp2) to be causative. This finding was confirmed by a complementation assay with the dorothy allele and an independent Nubp2 null allele (Nubp2null). We demonstrated that Nubp2 is indispensable for embryogenesis. NUBP2 is implicated in both the cytosolic iron/sulfur cluster assembly pathway and negative regulation of ciliogenesis. Conditional ablation of Nubp2 in the neural crest lineage with Wnt1-cre recapitulates the dorothy craniofacial phenotype. Using this model, we found that the proportion of ciliated cells in the craniofacial mesenchyme was unchanged, and that markers of the SHH, FGF, and BMP signaling pathways are unaltered. Finally, we show evidence that the phenotype results from a marked increase in apoptosis within the craniofacial mesenchyme.


Assuntos
Proteínas de Ligação ao GTP/genética , Crista Neural/embriologia , Animais , Desenvolvimento Embrionário/genética , Etilnitrosoureia , Feminino , Proteínas de Ligação ao GTP/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese/genética , Mutação/genética , Crista Neural/metabolismo , Fenótipo , Transdução de Sinais/fisiologia , Crânio/metabolismo , Proteína Wnt1/metabolismo
10.
Sci Rep ; 9(1): 18635, 2019 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-31819067

RESUMO

Disseminated tumor cells (DTCs) undergo a dormant state in the distant metastatic site(s) before becoming overt metastatic diseases. In prostate cancer (PCa), bone metastasis can occur years after prostatectomy, suggesting that bone may provide dormancy-inducing factors. To search for these factors, we prepared conditioned media (CM) from calvariae. Using live-cell imaging, we found that Calvarial-CM treatment increased cellular quiescence in C4-2B4 PCa cells. Mass spectrometry analysis of Calvarial-CM identified 132 secreted factors. Western blot and ELISA analyses confirmed the presence of several factors, including DKK3, BMP1, neogenin and vasorin in the Calvarial-CM. qRT-PCR analysis of total calvariae versus isolated osteoblasts showed that DKK3, BMP1, vasorin and neogenin are mainly expressed by osteoblasts, while MIA, LECT1, NGAL and PEDF are expressed by other calvarial cells. Recombinant human DKK3, BMP1, vasorin, neogenin, MIA and NGAL treatment increased cellular quiescence in both C4-2b and C4-2B4 PCa cells. Mechanistically, DKK3, vasorin and neogenin, but not BMP1, increased dormancy through activating the p38MAPK signaling pathway. Consistently, DKK3, vasorin and neogenin failed to induce dormancy in cells expressing dominant-negative p38αMAPK while BMP1 remained active, suggesting that BMP1 uses an alternative dormancy signaling pathway. Thus, bone secretes multiple dormancy-inducing factors that employ distinct signaling pathways to induce DTC dormancy in bone.


Assuntos
Proteína Morfogenética Óssea 1/genética , Neoplasias Ósseas/genética , Meios de Cultivo Condicionados/farmacologia , Neoplasias da Próstata/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Proteínas de Membrana/genética , Metástase Neoplásica , Osteoblastos/metabolismo , Osteoblastos/patologia , Próstata/metabolismo , Próstata/patologia , Prostatectomia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacos , Crânio/metabolismo , Crânio/patologia
11.
Cell Rep ; 29(13): 4362-4376.e6, 2019 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875546

RESUMO

Intramuscular motor innervation is an essential process in neuromuscular development. Recently, mutations in COL25A1, encoding CLAC-P/collagen XXV, have been linked to the development of a congenital cranial dysinnervation disorder (CCDD). Yet the molecular mechanisms of intramuscular innervation and the etiology of CCDD related to COL25A1 have remained elusive. Here, we report that muscle-derived collagen XXV is indispensable for intramuscular innervation. In developing skeletal muscles, Col25a1 expression is tightly regulated by muscle excitation. In vitro and cell-based assays reveal a direct interaction between collagen XXV and receptor protein tyrosine phosphatases (PTPs) σ and δ. Motor explant assays show that expression of collagen XXV in target cells attracts motor axons, but this is inhibited by exogenous PTPσ/δ. CCDD mutations attenuate motor axon attraction by reducing collagen XXV-PTPσ/δ interaction. Overall, our study identifies PTPσ/δ as putative receptors for collagen XXV, implicating collagen XXV and PTPσ/δ in intramuscular innervation and a developmental ocular motor disorder.


