No description available.

No description available.

<p>Both Notch and the Wnt pathways are key regulators in maintaining the homeostasis in the intestine. Defects on the key tumor suppressor adenomatous polyposis coli, APC a gene in the Wnt pathway is most frequently mutated in colorectal cancer. Previous studies have indicated that there is a crosstalk between these two pathways. We investigate if there is correlation by first using bioinformatics to find Lef1/Tcf sites in several of the Notch pathway gene promoters. Bioinformatically we found that a lot of the genes contained theses sites controlled by the APC's destruction target β-catenin. By using semi quantitative PCR and western blot we found that Hes 1, Hes 7, JAG 2, MAML 1, Notch 2, NUMB, NUMBL, RFNG and LFNG was downregulated in HT29 colon cancer cells carrying a vector containing wild type APC. All but JAG 2 contains at least one Lef1/Tcf site in their promoter region. The results were verified in HT29 cells transfected with siRNA against β-catenin. We also investigated what would happen to the Lef1/Tcf target gene program of the Wnt pathway, if the Notch pathway was inhibited with the gamma-secretase inhibitor DAPT. Results showed no downregulution of β-catenin or its target gene Cyclin D1.Taken together, these results demonstrate that the Wnt pathway can be placed upstream of the Notch pathway and regulates the latter through β-catenin and the Lef1/Tcf target gene program. However, preliminary results indicate that there is no regulation of APC/β-catenin by the Notch pathway.</p>

No description available.

The regenerative tissues such as hair follicles, intestine and teeth have a particular microenvironment known as “stem cell niche” which houses stem cells and act as a signaling center to control stem cell fate. The precise and timely regulation of stem cell renewal and differentiation is essential for tissue formation, growth and homeostasis over the course of a lifetime. However, the molecular underpinning to control this regulation is poorly understood. To address this issue, we use the continuously growing mouse incisor as a model to study the gene regulatory network which controls dental epithelial stem cell (DESC) maintenance, growth and craniofacial morphogenesis. We found FoxO6, a transcription factor mainly expressed in the brain and craniofacial region, control DESC proliferation by regulating Hippo signaling. FoxO6 loss-of-function mice undergo increases in cell proliferation which finally leads to lengthening of the incisors, expansion of the face and skull and enlargement of the mandible and maxilla. We have screened three human FOXO6 single nucleotide polymorphisms which are associated with facial morphology ranging from retrognathism to prognathism. Our study also reveals that Sox2 and Lef-1, two markers for early craniofacial development, are regulated by Pitx2 to control DESC maintenance, differentiation and craniofacial development. Conditional Sox2 deletion in the oral and dental epithelia results in severe craniofacial defects, including ankyloglossia, cleft palate, arrested incisor development and abnormal molar development. The loss of Sox2 in DESCs leads to impaired stem cell proliferation, migration and subsequent dissolution of the tooth germ. On the other hand, conditional overexpression of Lef-1 in oral and dental epithelial region increases DESC proliferation and creates a new labial cervical loop stem cell compartment in dental epithelial stem cell niche, which produces rapidly growing long “tusk-like” incisors. Interestingly, Lef-1 overexpression rescues the tooth arrest defects but not the ankyloglossia or cleft palate in Sox2 conditional deletion mice. Our data also reveal that miRNA and histone remodeler are involved in regulating DESC proliferation and craniofacial morphogenesis. We describe a miR-23a/b:Hmgn2:Pitx2 signaling pathway in regulating dental epithelial cell growth and differentiation. Pitx2 activates expression of amelogenin which is the major protein component for enamel deposition. This activation can be repressed by the chromatin-associated factor Hmgn2. miR-23a and miR-23b directly target Hmgn2, leading to the release of the Hmgn2 inhibition of Pitx2 transcriptional activity and thus enhance Amelogenin production. Phenotypically, ablation of Hmgn2 in mice results in an overgrowth of incisors with increased Amelogenin expression. The findings in this study increase our current understanding of the molecular regulation of dental epithelial stem cell fate. It not only highlights new gene regulatory network that controls dental stem cell maintenance, growth and craniofacial morphogenesis, but also sheds new light on developing novel stem cell therapy or gene therapy for tooth regeneration and dental diseases.

