Isolation and characterisation of GTF2IRD2, a novel fusion gene and member of the TFII-I family of transcription factors, deleted in Williams-Beuren syndrome

Williams-Beuren syndrome (WBS) is a developmental disorder with characteristic physical, cognitive and behavioural traits caused by a microdeletion of approximately 1.5 Mb on chromosome 7q11.23. In total, 24 genes have been described within the deleted region to date. We have isolated and characterised a novel human gene, GTF2IRD2, mapping to the WBS critical region thought to harbour genes important for the cognitive aspects of the disorder. GTF2IRD2 is the third member of the novel TFII-I family of genes clustered on 7q11.23. The GTF2IRD2 protein contains two putative helix-loop-helix regions (I-repeats) and an unusual C-terminal CHARLIE8 transposon-like domain, thought to have arisen as a consequence of the random insertion of a transposable element generating a functional fusion gene. The retention of a number of conserved transposase-associated motifs within the protein suggests that the CHARLIE8-like region may still have some degree of transposase functionality that could influence the stability of the region in a mechanism similar to that proposed for Charcot-Marie-Tooth neuropathy type 1A. GTF2IRD2 is highly conserved in mammals and the mouse ortholgue (Gtf2ird2) has also been isolated and maps to the syntenic WBS region on mouse chromosome 5G. Deletion mapping studies using somatic cell hybrids show that some WBS patients are hemizygous for this gene, suggesting that it could play a role in the pathogenesis of the disorder.     

Expression survey of genes critical for tooth development in the human embryonic tooth germ.

In the developing murine tooth, the expression patterns of numerous regulatory genes have been examined and their roles have begun to be revealed. To unveil the molecular mechanisms that regulate human tooth morphogenesis, we examined the expression patterns of several regulatory genes, including BMP4, FGF8, MSX1, PAX9, PITX2, and SHOX2, and compared them with that found in mice. All of these genes are known to play critical roles in murine tooth development. Our results show that these genes exhibit basically similar expression patterns in the human tooth germ compared with that in the mouse. However, slightly different expression patterns were also observed for some of the genes at certain stages. For example, MSX1 expression was detected in the inner enamel epithelium in addition to the dental mesenchyme at the bell stage of the human tooth. Moreover, FGF8 expression remained in the dental epithelium at the cap stage, while PAX9 and SHOX2 expression was detected in both dental epithelium and mesenchyme of the human tooth germ. Our results indicate that, although slight differences exist in the gene expression patterns, the human and mouse teeth not only share considerable homology in odontogenesis but also use similar underlying molecular networks.     

Expression of claudins in murine tooth development.

Claudins belong to a family of transmembrane proteins that were identified as components of tight junction strands. We carried out comparative in situ hybridization analysis of 11 claudin genes (claudin1 - claudin11) during murine odontogenesis from the formation of the epithelial thickening to the cytodifferentiation stage. We identify dynamic spatiotemporal expression of 9 of the 11 claudins. At the early bell stage, two claudins (claudin1 and 4) are specifically expressed in stratum intemedium, whereas only one claudin is expressed in each of the preameloblasts (claudin2) and preodontoblasts (claudin10). At the bud stage, when the first epithelial differentiation pathways are being established, localized expression of six claudins (claudin1, 3, 4, 6, 7, and 10) identify spatial specific interactions, suggesting a hitherto unobserved complexity of epithelial organization, within the early tooth primordium.     

A biallelic ANTXR1 variant expands the anthrax toxin receptor associated phenotype to tooth agenesis

Tooth development is regulated by multiple genetic pathways, which ultimately drive the complex interactions between the oral epithelium and mesenchyme. Disruptions at any time point during this process may lead to failure of tooth development, also known as tooth agenesis (TA). TA is a common craniofacial abnormality in humans and represents the failure to develop one or more permanent teeth. Many genes and potentially subtle variants in these genes contribute to the TA phenotype. We report the clinical and genetic impact of a rare homozygous ANTXR1 variant (c.1312C>T), identified by whole exome sequencing (WES), in a consanguineous Turkish family with TA. Mutations in ANTXR1 have been associated with GAPO (growth retardation, alopecia, pseudoanodontia, and optic atrophy) syndrome and infantile hemangioma, however no clinical characteristics associated with these conditions were observed in our study family. We detected the expression of Antxr1 in oral and dental tissues of developing mouse embryos, further supporting a role for this gene in tooth development. Our findings implicate ANTXR1 as a candidate gene for isolated TA, suggest the involvement of specific hypomorphic alleles, and expand the previously known ANTXR1‐associated phenotypes.