Assuntos
Músculo Esquelético/metabolismo , Colágenos não Fibrilares/genética , Transtornos da Motilidade Ocular/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Crânio/metabolismo , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Linhagem Celular , Diafragma/anormalidades , Diafragma/inervação , Diafragma/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Neurônios Motores/metabolismo , Neurônios Motores/ultraestrutura , Músculo Esquelético/anormalidades , Músculo Esquelético/inervação , Mioblastos/metabolismo , Mioblastos/patologia , Colágenos não Fibrilares/metabolismo , Transtornos da Motilidade Ocular/congênito , Transtornos da Motilidade Ocular/metabolismo , Transtornos da Motilidade Ocular/patologia , Ligação Proteica , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Neuropatia Ciática/cirurgia , Transdução de Sinais , Crânio/anormalidades , Crânio/inervação
12.
Stem Cell Res Ther ; 10(1): 335, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31747933

RESUMO

BACKGROUND: Mesenchymal stem cell (MSC)-derived exosomes have been recognized as new candidate agents for treating critical-sized bone defects; they promote angiogenesis and may be an alternative to cell therapy. In this study, we evaluated whether exosomes derived from bone marrow-derived MSCs (BMSCs) preconditioned with a low dose of dimethyloxaloylglycine (DMOG), DMOG-MSC-Exos, exert superior proangiogenic activity in bone regeneration and the underlying mechanisms involved. METHODS: To investigate the effects of these exosomes, scratch wound healing, cell proliferation, and tube formation assays were performed in human umbilical vein endothelial cells (HUVECs). To test the effects in vivo, a critical-sized calvarial defect rat model was established. Eight weeks after the procedure, histological/histomorphometrical analysis was performed to measure bone regeneration, and micro-computerized tomography was used to measure bone regeneration and neovascularization. RESULTS: DMOG-MSC-Exos activated the AKT/mTOR pathway to stimulate angiogenesis in HUVECs. This contributed to bone regeneration and angiogenesis in the critical-sized calvarial defect rat model in vivo. CONCLUSIONS: Low doses of DMOG trigger exosomes to exert enhanced proangiogenic activity in cell-free therapeutic applications.


Assuntos
Aminoácidos Dicarboxílicos/farmacologia , Células da Medula Óssea/metabolismo , Regeneração Óssea , Exossomos , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica , Transdução de Sinais , Crânio , Animais , Células da Medula Óssea/patologia , Exossomos/metabolismo , Exossomos/patologia , Exossomos/transplante , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Crânio/lesões , Crânio/metabolismo , Crânio/patologia , Serina-Treonina Quinases TOR/metabolismo
13.
Sci Rep ; 9(1): 16196, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31700010

RESUMO

Brain trauma was clinically associated with increased osteogenesis in the appendicular skeleton. We showed previously in C57BL/6J mice that mild traumatic brain injury (mTBI) transiently induced bone formation in the femur via the cannabinoid-1 (CB1) receptor. Here, we subjected ICR mice to mTBI and examined the bone response in the skull using microCT. We also measured mast cell degranulation (MCD)72 h post-injury. Finally, we measured brain and calvarial endocannabinoids levels post-mTBI. mTBI led to decreased bone porosity on the contralateral (untouched) side. This effect was apparent both in young and mature mice. Administration of rimonabant (CB1 inverse agonist) completely abrogated the effect of mTBI on calvarial porosity and significantly reduced MCD, compared with vehicle-treated controls. We also found that mTBI resulted in elevated levels of anandamide, but not 2-arachidonoylglycerol, in the contralateral calvarial bone, whereas brain levels remained unchanged. In C57BL/6J CB1 knockout mice, mTBI did not reduce porosity but in general the porosity was significantly lower than in WT controls. Our findings suggest that mTBI induces a strain-specific CB1-dependent bone anabolic response in the skull, probably mediated by anandamide, but seemingly unrelated to inflammation. The endocannabinoid system is therefore a plausible target in management of bone response following head trauma.


Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Crânio/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Lesões Encefálicas Traumáticas/patologia , Endocanabinoides/metabolismo , Glicerídeos/metabolismo , Masculino , Mastócitos , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Alcamidas Poli-Insaturadas/metabolismo , Rimonabanto/farmacologia , Crânio/patologia
14.
PLoS One ; 14(11): e0225472, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31751396

