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1.
Methods Mol Biol ; 2351: 165-179, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382189

RESUMO

Targeted chromatin capture (T2C) is a 3C-based method and is used to study the 3D chromatin organization, interactomes and structural changes associated with gene regulation, progression through the cell cycle, and cell survival and development. Low input targeted chromatin capture (low-T2C) is an optimized version of the T2C protocol for low numbers of cells. Here, we describe the protocol for low-T2C, including all experimental steps and bioinformatics tools in detail.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/genética , Biologia Computacional/métodos , Cromatina/química , Cromatina/metabolismo , Mapeamento Cromossômico , Regulação da Expressão Gênica , Biblioteca Gênica , Genômica/métodos , Reprodutibilidade dos Testes
2.
Cells ; 10(2)2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33513779

RESUMO

The ability to regenerate amputated or injured tissues and organs is a fascinating property shared by several invertebrates and, interestingly, some vertebrates. The mechanism of evolutionary loss of regeneration in mammals is not understood, yet from the biomedical and clinical point of view, it would be very beneficial to be able, at least partially, to restore that capability. The current availability of new experimental tools, facilitating the comparative study of models with high regenerative ability, provides a powerful instrument to unveil what is needed for a successful regeneration. The present review provides an updated overview of multiple aspects of appendage regeneration in three vertebrates: lizard, salamander, and zebrafish. The deep investigation of this process points to common mechanisms, including the relevance of Wnt/ß-catenin and FGF signaling for the restoration of a functional appendage. We discuss the formation and cellular origin of the blastema and the identification of epigenetic and cellular changes and molecular pathways shared by vertebrates capable of regeneration. Understanding the similarities, being aware of the differences of the processes, during lizard, salamander, and zebrafish regeneration can provide a useful guide for supporting effective regenerative strategies in mammals.


Assuntos
Extremidades/fisiologia , Regeneração/fisiologia , Vertebrados/fisiologia , Animais , Padronização Corporal/genética , Epigênese Genética , Filogenia , Regeneração/genética
3.
J Med Genet ; 57(6): 361-370, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31857429

RESUMO

Most of the human genome has a regulatory function in gene expression. The technological progress made in recent years permitted the revision of old and discovery of new mutations outside of the protein-coding regions that do affect human limb morphology. Steadily increasing discovery rate of such mutations suggests that until now the largely neglected part of the genome rises to its well-deserved prominence. In this review, we describe the recent technological advances permitting this unprecedented advance in identifying non-coding mutations. We especially focus on the mutations in cis-regulatory elements such as enhancers, and trans-regulatory elements such as miRNA and long non-coding RNA, linked to hereditary or inborn limb defects. We also discuss the role of chromatin organisation and enhancer-promoter interactions in the aetiology of limb malformations.


Assuntos
Deformidades Congênitas dos Membros/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Extremidades/crescimento & desenvolvimento , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Genoma Humano/genética , Humanos , Deformidades Congênitas dos Membros/patologia
5.
Clin Genet ; 96(5): 429-438, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31347140

RESUMO

RUNX2 (Runt-related transcription factor 2) is a master regulator of osteoblast differentiation, cartilage and bone development. Pathogenic variants in RUNX2 have been linked to the Cleidocranial dysplasia (CCD), which is characterized by hypoplasia or aplasia of clavicles, delayed fontanelle closure, and dental anomalies. Here, we report 11 unrelated Polish patients with CCD caused by pathogenic alterations located in the Runt domain of RUNX2. In total, we identified eight different intragenic variants, including seven missense and one splicing mutation. Three of them are novel: c.407T>A p.(Leu136Gln), c.480C>G p.(Asn160Lys), c.659C>G p.(Thr220Arg), additional three were not functionally tested: c.391C>T p.(Arg131Cys), c.580+1G>T p.(Lys195_Arg229del), c.652A>G p.(Lys218Glu), and the remaining two: c.568C>T p.(Arg190Trp), c.673C>T p.(Arg225Trp) were previously reported and characterized. The performed transactivation and localization studies provide evidence of decreased transcriptional activity of RUNX2 due to mutations targeting the Runt domain and prove that impairment of nuclear localization signal (NLS) affects the subcellular localization of the protein. Presented data show that pathogenic variants discovered in our patients have a detrimental effect on RUNX2, triggering the CCD phenotype.