Both Notch and the Wnt pathways are key regulators in maintaining the homeostasis in the intestine. Defects on the key tumor suppressor adenomatous polyposis coli, APC a gene in the Wnt pathway is most frequently mutated in colorectal cancer. Previous studies have indicated that there is a crosstalk between these two pathways. We investigate if there is correlation by first using bioinformatics to find Lef1/Tcf sites in several of the Notch pathway gene promoters. Bioinformatically we found that a lot of the genes contained theses sites controlled by the APC's destruction target β-catenin. By using semi quantitative PCR and western blot we found that Hes 1, Hes 7, JAG 2, MAML 1, Notch 2, NUMB, NUMBL, RFNG and LFNG was downregulated in HT29 colon cancer cells carrying a vector containing wild type APC. All but JAG 2 contains at least one Lef1/Tcf site in their promoter region. The results were verified in HT29 cells transfected with siRNA against β-catenin. We also investigated what would happen to the Lef1/Tcf target gene program of the Wnt pathway, if the Notch pathway was inhibited with the gamma-secretase inhibitor DAPT. Results showed no downregulution of β-catenin or its target gene Cyclin D1.Taken together, these results demonstrate that the Wnt pathway can be placed upstream of the Notch pathway and regulates the latter through β-catenin and the Lef1/Tcf target gene program. However, preliminary results indicate that there is no regulation of APC/β-catenin by the Notch pathway.

No description available.

La transició Epiteli-Mesènquima es dóna durant el desenvolupament embrionari i en els estadis tardans de la progressió tumoral permetent que es produeixi la metàstasi. Aquestes transicions necessiten una repressió de l'E-Cadherina i es pot reproduir en cèl·lules en cultiu amb l'expressió ectòpica de Snail1, un repressor de l'E-Cadherina. Durant la transició produïda per Snail es produeix la ràpida activació de gens mesenquimals com Fibronectina i LEF1. L'expressió forçada d'E-Cadherina fa disminuir els nivells de RNA de Fibronectina i LEF1, indicant que en l'activació d'aquests dos gens està implicat un cofactor sensible a l'E-Cadherina. En concordança, la transcripció de Fibronectina i LEF1 és depenent de &#61538;-Catenina i NF&#61547;B. La sobreexpressió d'E-Cadherina inhibeix l'activitat transcripcional d'aquests dos factors i disminueix la seva interacció amb el promotor de Fibronectina. De manera similar a la &#61538;-Catenina, NF&#61547;B es detecta associat a l'E-Cadherina i altres components dels contactes intercel·lulars. Quan es trenquen les unions adherents, com quan es sobreexpressa Snail, la interacció E-Cadherina-NF&#61547;B disminueix i augmenta l'activitat transcripcional de NF&#61547;B i&#61472;&#61472;&#61538;-Catenina. / Epithelial to mesenchymal transitions takes place during embryo development and in the late stages of tumorigenesis allowing metastasis formation. These transitions require E-Cadherin downregulation and can be reproduced in cell culture by ectopic expression of Snail1, an E-Cadherin gene repressor. During Snail-induced transition a rapid upregulation of mesenchymal genes such as Fibronectin and LEF1 has been characterized. Forced expression of E-Cadherin strongly down-regulates Fibronectin and LEF1 RNA levels, indicating that an E-Cadherin sensitive cofactor is involved in the activation of these genes. Accordingly, transcription of Fibronectin and LEF1 was dependent on &#61538;-Catenin and NF&#61547;B. E-Cadherin over-expression downregulated the transcriptional activity of both factors and decreased their interaction to Fibronectin promoter. Similarly to &#61538;-Catenin, NF&#61547;B was detected associated to E-Cadherin and other cell adhesion components. Association of NF&#61547;B to E-Cadherin required the integrity of this complex; conditions that disrupts adherens junctions, such as Snail over-expression, decreased E-Cadherin-NF&#61547;B interaction and up-regulates NF&#61547;B and &#61538;-Catenin transcriptional activity. Therefore, &#61538;-Catenin and NF&#61547;B transcriptional activities are required for expression of the studied mesenchymal genes and these activities are inactivated by immobilizing &#61538;-Catenin and NF&#61547;B to functional E-Cadherin structures.

