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1.
Proc Natl Acad Sci U S A ; 120(1): e2213846120, 2023 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-36574677

RESUMO

Piezo1 and 2 are evolutionarily conserved mechanosensory cation channels known to function on the cell surface by responding to external pressure and transducing a mechanically activated Ca2+ current. Here we show that both Piezo1 and 2 also exhibit concentrated intracellular localization at centrosomes. Both Piezo1 and 2 loss-of-function and Piezo1 activation by the small molecule Yoda1 result in supernumerary centrosomes, premature centriole disengagement, multi-polar spindles, and mitotic delay. By using a GFP, Calmodulin and M13 Protein fusion (GCaMP) Ca2+-sensitive reporter, we show that perturbations in Piezo modulate Ca2+ flux at centrosomes. Moreover, the inhibition of Polo-like-kinase 1 eliminates Yoda1-induced centriole disengagement. Because previous studies have implicated force generation by microtubules as essential for maintaining centrosomal integrity, we propose that mechanotransduction by Piezo maintains pericentrosomal Ca2+ within a defined range, possibly through sensing cell intrinsic forces from microtubules.


Assuntos
Centrossomo , Mecanotransdução Celular , Centrossomo/metabolismo , Centríolos , Microtúbulos
2.
Development ; 147(18)2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958507

RESUMO

The FaceBase Consortium was established by the National Institute of Dental and Craniofacial Research in 2009 as a 'big data' resource for the craniofacial research community. Over the past decade, researchers have deposited hundreds of annotated and curated datasets on both normal and disordered craniofacial development in FaceBase, all freely available to the research community on the FaceBase Hub website. The Hub has developed numerous visualization and analysis tools designed to promote integration of multidisciplinary data while remaining dedicated to the FAIR principles of data management (findability, accessibility, interoperability and reusability) and providing a faceted search infrastructure for locating desired data efficiently. Summaries of the datasets generated by the FaceBase projects from 2014 to 2019 are provided here. FaceBase 3 now welcomes contributions of data on craniofacial and dental development in humans, model organisms and cell lines. Collectively, the FaceBase Consortium, along with other NIH-supported data resources, provide a continuously growing, dynamic and current resource for the scientific community while improving data reproducibility and fulfilling data sharing requirements.


Assuntos
Pesquisa em Odontologia/métodos , Ossos Faciais/fisiologia , Crânio/fisiologia , Animais , Bases de Dados Factuais , Humanos , Reprodutibilidade dos Testes , Pesquisadores
3.
Nature ; 531(7594): 323-8, 2016 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-26958831

RESUMO

The repair and regeneration of tissues using endogenous stem cells represents an ultimate goal in regenerative medicine. To our knowledge, human lens regeneration has not yet been demonstrated. Currently, the only treatment for cataracts, the leading cause of blindness worldwide, is to extract the cataractous lens and implant an artificial intraocular lens. However, this procedure poses notable risks of complications. Here we isolate lens epithelial stem/progenitor cells (LECs) in mammals and show that Pax6 and Bmi1 are required for LEC renewal. We design a surgical method of cataract removal that preserves endogenous LECs and achieves functional lens regeneration in rabbits and macaques, as well as in human infants with cataracts. Our method differs conceptually from current practice, as it preserves endogenous LECs and their natural environment maximally, and regenerates lenses with visual function. Our approach demonstrates a novel treatment strategy for cataracts and provides a new paradigm for tissue regeneration using endogenous stem cells.


Assuntos
Catarata/terapia , Cristalino/citologia , Cristalino/fisiologia , Recuperação de Função Fisiológica , Regeneração/fisiologia , Células-Tronco/citologia , Visão Ocular/fisiologia , Animais , Catarata/congênito , Catarata/patologia , Catarata/fisiopatologia , Extração de Catarata , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Homeostase , Humanos , Macaca , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Células-Tronco/metabolismo
4.
Development ; 143(2): 356-66, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26681494