Assuntos
Displasia Cleidocraniana/genética , Subunidade alfa 1 de Fator de Ligação ao Core/química , Predisposição Genética para Doença , Conformação Proteica , Pré-Escolar , Displasia Cleidocraniana/epidemiologia , Displasia Cleidocraniana/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/ultraestrutura , Feminino , Humanos , Lactente , Masculino , Mutação/genética , Mutação de Sentido Incorreto/genética , Fenótipo , Polônia/epidemiologia , Isoformas de Proteínas/genética , Relação Estrutura-Atividade
6.
Sci Rep ; 9(1): 5782, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962493

RESUMO

The healing of large bone defects remains a major unmet medical need. Our developmental engineering approach consists of the in vitro manufacturing of a living cartilage tissue construct that upon implantation forms bone by recapitulating an endochondral ossification process. Key to this strategy is the identification of the cells to produce such cartilage intermediates efficiently. We applied a cell selection strategy based on published skeletal stem cell markers using mouse embryonic limb cartilage as cell source and analysed their potential to form bone in an in vivo ectopic assay. FGF2 supplementation to the culture media for expansion blocked dedifferentiation of the embryonic cartilage cells in culture and enriched for stem cells and progenitors as quantified using the recently published CD marker set. However, when the stem cells and progenitors were fractionated from expanded embryonic cartilage cells and assessed in the ectopic assay, a major loss of bone forming potential was observed. We conclude that cell expansion appears to affect the association between cell identity based on CD markers and in vivo bone forming capacity.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Murinas/citologia , Osteoblastos/citologia , Osteogênese , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Cartilagem/citologia , Células Cultivadas , Fêmur/citologia , Fêmur/embriologia , Camundongos , Células-Tronco Embrionárias Murinas/classificação , Células-Tronco Embrionárias Murinas/metabolismo , Osteoblastos/metabolismo
7.
PLoS One ; 13(6): e0198104, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29897942

RESUMO

Tissue calcification is an important physiological process required for the normal structure and function of bone. However, ectopic or excessive calcification contributes to diseases such as chondrocalcinosis, to calcium deposits in the skin or to vascular calcification. SMOC2 is a member of the BM-40/osteonectin family of calcium-binding secreted matricellular proteins. Using osteoprogenitor MC3T3-E1 cells stably overexpressing SMOC2, we show that SMOC2 inhibits osteogenic differentiation and extracellular matrix mineralization. Stable Smoc2 knockdown in these cells had no effect on mineralization suggesting that endogenous SMOC2 is not essential for the mineralization process. Mineralization in MC3T3-E1 cells overexpressing mutant SMOC2 lacking the extracellular calcium-binding domain was significantly increased compared to cells overexpressing full length SMOC2. When SMOC2 overexpressing cells were cultured in the presence of extracellular calcium supplementation, SMOC2's inhibitory effect on calcification was rescued. Our observations were translationally validated in primary human periosteal-derived cells. Furthermore, SMOC2 was able to impair mineralization in transdifferentiated human umbilical vein endothelial cells. Taken together, our data indicate that SMOC2 can act as an inhibitor of mineralization. We propose a possible role for SMOC2 to prevent calcification disorders.


Assuntos
Calcificação Fisiológica/genética , Proteínas de Ligação ao Cálcio/fisiologia , Diferenciação Celular/genética , Células Endoteliais/fisiologia , Osteoblastos/fisiologia , Animais , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Camundongos , Osteogênese/genética
8.
PLoS One ; 13(5): e0197535, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29771958

RESUMO

The elaborate anatomy of hands and feet is shaped by coordinated formation of digits and regression of the interdigital mesenchyme (IM). A failure of this process causes persistence of interdigital webbing and consequently cutaneous syndactyly. Bone morphogenetic proteins (BMPs) are key inductive factors for interdigital cell death (ICD) in vivo. NOGGIN (NOG) is a major BMP antagonist that can interfere with BMP-induced ICD when applied exogenously, but its in vivo role in this process is unknown. We investigated the physiological role of NOG in ICD and found that Noggin null mice display cutaneous syndactyly and impaired interdigital mesenchyme specification. Failure of webbing regression was caused by lack of cell cycle exit and interdigital apoptosis. Unexpectedly, Noggin null mutants also exhibit increased Indian hedgehog (Ihh) expression within cartilage condensations that leads to aberrant extension of IHH downstream signaling into the interdigital mesenchyme. A converse phenotype with increased apoptosis and reduced cell proliferation was found in the interdigital mesenchyme of Ihh mutant embryos. Our data point towards a novel role for NOG in balancing Ihh expression in the digits impinging on digit-interdigit cross talk. This suggests a so far unrecognized physiological role for IHH in interdigital webbing biology.