No description available.

This body of work is concerned with the genetics of craniofacial morphology and specifically with that of the cranial sutures which form fibrous articulations between the calvarial bones. The premature fusion of these sutures, known as craniosynostosis, is a common developmental abnormality and has been extensively utilised here as a tool through which to study the genetics of suture morphogenesis and craniofacial diversity. Investigations began with a search for polymorphisms associated with normal variation in human craniofacial characteristics. Denaturing High-Performance Liquid chromatography was used to identify polymorphisms in two genes causative for craniosynostosis by analysing DNA from a large cohort of individuals from four ethnogeographic populations. A single nucleotide polymorphism in fibroblast growth factor receptor 1 was identified as being associated with variation in the cephalic index, a common measure of cranial shape. To further, and specifically, investigate the molecular processes of suture morphogenesis gene expression was compared between unfused and prematurely fusing/fused suture tissues isolated from patients with craniosynostosis. Two approaches, both utilising Affymetrix gene expression microarrays, were used to identify genes differentially expressed during premature suture fusion. The first was a novel method which utilised the observation that explant cells from both fused and unfused suture tissue, cultured in minimal medium, produce a gene expression profile characteristic of minimally differentiated osteoblastic cells. Consequently, gene expression was compared between prematurely fused suture tissues and their corresponding in vitro de-differentiated cells. In addition to those genes known to be involved in suture morphogenesis, a large number of novel genes were identified which were up-regulated in the differentiated in vivo state and are thus implicated in premature suture fusion and in vivo osteoblast differentiation. The second microarray study involved an extensive analysis of 16 suture tissues and compared gene expression between unfused (n=9) and fusing/fused sutures (n=7). Again, both known genes and a substantially large number of novel genes were identified as being differentially expressed. Some of these novel genes included retinol binding protein 4 (RBP4), glypican 3 (GPC3), C1q tumour necrosis factor 3 (C1QTNF3), and WNT inhibitory factor 1 (WIF1). The known functions of these genes are suggestive of potential roles in suture morphogenesis. Realtime quantitative RT PCR (QRT-PCR) was used to verify the differential expression patterns observed for 11 genes and Western blot analysis and confocal microscopy was used to investigate the protein expression for 3 genes of interest. RBP4 was found to be localised on the ectocranial surface of unfused sutures and in cells lining the osteogenic fronts while GPC3 was localised to suture mesenchyme of unfused sutures. A comparison between each unfused suture (coronal, sagittal, metopic, and lambdoid) demonstrated that gene expression profiles are suture-specific which, based on the identification of differentially expressed genes, suggests possible molecular bases for the differential timing of normal fusion and the response of each suture to different craniosynostosis mutations. One observation of particular interest was the presence of cartilage in unfused lambdoid sutures, suggesting a role for chondrogenesis in posterior skull sutures which have generally been thought to develop by intramembranous ossification without a cartilage precursor. Finally, the effects of common media supplements used in in vitro experiments to stimulate differentiation of calvarial suture-derived cells were investigated with respect to their ability to induce in vivo-like gene expression. The response to standard differentiation medium (ascorbic acid + β-glycerophosphate) with and without dexamethasone was measured by both mineralisation and matrix formation assays and QRT-PCR of genes identified in the above described microarray studies. Both media induced collagen matrix and bone nodule formation indicative of differentiating osteoblasts. However, the genes expression profiles induced by both media differed and neither recapitulated the levels and profiles of gene expression observed in vivo for cells isolated from both fused and unfused suture tissues. This study has implications for translating results from in vitro work to the in vivo situation. Significantly, the dedifferentiation microarray study identified differentially expressed genes whose products may be considered candidates as more appropriate osteogenic supplements that may be used during in vitro experiments to better induce in vivo-like osteoblast differentiation. This study has made a substantial contribution to the identification of novel genes and pathways involved in controlling human suture morphogenesis and craniofacial diversity. The results from this research will stimulate new areas of inquiry which will one day aid in the development of better diagnostics and therapeutics for craniosynostosis, and other craniofacial and more general skeletal abnormalities.