RESUMO

The developing lens is a powerful system for investigating the molecular basis of inductive tissue interactions and for studying cataract, the leading cause of blindness. The formation of tightly controlled cell-cell adhesions and cell-matrix junctions between lens epithelial (LE) cells, between lens fiber (LF) cells, and between these two cell populations enables the vertebrate lens to adopt a highly ordered structure and acquire optical transparency. Adhesion molecules are thought to maintain this ordered structure, but little is known about their identity or interactions. Cysteine-rich motor neuron 1 (Crim1), a type I transmembrane protein, is strongly expressed in the developing lens and its mutation causes ocular disease in both mice and humans. How Crim1 regulates lens morphogenesis is not understood. We identified a novel ENU-induced hypomorphic allele of Crim1, Crim1(glcr11), which in the homozygous state causes cataract and microphthalmia. Using this and two other mutant alleles, Crim1(null) and Crim1(cko), we show that the lens defects in Crim1 mouse mutants originate from defective LE cell polarity, proliferation and cell adhesion. Crim1 adhesive function is likely to be required for interactions both between LE cells and between LE and LF cells. We show that Crim1 acts in LE cells, where it colocalizes with and regulates the levels of active ß1 integrin and of phosphorylated FAK and ERK. The RGD and transmembrane motifs of Crim1 are required for regulating FAK phosphorylation. These results identify an important function for Crim1 in the regulation of integrin- and FAK-mediated LE cell adhesion during lens development.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Cristalino/citologia , Animais , Receptores de Proteínas Morfogenéticas Ósseas/genética , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Cristalino/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Organogênese/genética , Organogênese/fisiologia , Fosforilação , Transdução de Sinais/fisiologia
5.
Development ; 143(14): 2677-88, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27287806

RESUMO

The FaceBase Consortium, funded by the National Institute of Dental and Craniofacial Research, National Institutes of Health, is designed to accelerate understanding of craniofacial developmental biology by generating comprehensive data resources to empower the research community, exploring high-throughput technology, fostering new scientific collaborations among researchers and human/computer interactions, facilitating hypothesis-driven research and translating science into improved health care to benefit patients. The resources generated by the FaceBase projects include a number of dynamic imaging modalities, genome-wide association studies, software tools for analyzing human facial abnormalities, detailed phenotyping, anatomical and molecular atlases, global and specific gene expression patterns, and transcriptional profiling over the course of embryonic and postnatal development in animal models and humans. The integrated data visualization tools, faceted search infrastructure, and curation provided by the FaceBase Hub offer flexible and intuitive ways to interact with these multidisciplinary data. In parallel, the datasets also offer unique opportunities for new collaborations and training for researchers coming into the field of craniofacial studies. Here, we highlight the focus of each spoke project and the integration of datasets contributed by the spokes to facilitate craniofacial research.


Assuntos
Bases de Dados Factuais , Face/embriologia , Pesquisadores , Crânio/embriologia , Animais , Imunoprecipitação da Cromatina , Biologia Computacional , Genômica , Humanos , Camundongos , Modelos Animais , Peixe-Zebra
7.
Hum Mol Genet ; 25(7): 1255-70, 2016 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-26758871

RESUMO

CAPZB is an actin-capping protein that caps the growing end of F-actin and modulates the cytoskeleton and tethers actin filaments to the Z-line of the sarcomere in muscles. Whole-genome sequencing was performed on a subject with micrognathia, cleft palate and hypotonia that harbored a de novo, balanced chromosomal translocation that disrupts the CAPZB gene. The function of capzb was analyzed in the zebrafish model. capzb(-/-) mutants exhibit both craniofacial and muscle defects that recapitulate the phenotypes observed in the human subject. Loss of capzb affects cell morphology, differentiation and neural crest migration. Differentiation of both myogenic stem cells and neural crest cells requires capzb. During palate morphogenesis, defective cranial neural crest cell migration in capzb(-/-) mutants results in loss of the median cell population, creating a cleft phenotype. capzb is also required for trunk neural crest migration, as evident from melanophores disorganization in capzb(-/-) mutants. In addition, capzb over-expression results in embryonic lethality. Therefore, proper capzb dosage is important during embryogenesis, and regulates both cell behavior and tissue morphogenesis.