     

Immunohistochemical examination for the distribution of podoplanin-expressing cells in developing mouse molar tooth germs

We recently reported the expression of podoplanin in the apical bud of adult mouse incisal tooth. This study was aimed to investigate the distribution of podoplanin-expressing cells in mouse tooth germs at several developing stages. At the bud stage podoplanin was expressed in oral mucous epithelia and in a tooth bud. At the cap stage podoplanin was expressed on inner and outer enamel epithelia but not in mesenchymal cells expressing the neural crest stem cell marker nestin. At the early bell stage nestin and podoplanin were expressed in cervical loop and odontoblasts. At the root formation stage both nestin and podoplanin were weakly expressed in odontoblasts generating radicular dentin. Podoplanin expression was also found in the Hertwig epithelial sheath. These results suggest that epithelial cells of developing tooth germ acquire the ability to express nestin, and that tooth germ epithelial cells maintain the ability to express podoplanin in oral mucous epithelia. The expression of podoplanin in odontoblasts was induced as tooth germ development advanced, but was suppressed with the completion of the primary dentin, suggesting that podoplanin may be involved in the cell growth of odontoblasts. Nestin may function as an intermediate filament that binds podoplanin in odontoblasts.     

Autosomal dominant supravalvular aortic stenosis: large three-generation family

Supravalvular aortic stenosis (SVAS) can be inherited as an isolated autosomal dominant trait or can be a component manifestation of the Williams syndrome. Some consider the Williams syndrome to be due to more severe expression of the gene defect that causes isolated SVAS. We describe a family with isolated SVAS that is the largest thoroughly studied family with this disorder to our knowledge; no patients in this family had Williams syndrome. Five members of this family were reported by Lewis et al. (Dis Chest 55:372-379, 1969). We reevaluated this family and now include examinations of the parents, additional sibs and children of the original 5 patients. Twenty relatives had physical and echocardiographic examinations. In addition, information from outside sources was obtained on 7 relatives not personally evaluated. The SVAS showed marked variability of expression and was not associated with mental retardation or with the facial manifestations of Williams syndrome. We think that previous reports of Williams syndrome reputedly occurring within the same family as isolated autosomal dominant SVAS were inadequately documented. Based on our family and review of the literature, we suggest that isolated SVAS and Williams syndrome represent clinically distinct entities.     

Evaluation of candidate genes for familial brachydactyly.

Type A1 brachydactyly in humans is a recognisable syndrome characterised by shortening of the middle phalanx of all digits with occasional fusion of the middle and terminal phalanges. The purpose of this study was to evaluate candidate genes for type A1 brachydactyly in two families with multiple affected members. Several classes of genes have been implicated in the control of distal limb development including homeobox containing genes (MSX1, MSX2) some members of the homeobox gene family, and genes encoding growth factors of the FGF, TGF, and PDGF families. Homeobox (Hox) genes are a family of developmental control genes activated early in embryogenesis that encode positional information along the anterior-posterior body axis and specify distinct spatial domains within developing limbs. Growth factor genes can regulate the proliferation and differentiation of various embryonic structures including limb buds and have been shown to influence Hox gene expression. Candidate genes HOXD, MSX1, MSX2, FGF-1, and FGF-2 were excluded in one family. The brachydactyly type A1 gene or locus was not found in either of the two families studied.     