Assuntos
Apoptose/fisiologia , Proteínas Morfogenéticas Ósseas/fisiologia , Proteínas de Transporte/fisiologia , Proteínas Hedgehog/fisiologia , Mesoderma/embriologia , Transdução de Sinais/fisiologia , Sindactilia/fisiopatologia , Animais , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas de Transporte/genética , Cartilagem/embriologia , Ciclo Celular , Ectoderma/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/deficiência , Proteínas Hedgehog/genética , Mesoderma/citologia , Mesoderma/patologia , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Organismos Livres de Patógenos Específicos , Sindactilia/embriologia , Sindactilia/patologia , Dedos do Pé/embriologia
10.
Mol Syndromol ; 8(5): 253-260, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28878609

RESUMO

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder linked to mutations in the Runt-related transcription factor 2, encoded by the RUNX2 gene, which is essential for osteoblast differentiation and skeletal development. Here, we describe a novel nonsense mutation (c.532C>T; p.Q178X) in RUNX2 identified in 3 affected members of a Polish family with CCD. The localization and transcriptional transactivation studies show that the mutated form of the protein has altered the subcellular localization and significantly decreased transactivation properties, respectively. Consequently, our data show that the c.532C>T mutation generates a defective RUNX2 protein and is genetically linked to the CCD phenotype.

11.
Sci Rep ; 7: 42583, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28198397

RESUMO

The human ubiquitous protein cystinosin is responsible for transporting the disulphide amino acid cystine from the lysosomal compartment into the cytosol. In humans, Pathogenic mutations of CTNS lead to defective cystinosin function, intralysosomal cystine accumulation and the development of cystinosis. Kidneys are initially affected with generalized proximal tubular dysfunction (renal Fanconi syndrome), then the disease rapidly affects glomeruli and progresses towards end stage renal failure and multiple organ dysfunction. Animal models of cystinosis are limited, with only a Ctns knockout mouse reported, showing cystine accumulation and late signs of tubular dysfunction but lacking the glomerular phenotype. We established and characterized a mutant zebrafish model with a homozygous nonsense mutation (c.706 C > T; p.Q236X) in exon 8 of ctns. Cystinotic mutant larvae showed cystine accumulation, delayed development, and signs of pronephric glomerular and tubular dysfunction mimicking the early phenotype of human cystinotic patients. Furthermore, cystinotic larvae showed a significantly increased rate of apoptosis that could be ameliorated with cysteamine, the human cystine depleting therapy. Our data demonstrate that, ctns gene is essential for zebrafish pronephric podocyte and proximal tubular function and that the ctns-mutant can be used for studying the disease pathogenic mechanisms and for testing novel therapies for cystinosis.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Cistinose/genética , Cistinose/metabolismo , Glomérulos Renais/metabolismo , Túbulos Renais Proximais/metabolismo , Mutação , Sequência de Aminoácidos , Animais , Apoptose/genética , Cistina/metabolismo , Cistinose/mortalidade , Cistinose/patologia , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Taxa de Filtração Glomerular , Humanos , Glomérulos Renais/patologia , Glomérulos Renais/ultraestrutura , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/ultraestrutura , Locomoção , Lisossomos/metabolismo , Fenótipo , Podócitos/metabolismo , Podócitos/patologia , Podócitos/ultraestrutura , Peixe-Zebra
12.
Sci Rep ; 6: 31949, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27573479

RESUMO

Inactivation of Noggin, a secreted antagonist of Bone Morphogenetic Proteins (BMPs), in mice leads, among others, to severe malformations of the appendicular skeleton and defective skeletal muscle fibers. To determine the molecular basis of the phenotype, we carried out a histomorphological and molecular analysis of developing muscles Noggin(-/-) mice. We show that in 18.5 dpc embryos there is a marked reduction in muscle fiber size and a failure of nuclei migration towards the cell membrane. Molecularly, the absence of Noggin results in an increased BMP signaling in muscle tissue as shown by the increase in SMAD1/5/8 phosphorylation, concomitant with the induction of BMP target genes such as Id1, 2, 3 as well as Msx1. Finally, upon removal of Noggin, the number of mesenchymal Pax7(+) muscle precursor cells is reduced and they are more prone to differentiate into adipocytes in vitro. Thus, our results highlight the importance of Noggin/BMP balance for myogenic commitment of early fetal progenitor cells.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Mioblastos/citologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas de Transporte/genética , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/citologia , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Transdução de Sinais
13.
PLoS One ; 9(10): e110484, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25335082