Assuntos
Proteína de Capeamento de Actina CapZ/genética , Diferenciação Celular , Cabeça/embriologia , Morfogênese , Crista Neural/embriologia , Animais , Fissura Palatina/genética , Fissura Palatina/metabolismo , Modelos Animais de Doenças , Feminino , Cabeça/fisiologia , Humanos , Lactente , Micrognatismo/genética , Micrognatismo/metabolismo , Hipotonia Muscular/genética , Hipotonia Muscular/metabolismo , Mutação , Crista Neural/metabolismo , Crista Neural/fisiologia , Análise de Sequência de DNA , Síndrome , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia
8.
Am J Hum Genet ; 97(4): 608-15, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26365341

RESUMO

Skeletal dysplasias are highly variable Mendelian phenotypes. Molecular diagnosis of skeletal dysplasias is complicated by their extreme clinical and genetic heterogeneity. We describe a clinically recognizable autosomal-recessive disorder in four affected siblings from a consanguineous Saudi family, comprising progressive spondyloepimetaphyseal dysplasia, short stature, facial dysmorphism, short fourth metatarsals, and intellectual disability. Combined autozygome/exome analysis identified a homozygous frameshift mutation in RSPRY1 with resulting nonsense-mediated decay. Using a gene-centric "matchmaking" system, we were able to identify a Peruvian simplex case subject whose phenotype is strikingly similar to the original Saudi family and whose exome sequencing had revealed a likely pathogenic homozygous missense variant in the same gene. RSPRY1 encodes a hypothetical RING and SPRY domain-containing protein of unknown physiological function. However, we detect strong RSPRY1 protein localization in murine embryonic osteoblasts and periosteal cells during primary endochondral ossification, consistent with a role in bone development. This study highlights the role of gene-centric matchmaking tools to establish causal links to genes, especially for rare or previously undescribed clinical entities.


Assuntos
Doenças do Desenvolvimento Ósseo/genética , Genes Recessivos/genética , Anormalidades Musculoesqueléticas/genética , Mutação/genética , Ossificação Heterotópica/genética , Osteocondrodisplasias/genética , Adolescente , Animais , Doenças do Desenvolvimento Ósseo/patologia , Criança , Consanguinidade , Desoxirribonucleases de Sítio Específico do Tipo II , Nanismo/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Exoma , Feminino , Homozigoto , Humanos , Deficiência Intelectual/genética , Masculino , Camundongos , Anormalidades Musculoesqueléticas/patologia , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteocondrodisplasias/patologia , Linhagem , Periósteo/metabolismo , Periósteo/patologia , Fenótipo , Análise de Sequência de DNA
10.
Genes Dev ; 24(10): 980-5, 2010 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-20413611

RESUMO

How transcription factors interpret the cis-regulatory logic encoded within enhancers to mediate quantitative changes in spatiotemporally restricted expression patterns during animal development is not well understood. Pax6 is a dosage-sensitive gene essential for eye development. Here, we identify the Prep1 (pKnox1) transcription factor as a critical dose-dependent upstream regulator of Pax6 expression during lens formation. We show that Prep1 activates the Pax6 lens enhancer by binding to two phylogenetically conserved lower-affinity DNA-binding sites. Finally, we describe a mechanism whereby Pax6 levels are determined by transcriptional synergy of Prep1 bound to the two sites, while timing of enhancer activation is determined by binding site affinity.


Assuntos
Elementos Facilitadores Genéticos/genética , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Cristalino/embriologia , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fator de Transcrição PAX6 , Alinhamento de Sequência , Fatores de Tempo
11.
Hum Mol Genet ; 24(8): 2375-89, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25574029

RESUMO

Cardiac left ventricular outflow tract (LVOT) defects represent a common but heterogeneous subset of congenital heart disease for which gene identification has been difficult. We describe a 46,XY,t(1;5)(p36.11;q31.2)dn translocation carrier with pervasive developmental delay who also exhibited LVOT defects, including bicuspid aortic valve (BAV), coarctation of the aorta (CoA) and patent ductus arteriosus (PDA). The 1p breakpoint disrupts the 5' UTR of AHDC1, which encodes AT-hook DNA-binding motif containing-1 protein, and AHDC1-truncating mutations have recently been described in a syndrome that includes developmental delay, but not congenital heart disease [Xia, F., Bainbridge, M.N., Tan, T.Y., Wangler, M.F., Scheuerle, A.E., Zackai, E.H., Harr, M.H., Sutton, V.R., Nalam, R.L., Zhu, W. et al. (2014) De Novo truncating mutations in AHDC1 in individuals with syndromic expressive language delay, hypotonia, and sleep apnea. Am. J. Hum. Genet., 94, 784-789]. On the other hand, the 5q translocation breakpoint disrupts the 3' UTR of MATR3, which encodes the nuclear matrix protein Matrin 3, and mouse Matr3 is strongly expressed in neural crest, developing heart and great vessels, whereas Ahdc1 is not. To further establish MATR3 3' UTR disruption as the cause of the proband's LVOT defects, we prepared a mouse Matr3(Gt-ex13) gene trap allele that disrupted the 3' portion of the gene. Matr3(Gt-ex13) homozygotes are early embryo lethal, but Matr3(Gt-ex13) heterozygotes exhibit incompletely penetrant BAV, CoA and PDA phenotypes similar to those in the human proband, as well as ventricular septal defect (VSD) and double-outlet right ventricle (DORV). Both the human MATR3 translocation breakpoint and the mouse Matr3(Gt-ex13) gene trap insertion disturb the polyadenylation of MATR3 transcripts and alter Matrin 3 protein expression, quantitatively or qualitatively. Thus, subtle perturbations in Matrin 3 expression appear to cause similar LVOT defects in human and mouse.