Immunolocalization of Alk8 during replacement tooth development in zebrafish

The novel type I transforming growth factor-beta (TGF-beta) family member receptor Alk8 was previously identified in a degenerate RT-PCR screen for zebrafish type I and II TGF-beta family member receptors. Functional analyses revealed that Alk8 acts through Bmp signaling pathways in early embryonic dorsoventral patterning, in neural crest cell specification, and in patterning and differentiation of neural crest cell-derived pharyngeal arch cartilages. In addition, Alk8 forms active signaling complexes with TGF-beta1 and the TGF-beta RII receptor, suggesting that Alk8 mediates cross talk between Bmp and TGF-beta subfamily members. In this study, immunohistochemical analysis was performed on zebrafish aged 2 days postfertilization to 1 year, revealing immunolocalization of Alk8 to tissues of the tooth-bearing ceratobranchial 5 (cb5) arch including dental epithelial and mesenchymal tooth tissues of developing primary and replacement teeth, mucous-producing crypt epithelium, keratinized bite plate, and developing taste buds. These results suggest roles for Alk8 in patterning tooth-bearing pharyngeal epithelium, in the initiation of tooth development, in odontoblast and ameloblast differentiation, and in osteoblast maturation. The ability for zebrafish to continuously form teeth throughout their lives allows for the comparison of Alk8 expression in both primary and replacement tooth development, revealing identical Alk8 expression profiles. This study advances our current understanding of the functions of Alk8, particularly with respect to primary and replacement tooth formation, reveals additional roles for Alk8 in dental epithelial patterning and in odontoblast, ameloblast and osteoblast differentiation, and demonstrates the utility of the zebrafish as a model for primary and replacement tooth development.     

Expression analysis of RSK gene family members: the RSK2 gene,mutated in Coffin-Lowry syndrome,is prominently expressed in brain structures essential for cognitive function and learning

Coffin-Lowry syndrome (CLS) is characterized by cognitive impairment, characteristic facial and digital findings and skeletal anomalies. The gene implicated in CLS encodes RSK2, a serine/threonine kinase acting in the Ras/MAPK signalling pathway. In humans, RSK2 belongs to a family of four highly homologous proteins (RSK1-RSK4), encoded by distinct genes. RSK2 mutations in CLS patients are extremely heterogeneous. No consistent relationship between specific mutations and the severity of the disease or the expression of uncommon features has been established. Together, the data suggest an influence of environmental and/or other genetic components on the presentation of the disease. Obvious modifying genes include those encoding other RSK family members. In this study we have determined the expression of RSK1, 2 and 3 genes in various human tissues, during mouse embryogenesis and in mouse brain. The three RSK mRNAs were expressed in all human tissues and brain regions tested, supporting functional redundancy. However, tissue specific variations in levels suggest that they may also serve specific roles. The mouse Rsk3 gene was prominently expressed in the developing neural and sensory tissues, whereas Rsk1 gene expression was the strongest in various other tissues with high proliferative activity, suggesting distinct roles during development. In adult mouse brain, the highest levels of Rsk2 expression were observed in regions with high synaptic activity, including the neocortex, the hippocampus and Purkinje cells. These structures are essential components in cognitive function and learning. Based on the expression levels, our results suggest that in these areas, the Rsk1 and Rsk3 genes may not be able to fully compensate for a lack of Rsk2 function.     

牙胚细胞体外培养过程中成牙基因的表达

背景：多项实验表明牙胚组织在不同的时间点有不同的基因发挥作用,共同促进牙胚发育。 目的：观察牙本质基质蛋白1、成釉蛋白、Ⅰ型胶原蛋白和同源异型盒基因1在大鼠牙胚细胞体外培养的不同时间的表达。 方法：对体外培养后第1,3,6天的牙胚细胞提取RNA,反转录后采用实时定量PCR的方法检测牙本质基质蛋白1、成釉蛋白、Ⅰ型胶原蛋白和同源异型盒基因1mRNA相对表达水平的变化。 结果与结论：牙胚细胞中牙本质基质蛋白1、成釉蛋白和Ⅰ型胶原蛋白mRNA的表达随培养时间的延长而增加,在培养3 d时达到峰值(P < 0.05),而同源异型盒基因1 mRNA的表达随培养时间的延长而下降(P < 0.05)。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Microdeletion 20p12.3 involving BMP2 contributes to syndromic forms of cleft palate