RESUMO

GPR22 is an orphan G protein-coupled receptor (GPCR). Since the ligand of the receptor is currently unknown, its biological function has not been investigated in depth. Many GPCRs and their intracellular effectors are targeted to cilia. Cilia are highly conserved eukaryotic microtubule-based organelles that protrude from the membrane of most mammalian cells. They are involved in a large variety of physiological processes and diseases. However, the details of the downstream pathways and mechanisms that maintain cilia length and structure are poorly understood. We show that morpholino knock down or overexpression of gpr22 led to defective left-right (LR) axis formation in the zebrafish embryo. Specifically, defective LR patterning included randomization of the left-specific lateral plate mesodermal genes (LPM) (lefty1, lefty2, southpaw and pitx2a), resulting in randomized cardiac looping. Furthermore, gpr22 inactivation in the Kupffer's vesicle (KV) alone was still able to generate the phenotype, indicating that Gpr22 mainly regulates LR asymmetry through the KV. Analysis of the KV cilia by immunofluorescence and transmission electron microscopy (TEM), revealed that gpr22 knock down or overexpression resulted in changes of cilia length and structure. Further, we found that Gpr22 does not act upstream of the two cilia master regulators, Foxj1a and Rfx2. To conclude, our study characterized a novel player in the field of ciliogenesis.


Assuntos
Cílios/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Padronização Corporal , Cílios/química , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/fisiologia , Fatores de Transcrição Forkhead/metabolismo , Microscopia Eletrônica de Transmissão , Oligonucleotídeos Antissenso/metabolismo , Fenótipo , Interferência de RNA , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
14.
Dev Dyn ; 243(11): 1375-90, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25044883

RESUMO

BACKGROUND: SMOC2 is a member of the BM-40 (SPARC) family of matricellular proteins, reported to influence signaling in the extracellular compartment. In mice, Smoc2 is expressed in many different tissues and was shown to enhance the response to angiogenic growth factors, mediate cell adhesion, keratinocyte migration, and metastasis. Additionally, SMOC2 is associated with vitiligo and craniofacial and dental defects. The function of Smoc2 during early zebrafish development has not been determined to date. RESULTS: In pregastrula zebrafish embryos, smoc2 is expressed ubiquitously. As development progresses, the expression pattern becomes more anteriorly restricted. At the onset of blood cell circulation, smoc2 morphants presented a mild ventralization of posterior structures. Molecular analysis of the smoc2 morphants indicated myelopoietic defects in the rostral blood islands during segmentation stages. Hemangioblast development and further specification of the myeloid progenitor cells were shown to be impaired. Additional experiments indicated that Bmp target genes were down-regulated in smoc2 morphants. CONCLUSIONS: Our findings reveal that Smoc2 is an essential player in the development of myeloid cells of the anterior lateral plate mesoderm during embryonic zebrafish development. Furthermore, our data show that Smoc2 affects the transcription of Bmp target genes without affecting initial dorsoventral patterning or mesoderm development.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mielopoese/genética , Mielopoese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/fisiologia , Mesoderma/metabolismo , Mielopoese/efeitos dos fármacos
15.
Dev Dyn ; 243(1): 37-48, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24038517

RESUMO

Synpolydactyly (SPD, OMIM 186000) is a rare congenital limb disorder characterized by syndactyly between the third and fourth fingers and between the fourth and fifth toes, with partial or complete digit duplication in the syndactylous web. The majority of these anomalies co-segregate with mutations in the HOXD13 gene,a homeobox transcription factor crucial for distal limb development. Different classes of HOXD13 mutations are involved in the pathogenesis of synpolydactyly, but an unequivocal genotype­phenotype correlation cannot always be achieved due to the clinical heterogeneity and reduced penetrance of SPD. All mutations identified so far mapped to the N-terminal polyalanine tract or to the C-terminal homeodomain of HOXD13,causing typical or atypical features of SPD, respectively. However, mutations outside of these domains cause a broad variety of clinical features that complicate the differential diagnosis. The existing animal models that are currently used to study HOXD13 (mal)function are therefore instrumental in unraveling potential genotype-phenotype correlations. Both mouse- and chick-based approaches allow the in vivo study of the pathogenic mechanism by which HOXD13 mutations cause SPD phenotypes as well as help in identifying the transcriptional targets.