Assuntos
Coartação Aórtica/genética , Valva Aórtica/anormalidades , Permeabilidade do Canal Arterial/genética , Doenças das Valvas Cardíacas/genética , Proteínas Associadas à Matriz Nuclear/genética , Proteínas de Ligação a RNA/genética , Adolescente , Animais , Coartação Aórtica/metabolismo , Valva Aórtica/metabolismo , Doença da Válvula Aórtica Bicúspide , Pré-Escolar , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Permeabilidade do Canal Arterial/metabolismo , Feminino , Inativação Gênica , Doenças das Valvas Cardíacas/metabolismo , Ventrículos do Coração/anormalidades , Ventrículos do Coração/metabolismo , Humanos , Recém-Nascido , Masculino , Camundongos , Mutagênese Insercional , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Translocação Genética
12.
J Biol Chem ; 290(33): 20448-54, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26045558

RESUMO

PAX6 is a master regulatory gene involved in neuronal cell fate specification. It also plays a critical role in early eye field and subsequent limbal stem cell (LSC) determination during eye development. Defects in Pax6 cause aniridia and LSC deficiency in humans and the Sey (Small eye) phenotype in mice (Massé, K., Bhamra, S., Eason, R., Dale, N., and Jones, E. A. (2007) Nature 449, 1058-1062). However, how PAX6 specifies LSC and corneal fates during eye development is not well understood. Here, we show that PAX6 is expressed in the primitive eye cup and later in corneal tissue progenitors in early embryonic development. In contrast, p63 expression commences after that of PAX6 in ocular adnexal and skin tissue progenitors and later in LSCs. Using an in vitro feeder-free culture system, we show that PAX6 knockdown in LSCs led to up-regulation of skin epidermis-specific keratins concomitant with differentiation to a skin fate. Using gene expression analysis, we identified the involvement of Notch, Wnt, and TGF-ß signaling pathways in LSC fate determination. Thus, loss of PAX6 converts LSCs to epidermal stem cells, as demonstrated by a switch in the keratin gene expression profile and by the appearance of congenital dermoid tissue.


Assuntos
Linhagem da Célula/fisiologia , Proteínas do Olho/fisiologia , Proteínas de Homeodomínio/fisiologia , Sistema Límbico/citologia , Fatores de Transcrição Box Pareados/fisiologia , Proteínas Repressoras/fisiologia , Células-Tronco/citologia , Animais , Córnea/embriologia , Proteínas do Olho/genética , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Humanos , Sistema Límbico/metabolismo , Proteínas de Membrana/genética , Camundongos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Transdução de Sinais , Células-Tronco/metabolismo
13.
Kidney Int ; 90(6): 1262-1273, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27591083

RESUMO

Primary glomerulocystic kidney disease is a special form of renal cystic disorder characterized by Bowman's space dilatation in the absence of tubular cysts. ZEB2 is a SMAD-interacting transcription factor involved in Mowat-Wilson syndrome, a congenital disorder with an increased risk for kidney anomalies. Here we show that deletion of Zeb2 in mesenchyme-derived nephrons with either Pax2-cre or Six2-cre causes primary glomerulocystic kidney disease without tubular cysts in mice. Glomerulotubular junction analysis revealed many atubular glomeruli in the kidneys of Zeb2 knockout mice, which explains the presence of glomerular cysts in the absence of tubular dilatation. Gene expression analysis showed decreased expression of early proximal tubular markers in the kidneys of Zeb2 knockout mice preceding glomerular cyst formation, suggesting that defects in proximal tubule development during early nephrogenesis contribute to the formation of congenital atubular glomeruli. At the molecular level, Zeb2 deletion caused aberrant expression of Pkd1, Hnf1ß, and Glis3, three genes causing glomerular cysts. Thus, Zeb2 regulates the morphogenesis of mesenchyme-derived nephrons and is required for proximal tubule development and glomerulotubular junction formation. Our findings also suggest that ZEB2 might be a novel disease gene in patients with primary glomerular cystic disease.