RESUMO

Growth factors in serum-free conditioned media from human bone marrow-derived mesenchymal stem cells (MSC-CM) are known to be effective in bone regeneration. However, the secretomes in MSC-CM that act as active ingredients for bone regeneration, as well as their mechanisms, remains unclear. Exosomes, components of MSC-CM, provide the recipient cells with genetic information and enhance the recipient cellular paracrine stimulation, which contributes to tissue regeneration. We hypothesized that MSC-CM-derived exosomes (MSC-Exo) promoted bone regeneration, and that angiogenesis was a key step. Here, we prepared an MSC-Exo group, MSC-CM group, and Exo-antiVEGF group (MSC-Exo with angiogenesis inhibitor), and examined the osteogenic and angiogenic potential in MSCs. Furthermore, we used a rat model of calvaria bone defect and implanted each sample to evaluate bone formation weekly, until week 4 after treatment. Results showed that MSC-Exo enhanced cellular migration and osteogenic and angiogenic gene expression in MSCs compared to that in other groups. In vivo, early bone formation by MSC-Exo was also confirmed. Two weeks after implantation, the newly formed bone area was 31.5 ± 6.5% in the MSC-Exo group while those in the control and Exo-antiVEGF groups were 15.4 ± 4.4% and 8.7 ± 1.1%, respectively. Four weeks after implantation, differences in the area between the MSC-Exo group and the Exo-antiVEGF or control groups were further broadened. Histologically, notable accumulation of osteoblast-like cells and vascular endothelial cells was observed in the MSC-Exo group; however, fewer cells were found in the Exo-antiVEGF and control groups. In conclusion, MSC-Exo promoted bone regeneration during early stages, as well as enhanced angiogenesis. Considering the tissue regeneration with transplanted cells and their secretomes, this study suggests that exosomes might play an important role, especially in angiogenesis.


Assuntos
Regeneração Óssea , Meios de Cultivo Condicionados/metabolismo , Exossomos/metabolismo , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Crânio/lesões , Inibidores da Angiogênese/farmacologia , Animais , Regeneração Óssea/efeitos dos fármacos , Técnicas de Cultura de Células , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Humanos , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Comunicação Parácrina , Ratos , Crânio/metabolismo
15.
PLoS One ; 14(10): e0223052, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31613887

RESUMO

To further investigate the role of the phosphate (Pi) transporter PIT1 in Pi homeostasis and tissue mineralization, we developed a transgenic mouse expressing the C-terminal influenza hemagglutinin (HA) epitope-tagged human PIT1 transporter under control of the cytomegalovirus/chicken beta actin/rabbit beta-globin gene (CAG) promotor and a loxP-stop-loxP (LSL) cassette permitting conditional activation of transgene expression (LSL-HA-hPITtg/+). For an initial characterization of this conditional mouse model, germline excision of the LSL cassette was performed to induce expression of the transgene in all mouse tissues (HA-hPIT1tg/+). Recombination was confirmed using genomic DNA obtained from blood samples of these mice. Furthermore, expression of HA-hPIT1 was found to be at least 10-fold above endogenous mouse Pit1 in total RNA isolated from multiple tissues and from cultured primary calvaria osteoblasts (PCOB) estimated by semi-quantitative RT-PCR. Robust expression of the HA-hPIT1 protein was also observed upon immunoblot analysis in most tissues and permits HA-mediated immunoprecipitation of the transporter. Characterization of the phenotype of HA-hPIT1tg/+ mice at 80 days of age when fed a standard chow (0.7% Pi and 1% calcium) showed elevated plasma Pi, but normal plasma iPTH, iFGF23, serum calcium, BUN, 1,25-dihydroxy vitamin D levels and urine Pi, calcium and protein excretion when compared to WT littermates. Likewise, no change in bone mineral density was observed upon uCT analysis of the distal femur obtained from these mice. In conclusion, heterozygous overexpression of HA-hPIT1 is compatible with life and causes hyperphosphatemia while bone and mineral metabolism of these mice are otherwise normal.


Assuntos
Efeito Fundador , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Camundongos Transgênicos/genética , Fosfatos/metabolismo , Fator de Transcrição Pit-1/genética , Transgenes , Actinas/genética , Actinas/metabolismo , Animais , Transporte Biológico , Densidade Óssea , Calcitriol/sangue , Galinhas , Citomegalovirus/genética , Citomegalovirus/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Masculino , Camundongos , Camundongos Transgênicos/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Hormônio Paratireóideo/genética , Hormônio Paratireóideo/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Crânio/citologia , Crânio/metabolismo , Fator de Transcrição Pit-1/metabolismo , Globinas beta/genética , Globinas beta/metabolismo
16.
Stem Cells Transl Med ; 8(12): 1306-1317, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31631568