Assuntos
Proteínas de Homeodomínio/metabolismo , Sindactilia/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas de Homeodomínio/genética , Mutação , Sindactilia/genética , Fatores de Transcrição/genética
16.
J Cell Biol ; 200(6): 709-20, 2013 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-23479743

RESUMO

Cilia project from the surface of most vertebrate cells and are important for several physiological and developmental processes. Ciliary defects are linked to a variety of human diseases, named ciliopathies, underscoring the importance of understanding signaling pathways involved in cilia formation and maintenance. In this paper, we identified Rer1p as the first endoplasmic reticulum/cis-Golgi-localized membrane protein involved in ciliogenesis. Rer1p, a protein quality control receptor, was highly expressed in zebrafish ciliated organs and regulated ciliary structure and function. Both in zebrafish and mammalian cells, loss of Rer1p resulted in the shortening of cilium and impairment of its motile or sensory function, which was reflected by hearing, vision, and left-right asymmetry defects as well as decreased Hedgehog signaling. We further demonstrate that Rer1p depletion reduced ciliary length and function by increasing γ-secretase complex assembly and activity and, consequently, enhancing Notch signaling as well as reducing Foxj1a expression.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Fatores de Transcrição Forkhead/biossíntese , Regulação da Expressão Gênica/fisiologia , Glicoproteínas de Membrana/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transporte Vesicular , Secretases da Proteína Precursora do Amiloide/genética , Animais , Linhagem Celular , Cílios/genética , Cílios/metabolismo , Fatores de Transcrição Forkhead/genética , Humanos , Glicoproteínas de Membrana/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Suínos , Peixe-Zebra , Proteínas de Peixe-Zebra
17.
Hum Mol Genet ; 21(11): 2464-75, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22373878

RESUMO

Synpolydactyly (SPD) is a distal limb anomaly characterized by incomplete digit separation and the presence of supernumerary digits in the syndactylous web. This phenotype has been associated with mutations in the homeodomain or polyalanine tract of the HOXD13 gene. We identified a novel mutation (G11A) in HOXD13 that is located outside the previously known domains and affects the intracellular half life of the protein. Misexpression of HOXD13(G11A) in the developing chick limb phenocopied the human SPD phenotype. Finally, we demonstrated through in vitro studies that this mutation has a destabilizing effect on GLI3R uncovering an unappreciated mechanism by which HOXD13 determines the patterning of the limb.


Assuntos
Padronização Corporal/genética , Proteínas de Homeodomínio/genética , Mutação , Sindactilia/genética , Fatores de Transcrição/genética , Animais , Células COS , Embrião de Galinha , Chlorocebus aethiops , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Sindactilia/metabolismo , Fatores de Transcrição/metabolismo , Transfecção , Proteína Gli3 com Dedos de Zinco
18.
Eur J Med Genet ; 55(1): 1-7, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21782042

RESUMO

Synpolydactyly (SPD) is a rare congenital limb disorder caused by mutations in the HOXD13 gene, a homeobox transcription factor crucial for autopod development. The hallmarks of SPD are the webbing between the third and the fourth finger and the fourth and the fifth toe, with a partial or complete digit duplication in the syndactylous web. Different classes of HOXD13 mutations are involved in the pathogenesis of synpolydactyly, but an unequivocal genotype-phenotype correlation cannot always be achieved due to the lack of structure-function data of HOXD13. Mutations in DNA binding or polyalanine tract domains of HOXD13 result in predictable clinical outcomes. However, mutations outside of these domains cause a broad variety of clinical features that complicate the differential diagnosis. In this review, we summarize the different classes of HOXD13 mutations causing synpolydactyly phenotypes with respect to their underlying pathogenic mechanism of action. In addition, we emphasize the importance of the chicken embryo as an animal model system for the study of (limb) development and potential genotype-phenotype correlations in SPD or other human malformation syndromes.