Assuntos
Doenças do Sistema Nervoso Central/genética , Esmalte Dentário/anormalidades , Diabetes Mellitus Tipo 2/genética , Proteínas de Homeodomínio/fisiologia , Doenças Renais Císticas/genética , Rim/embriologia , Proteínas Repressoras/fisiologia , Animais , Proteínas de Ligação a DNA , Fator 1-beta Nuclear de Hepatócito/metabolismo , Rim/metabolismo , Camundongos Knockout , Proteínas Repressoras/metabolismo , Canais de Cátion TRPP/metabolismo , Transativadores/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco
14.
Proc Natl Acad Sci U S A ; 110(12): 4667-72, 2013 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-23487782

RESUMO

Mechanotransduction, the pathway by which mechanical forces are translated to biological signals, plays important but poorly characterized roles in physiology. PIEZOs are recently identified, widely expressed, mechanically activated ion channels that are hypothesized to play a role in mechanotransduction in mammals. Here, we describe two distinct PIEZO2 mutations in patients with a subtype of Distal Arthrogryposis Type 5 characterized by generalized autosomal dominant contractures with limited eye movements, restrictive lung disease, and variable absence of cruciate knee ligaments. Electrophysiological studies reveal that the two PIEZO2 mutations affect biophysical properties related to channel inactivation: both E2727del and I802F mutations cause the PIEZO2-dependent, mechanically activated currents to recover faster from inactivation, while E2727del also causes a slowing of inactivation. Both types of changes in kinetics result in increased channel activity in response to a given mechanical stimulus, suggesting that Distal Arthrogryposis Type 5 can be caused by gain-of-function mutations in PIEZO2. We further show that overexpression of mutated PIEZO2 cDNAs does not cause constitutive activity or toxicity to cells, indicating that the observed phenotype is likely due to a mechanotransduction defect. Our studies identify a type of channelopathy and link the dysfunction of mechanically activated ion channels to developmental malformations and joint contractures.


Assuntos
Artrogripose , Doenças Genéticas Inatas , Canais Iônicos/genética , Canais Iônicos/metabolismo , Mecanotransdução Celular/genética , Mutação , Adulto , Artrogripose/genética , Artrogripose/metabolismo , Artrogripose/patologia , Artrogripose/fisiopatologia , Linhagem Celular , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Doenças Genéticas Inatas/patologia , Doenças Genéticas Inatas/fisiopatologia , Humanos , Lactente , Recém-Nascido , Masculino
15.
Nucleic Acids Res ; 41(2): e38, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23143268

RESUMO

Interactions between DNA and transcription factors (TFs) guide cellular function and development, yet the complexities of gene regulation are still far from being understood. Such understanding is limited by a paucity of techniques with which to probe DNA-protein interactions. We have devised magnetic protein immobilization on enhancer DNA (MagPIE), a simple, rapid, multi-parametric assay using flow cytometric immunofluorescence to reveal interactions among TFs, chromatin structure and DNA. In MagPIE, synthesized DNA is bound to magnetic beads, which are then incubated with nuclear lysate, permitting sequence-specific binding by TFs, histones and methylation by native lysate factors that can be optionally inhibited with small molecules. Lysate protein-DNA binding is monitored by flow cytometric immunofluorescence, which allows for accurate comparative measurement of TF-DNA affinity. Combinatorial fluorescent staining allows simultaneous analysis of sequence-specific TF-DNA interaction and chromatin modification. MagPIE provides a simple and robust method to analyze complex epigenetic interactions in vitro.


Assuntos
DNA/metabolismo , Citometria de Fluxo/métodos , Fatores de Transcrição/metabolismo , Animais , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Histonas/metabolismo , Camundongos
16.
Am J Hum Genet ; 89(1): 44-55, 2011 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-21703590

RESUMO

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Specc1l is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.