RESUMO

Despite the great advance of bone tissue engineering in the last few years, repair of bone defects remains a major problem. Low cell engraftment and dose-dependent side effects linked to the concomitant administration of bone morphogenetic proteins (BMPs) are the main problems currently hindering the clinical use of mesenchymal stem cell (MSC)-based therapies in this field. We have managed to bypass these drawbacks by combining the silencing the Smurf1 ubiquitin ligase in MSCs with the use of a scaffold that sustainably releases low doses of BMP-2. In this system, Smurf1 silencing is achieved by using GapmeRs, a clinically safe method that avoids the use of viral vectors, facilitating its translation to the clinic. Here, we show that a single transient transfection with a small quantity of a Smurf1-specific GapmeR is able to induce a significant level of silencing of the target gene, enough to prime MSCs for osteogenic differentiation. Smurf1 silencing highly increases MSCs responsiveness to BMP-2, allowing a dramatic reduction of the dose needed to achieve the desired therapeutic effect. The combination of these primed cells with alginate scaffolds designed to sustainably and locally release low doses of BMP-2 to the defect microenvironment is able to induce the formation of a mature bone matrix both in an osteoporotic rat calvaria system and in a mouse ectopic model. Importantly, this approach also enhances osteogenic differentiation in MSCs from osteoporotic patients, characterized by a reduced bone-forming potential, even at low BMP doses, underscoring the regenerative potential of this system. Stem Cells Translational Medicine 2019;8:1306&1317.


Assuntos
Regeneração Óssea , Lipídeos/química , Células-Tronco Mesenquimais/citologia , Nanopartículas/administração & dosagem , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos/genética , Crânio/crescimento & desenvolvimento , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Alginatos/química , Animais , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular , Células Cultivadas , Feminino , Inativação Gênica , Humanos , Células-Tronco Mesenquimais/metabolismo , Ratos , Ratos Sprague-Dawley , Crânio/lesões , Crânio/metabolismo , Engenharia Tecidual/métodos , Ubiquitina-Proteína Ligases/genética
17.
Dev Dyn ; 248(10): 1009-1019, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31397024

RESUMO

BACKGROUND: The murine calvaria has several membrane bones with different tissue origins (e.g., neural crest-derived frontal bone vs. mesoderm-derived parietal bone). Neural crest-derived frontal bone exhibits superior osteogenic activities and bone regeneration. MicroRNA (miRNA) has been emerged as a crucial regulator during organogenesis and is involved in a range of developmental processes. However, the underlying roles of miRNA regulation in frontal bone and parietal bone is unknown. RESULTS: Total of 83 significantly expressed known miRNAs were identified in frontal bones versus parietal bones. The significantly enriched gene ontology and KEGG pathway that were predicted by the enrichment miRNAs were involved in several biological processes (cell differentiation, cell adhesion, and transcription), and multiple osteogenic pathways (e.g., focal adhesion, MAPK, VEGF, Wnt, and insulin signaling pathway. Focal adhesion and insulin signaling pathway were selected for target verification and functional analysis, and several genes were predicted to be targets genes by the differentially expressed miRNAs, and these targets genes were tested with significant expressions. CONCLUSIONS: Our results revealed a novel pattern of miRNAs in murine calvaria with dual tissue origins, and explorations of these miRNAs will be valuable for the translational studies to enhance osteogenic potential and bone regeneration in the clinic.


Assuntos
Osso Frontal/metabolismo , MicroRNAs/análise , Osso Parietal/metabolismo , Crânio/metabolismo , Animais , Regeneração Óssea , Adesões Focais , Insulina/metabolismo , Camundongos , MicroRNAs/fisiologia , Osteogênese , Transdução de Sinais
18.
Dev Dyn ; 248(12): 1264-1272, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31464047

RESUMO

BACKGROUND: Six1 is a transcriptional factor that plays an important role in embryonic development. Mouse and chick embryos deficient for Six1 have multiple craniofacial anomalies in the facial bones and cartilages. Multiple Six1 enhancers have been identified, but none of them has been reported to be active in the maxillary and mandibular process. RESULTS: We studied two Six1 enhancers in the chick neural crest tissues during craniofacial development. We showed that two evolutionarily conserved enhancers, Six1E1 and Six1E2, act synergistically. Neither Six1E1 nor Six1E2 alone can drive enhancer reporter signal in the maxillary or mandibular processes. However, their combination, Six1E, showed robust enhancer activity in these tissues. Similar reporter signal can also be driven by the mouse homolog of Six1E. Mutations of multiple conserved transcriptional factor binding sites altered the enhancer activity of Six1E, especially mutation of the LIM homeobox binding site, dramatically reduced the enhancer activity, implying that the Lhx protein family be an important regulator of Six1 expression. CONCLUSION: This study, for the first time, described the synergistic activation of two Six1 enhancers in the maxillary and mandibular processes and will facilitate more detailed studies of the regulation of Six1 in craniofacial development.