Assuntos
Proteínas de Homeodomínio/metabolismo , Mutação , Sindactilia/genética , Fatores de Transcrição/metabolismo , Substituição de Aminoácidos , Animais , Embrião de Galinha , Modelos Animais de Doenças , Estudos de Associação Genética , Proteínas de Homeodomínio/genética , Humanos , Estrutura Terciária de Proteína , Sindactilia/patologia , Fatores de Transcrição/genética
19.
Mol Cell Endocrinol ; 349(2): 289-97, 2012 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-22120204

RESUMO

We used the chick embryo to study the mechanisms regulating intracellular TH availability in developing brain. TH-transporters OATP1C1 and MCT8, and deiodinases D1, D2, and D3 were expressed in a region-specific way, well before the onset of endogenous TH secretion. Between day 4 and 10 of development MCT8 and D2 mRNA levels increased, while OATP1C1 and D3 mRNA levels decreased. D2 and D3 mRNAs were translated into active protein, while no D1 activity was detectable. Injection of THs into the yolk 24h before sampling increased TH levels in the brain and resulted in decreased OATP1C1 and increased MCT8 expression in 4-day-old embryos. A compensatory response in deiodinase activity was only observed at day 8. We conclude that THs are active in the early embryonic brain and TH-transporters and deiodinases can regulate their availability. However, the absence of clear compensatory mechanisms at day 4 makes the brain more vulnerable for changes in maternal TH supply.


Assuntos
Encéfalo/metabolismo , Iodeto Peroxidase/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Glândula Tireoide/metabolismo , Tiroxina/metabolismo , Tri-Iodotironina/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/embriologia , Embrião de Galinha , Galinhas , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Iodeto Peroxidase/genética , Proteínas de Membrana Transportadoras/genética , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Glândula Tireoide/embriologia , Tiroxina/farmacologia , Fatores de Tempo , Tri-Iodotironina/farmacologia
20.
J Bone Miner Res ; 27(2): 413-28, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22028304

RESUMO

Progeny of mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) revealed a mouse, designated Longpockets (Lpk), with short humeri, abnormal vertebrae, and disorganized growth plates, features consistent with spondyloepiphyseal dysplasia congenita (SEDC). The Lpk phenotype was inherited as an autosomal dominant trait. Lpk/+ mice were viable and fertile and Lpk/Lpk mice died perinatally. Lpk was mapped to chromosome 15 and mutational analysis of likely candidates from the interval revealed a Col2a1 missense Ser1386Pro mutation. Transient transfection of wild-type and Ser1386Pro mutant Col2a1 c-Myc constructs in COS-7 cells and CH8 chondrocytes demonstrated abnormal processing and endoplasmic reticulum retention of the mutant protein. Histology revealed growth plate disorganization in 14-day-old Lpk/+ mice and embryonic cartilage from Lpk/+ and Lpk/Lpk mice had reduced safranin-O and type-II collagen staining in the extracellular matrix. The wild-type and Lpk/+ embryos had vertical columns of proliferating chondrocytes, whereas those in Lpk/Lpk mice were perpendicular to the direction of bone growth. Electron microscopy of cartilage from 18.5 dpc wild-type, Lpk/+, and Lpk/Lpk embryos revealed fewer and less elaborate collagen fibrils in the mutants, with enlarged vacuoles in the endoplasmic reticulum that contained amorphous inclusions. Micro-computed tomography (CT) scans of 12-week-old Lpk/+ mice revealed them to have decreased bone mineral density, and total bone volume, with erosions and osteophytes at the joints. Thus, an ENU mouse model with a Ser1386Pro mutation of the Col2a1 C-propeptide domain that results in abnormal collagen processing and phenotypic features consistent with SEDC and secondary osteoarthritis has been established.


Assuntos
Colágeno Tipo II/genética , Mutação de Sentido Incorreto/genética , Osteoartrite/complicações , Osteoartrite/genética , Osteocondrodisplasias/congênito , Sequência de Aminoácidos , Animais , Sequência de Bases , Condrócitos/metabolismo , Condrócitos/patologia , Condrócitos/ultraestrutura , Cromossomos de Mamíferos/genética , Colágeno Tipo II/química , Modelos Animais de Doenças , Embrião de Mamíferos/anormalidades , Embrião de Mamíferos/patologia , Loci Gênicos/genética , Lâmina de Crescimento/anormalidades , Lâmina de Crescimento/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Tamanho do Órgão , Osteocondrodisplasias/complicações , Osteocondrodisplasias/genética , Osteogênese , Fenótipo , Mapeamento Físico do Cromossomo , Processamento de Proteína Pós-Traducional
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