Assuntos
Fissura Palatina/genética , Disostose Craniofacial/genética , Proteínas do Citoesqueleto/deficiência , Anormalidades do Olho/genética , Anormalidades Maxilofaciais/genética , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Actinas/genética , Animais , Adesão Celular , Linhagem Celular , Movimento Celular/genética , Proliferação de Células , Fissura Palatina/patologia , Disostose Craniofacial/patologia , Drosophila/genética , Drosophila/metabolismo , Anormalidades do Olho/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Hibridização In Situ , Masculino , Anormalidades Maxilofaciais/patologia , Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Tubulina (Proteína)/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
17.
Hum Mol Genet ; 20(5): 962-74, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21156717

RESUMO

Clinical barriers to stem-cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem-cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short- or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5-7 days with 0.5% efficiency. This efficiency is greater than that reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4(+) or c-Kit(+) amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human embryonic stem (ES) cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture.


Assuntos
Âmnio/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição de Octâmero/genética , Fatores de Transcrição de Octâmero/metabolismo
18.
Small ; 9(20): 3374-84, 2013 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23677651

RESUMO

Fertilization is central to the survival and propagation of a species, however, the precise mechanisms that regulate the sperm's journey to the egg are not well understood. In nature, the sperm has to swim through the cervical mucus, akin to a microfluidic channel. Inspired by this, a simple, cost-effective microfluidic channel is designed on the same scale. The experimental results are supported by a computational model incorporating the exhaustion time of sperm.


Assuntos
Movimento Celular , Microfluídica/métodos , Espermatozoides/citologia , Animais , Separação Celular , Simulação por Computador , Humanos , Masculino , Camundongos , Fatores de Tempo
19.
Bioinformatics ; 28(23): 3115-22, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23060611

RESUMO

MOTIVATION: Proteomics presents the opportunity to provide novel insights about the global biochemical state of a tissue. However, a significant problem with current methods is that shotgun proteomics has limited success at detecting many low abundance proteins, such as transcription factors from complex mixtures of cells and tissues. The ability to assay for these proteins in the context of the entire proteome would be useful in many areas of experimental biology. RESULTS: We used network-based inference in an approach named SNIPE (Software for Network Inference of Proteomics Experiments) that selectively highlights proteins that are more likely to be active but are otherwise undetectable in a shotgun proteomic sample. SNIPE integrates spectral counts from paired case-control samples over a network neighbourhood and assesses the statistical likelihood of enrichment by a permutation test. As an initial application, SNIPE was able to select several proteins required for early murine tooth development. Multiple lines of additional experimental evidence confirm that SNIPE can uncover previously unreported transcription factors in this system. We conclude that SNIPE can enhance the utility of shotgun proteomics data to facilitate the study of poorly detected proteins in complex mixtures. AVAILABILITY AND IMPLEMENTATION: An implementation for the R statistical computing environment named snipeR has been made freely available at http://genetics.bwh.harvard.edu/snipe/. CONTACT: ssunyaev@rics.bwh.harvard.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Algoritmos , Proteoma/análise , Proteômica/métodos , Software , Animais , Biologia Computacional/métodos , Camundongos , Dente/metabolismo
20.
Proc Natl Acad Sci U S A ; 107(10): 4596-600, 2010 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-20176969

RESUMO

The vitrification of a liquid occurs when ice crystal formation is prevented in the cryogenic environment through ultrarapid cooling. In general, vitrification entails a large temperature difference between the liquid and its surrounding medium. In our droplet vitrification experiments, we observed that such vitrification events are accompanied by a Leidenfrost phenomenon, which impedes the heat transfer to cool the liquid, when the liquid droplet comes into direct contact with liquid nitrogen. This is distinct from the more generally observed Leidenfrost phenomenon that occurs when a liquid droplet is self-vaporized on a hot plate. In the case of rapid cooling, the phase transition from liquid to vitrified solid (i.e., vitrification) and the levitation of droplets on liquid nitrogen (i.e., Leidenfrost phenomenon) take place simultaneously. Here, we investigate these two simultaneous physical events by using a theoretical model containing three dimensionless parameters (i.e., Stefan, Biot, and Fourier numbers). We explain theoretically and observe experimentally a threshold droplet radius during the vitrification of a cryoprotectant droplet in the presence of the Leidenfrost effect.


Assuntos
Algoritmos , Crioprotetores/química , Nitrogênio/química , Simulação por Computador , Cristalização , Modelos Químicos , Volatilização
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