RESUMO
Osteogenic supplements are a requirement for osteoblastic cell differentiation during in vitro culture of human calvarial suture-derived cell populations. We investigated the ability of ascorbic acid and beta-glycerophosphate with and without the addition of dexamethasone to stimulate in vivo-like osteoblastic differentiation. Cells were isolated from unfused and prematurely fused suture tissue from patients with syndromic and non-syndromic craniosynostosis and cultured in each osteogenic medium for varying lengths of time. The effect of media supplementation was investigated with respect to the ability of cells to form mineralised bone nodules and the expression of five osteodifferentiation marker genes (COL1A1, ALP, BSP, OC and RUNX2), and five genes that are differentially expressed during human premature suture fusion (GPC3, RBP4, C1QTNF3, WIF1 and FGF2). Cells from unfused sutures responded more slowly to osteogenic media but formed comparable bone nodules to fused suture-derived cells after 16 days of culture in either osteogenic media. However, gene expression differed between unfused and fused suture-derived cells, as did expression in each osteogenic medium. When compared to expression in the explant tissue of origin, neither medium induced a level or profile of gene expression similar to that seen in vivo. Overall, our results demonstrate that cells from the same suture that are isolated during different stages of morphogenesis in vivo, despite being de-differentiated to a similar level in vitro, respond uniquely and differently to each osteogenic medium. Further, we suggest that neither cell culture medium recapitulates differentiation via activation of the same genetic cascades as occurs in vivo.
Assuntos
Suturas Cranianas/citologia , Ácido Ascórbico/farmacologia , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Suturas Cranianas/efeitos dos fármacos , Suturas Cranianas/metabolismo , Craniossinostoses/genética , Craniossinostoses/metabolismo , Craniossinostoses/patologia , Meios de Cultura , Primers do DNA/genética , Dexametasona/farmacologia , Fator 2 de Crescimento de Fibroblastos/genética , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Glicerofosfatos/farmacologia , Humanos , Técnicas In Vitro , Lactente , Masculino , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Fenótipo , Fatores de Necrose Tumoral/genéticaRESUMO
Some of the zinc finger proteins of the snail family are essential in the formation of mesoderm during gastrulation and the development of neural crest and its derivatives. We have isolated the human SNAIL gene (HGMW-approved symbol SNAI1) and describe its genomic organization, having sequenced a region spanning more than 5882 bp. The human SNAIL gene contains three exons. The SNAIL transcript is 2. 0 kb and is found in placenta and adult heart, lung, brain, liver, and skeletal muscle. It codes for a protein of 264 amino acids and 29.1 kDa. This protein contains three classic zinc fingers and one atypical zinc finger. The human SNAIL protein is 87.5, 58.7, 50.9, 50.7, 55.4, and 31.5% identical to mouse Snail, chicken snail-like, zebrafish snail1, zebrafish snail2, Xenopus snail, and Drosophila snail proteins, respectively. The zinc finger region is 95.5% identical between human and mouse Snail. Because Drosophila snail and twist are important regulators during mesoderm development and because human TWIST mutations have been implicated in craniosynostosis, a cohort of 59 patients with craniosynostosis syndromes were screened for SNAIL mutations. None were found. By somatic cell and radiation hybrid mapping panels, SNAIL was localized to human chromosome 20q13.2, between markers D20S886 and D20S109. A SNAIL-related, putative processed pseudogene (HGMW-approved symbol SNAI1P) was also isolated and maps to human chromosome 2q33-q37.
Assuntos
Cromossomos Humanos Par 20/genética , Cromossomos Humanos Par 2/genética , Craniossinostoses/genética , Proteínas de Ligação a DNA/genética , Genes , Pseudogenes , Fatores de Transcrição/genética , Dedos de Zinco/genética , Adulto , Sequência de Aminoácidos , Animais , Estudos de Coortes , Análise Mutacional de DNA , DNA Complementar/genética , Drosophila melanogaster/genética , Desenvolvimento Embrionário e Fetal/genética , Éxons/genética , Etiquetas de Sequências Expressas , Proteínas Fetais/genética , Humanos , Células Híbridas , Masculino , Mesoderma/fisiologia , Dados de Sequência Molecular , Morfogênese/genética , Crista Neural/fisiologia , Especificidade de Órgãos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de Transcrição da Família Snail , Especificidade da Espécie , Vertebrados/genética , Peixe-Zebra/genéticaRESUMO
The Snail family of proteins in vertebrates comprises two zinc-finger transcription factors, Snail and Slug, which are thought to be involved in the formation of the mesoderm and neural crest. Here, we describe the isolation and characterisation of the human Snail (SNAI1) gene and a related Snail-like pseudogene, SNAI1P. SNAI1 spans approximately 6.4kb, contains three exons and has a CpG island upstream of the coding sequence. A single transcript of 1.9 kb was detected in several human foetal tissues, with the highest expression in the kidney. The SNAI1 open reading frame encodes a protein of 264 amino acids containing four zinc-finger motifs that show 87.1% identity to mouse Snail (mSna). SNAI1 was mapped to chromosome band 20q13.1 and is likely to lie between markers D20S109 and D20S196. Investigation of SNAI1 coding sequences by single-strand conformation polymorphism analysis excluded SNAI1 as a major disease gene in craniosynostosis. Two single nucleotide polymorphisms encoding synonymous amino acids were identified in exon 2. The SNAI1P pseudogene was isolated, sequenced and mapped to chromosome band 2q34.
Assuntos
Craniossinostoses/genética , Proteínas de Ligação a DNA/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 2/genética , Primers do DNA/genética , DNA Complementar/genética , Feto/metabolismo , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Polimorfismo Conformacional de Fita Simples , Pseudogenes , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição da Família Snail , Dedos de Zinco/genéticaRESUMO
The identification in craniosynostosis syndromes of mutations in genes belonging to the fibroblast growth factor signalling pathway and the transcriptional regulator MSX2 provides important clues to the pathogenesis of these disorders. Although surgery continues to be the mainstay of treatment, new animal models and improved uncerstanding of cranial suture biology and pathology may lead to complementary therapies.