Assuntos
Elementos Facilitadores Genéticos/fisiologia , Ossos Faciais/embriologia , Proteínas de Homeodomínio/genética , Crista Neural/embriologia , Crânio/embriologia , Animais , Animais Geneticamente Modificados , Embrião de Galinha , Anormalidades Craniofaciais/genética , Desenvolvimento Embrionário/genética , Ossos Faciais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mandíbula/embriologia , Mandíbula/metabolismo , Maxila/embriologia , Maxila/metabolismo , Crista Neural/metabolismo , Crânio/metabolismo
19.
PLoS One ; 14(8): e0221402, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31442251

RESUMO

Craniosynostosis is the premature fusion of the sutures of the calvaria and is principally designated as being either syndromic (demonstrating characteristic extracranial malformations) or non-syndromic. While many forms of syndromic craniosynostosis are known to be caused by specific mutations, the genetic etiology of non-syndromic, single-suture craniosynostosis (SSC) is poorly understood. Based on the low recurrence rate (4-7%) and the fact that recurrent mutations have not been identified for most cases of SSC, we propose that some cases of isolated, single suture craniosynostosis may be polygenic. Previous work in our lab identified a disproportionately high number of rare and novel gain-of-function IGF1R variants in patients with SSC as compared to controls. Building upon this result, we used expression array data from calvarial osteoblasts isolated from infants with and without SSC to ascertain correlations between high IGF1 expression and expression of other osteogenic genes of interest. We identified a positive correlation between increased expression of IGF1 and RUNX2, a gene known to cause SSC with increased gene dosage. Subsequent phosphorylation assays revealed that osteoblast cell lines from cases with high IGF1 expression demonstrated inhibition of GSK3ß, a serine/threonine kinase known to inhibit RUNX2, thus activating osteogenesis through the IRS1-mediated Akt pathway. With these findings, we have utilized established mouse strains to examine a novel model of polygenic inheritance (a phenotype influenced by more than one gene) of SSC. Compound heterozygous mice with selective disinhibition of RUNX2 and either overexpression of IGF1 or loss of function of GSK3ß demonstrated an increase in the frequency and severity of synostosis as compared to mice with the RUNX2 disinhibition alone. These polygenic mouse models reinforce, in-vivo, that the combination of activation of the IGF1 pathway and disinhibition of the RUNX2 pathway leads to an increased risk of developing craniosynostosis and serves as a model of human SSC.


Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core/genética , Craniossinostoses/genética , Glicogênio Sintase Quinase 3 beta/genética , Fator de Crescimento Insulin-Like I/genética , Animais , Diferenciação Celular/genética , Craniossinostoses/patologia , Modelos Animais de Doenças , Mutação com Ganho de Função/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese/genética , Receptor IGF Tipo 1/genética , Crânio/metabolismo , Crânio/patologia
20.
Mol Cell Proteomics ; 18(10): 2078-2088, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31434710

RESUMO

Aging is characterized by a gradual deterioration in proteome. However, how protein dynamics that changes with normal aging and in disease is less well understood. Here, we profiled the snapshots of aging proteome in Drosophila, from head and muscle tissues of post-mitotic somatic cells, and the testis of mitotically-active cells. Our data demonstrated that dysregulation of proteome homeostasis, or proteostasis, might be a common feature associated with age. We further used pulsed metabolic stable isotope labeling analysis to characterize protein synthesis. Interestingly, this study determined an age-modulated decline in protein synthesis with age, particularly in the pathways related to mitochondria, neurotransmission, and proteostasis. Importantly, this decline became dramatically accelerated in Pink1 mutants, a Drosophila model of human age-related Parkinson's disease. Taken together, our multidimensional proteomic study revealed tissue-specific protein dynamics with age, highlighting mitochondrial and proteostasis-related proteins. We suggest that declines in proteostasis and mitochondria early in life are critical signals prior to the onset of aging and aging-associated diseases.


Assuntos
Envelhecimento/metabolismo , Regulação para Baixo , Proteínas de Drosophila/metabolismo , Mitocôndrias/metabolismo , Doença de Parkinson/metabolismo , Proteômica/métodos , Idade de Início , Animais , Modelos Animais de Doenças , Drosophila , Proteínas de Drosophila/genética , Masculino , Músculo Esquelético/metabolismo , Mutação , Especificidade de Órgãos , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/genética , Proteostase , Crânio/metabolismo , Testículo/metabolismo
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