Orofacial clefts of the lip and/or palate comprise one of the most common craniofacial birth defects in humans. Though a majority of cleft lip and/or cleft palate (CL/P) occurs as isolated congenital anomalies, there exist a large number of Mendelian disorders in which orofacial clefting is part of the clinical phenotype. Here we report on two individuals and one multi-generational family with microdeletions at 20p12.3 that include the bone morphogenetic protein 2 (BMP2) gene. In two propositi the deletion was almost identical at ～600 kb in size, and BMP2 was the only gene deleted; the third case had a ～5.5-Mb deletion (20p13p12.2) that encompassed at least 20 genes including BMP2. Clinical features were significant for cleft palate and facial dysmorphism in all three patients, including Pierre-Robin sequence in two. Microdeletion 20p13p12 involving BMP2 is rare and has been implicated in Wolff-Parkinson-White (WPW) syndrome with neurocognitive deficits and with Alagille syndrome when the deletion includes the neighboring JAG1 gene in addition to BMP2. Despite a significant role for the BMPs in orofacial development, heterozygous loss of BMP2 has not been previously reported in patients with syndromic clefting defects. Because BMP2 was the sole deleted gene in Patients 1 and 2 and one of the genes deleted in Patient 3, all of whom had clinical features in common, we suggest that haploinsufficiency for BMP2 is a crucial event that predisposes to cleft palate and additional anomalies. Lack of significant phenotypic components in family members of Patient 1 suggests variable expressivity for the phenotype.     

Isolation of a new homeobox gene belonging to the Pitx/Rieg family: expression during lens development and mapping to the aphakia region on mouse chromosome 19

We recently reported the positional cloning of a homeobox gene involved in the pathogenesis of Rieger syndrome, RIEG1 , and its mouse homolog, Rieg1 . Rieg1 (also independently described as Pitx2) is highly homologous to the Ptx1/Potx gene product, suggesting that there may be additional members of this novel Pitx family. The Pitx genes play an important role in eye, tooth, pituitary and umbilical region development as evidenced by Rieger syndrome and iris hypoplasia phenotypes, resulting from mutations in the RIEG1 gene and by expression studies. In order to characterize further the Pitx gene family we searched mouse cDNA libraries to identify additional members. A new gene was isolated which encodes a homeoprotein with strong homology to the other Pitx proteins and 97-100% identity in the homeodomain itself, suggesting that this is a third member of the family, Pitx3 . In whole mount in situ hybridization on mouse embryos ranging from 8.5 to 11.5 days post-coitum (d.p.c.), Pitx3 mRNA was seen only in the developing lens starting at day 11. Hybridization on cross- sections revealed strong signals in the lens vesicle in 11 d.p.c. embryos and throughout the lens, particularly in the anterior epithelium and equator region in 15 d.p.c. embryos. Pitx3 was mapped close to aphakia on mouse chromosome 19. The aphakia homozygous mouse is characterized by small eyes lacking a lens, which fail to develop beyond 11 d.p.c. These data make Pitx3 a strong candidate gene for the aphakia phenotype in the mouse and suggest a role for the human homolog in congenital lens malformations.      

Application of lentivirus-mediated RNAi in studying gene function in mammalian tooth development.

RNA interference (RNAi) has recently become a powerful tool to silence gene expression in mammalian cells, but its application in assessing gene function in mammalian developing organs remains highly limited. Here we describe several unique developmental properties of the mouse molar germ. Embryonic molar mesenchyme, but not the incisor mesenchyme, once dissociated into single cell suspension and re-aggregated, retains its odontogenic potential, the capability of a tissue to instruct an adjacent tissue to initiate tooth formation. Dissociated molar mesenchymal cells, even after being plated in cell culture, retain odontogenic competence, the capability of a tissue to respond to odontogenic signals and to support tooth formation. Most interestingly, while dissociated epithelial and mesenchymal cells of molar tooth germ are mixed and re-aggregated, the epithelial cells are able to sort out from the mesenchymal cells and organize into a well-defined dental epithelial structure, leading to the formation of a well-differentiated tooth organ after sub-renal culture. These unique molar developmental properties allow us to develop a strategy using a lentivirus-mediated RNAi approach to silence gene expression in dental mesenchymal cells and assess gene function in tooth development. We show that knockdown of Msx1 or Dlx2 expression in the dental mesenchyme faithfully recapitulates the tooth phenotype of their targeted mutant mice. Silencing of Barx1 expression in the dental mesenchyme causes an arrest of tooth development at the bud stage, demonstrating a crucial role for Barx1 in tooth formation. Our studies have established a reliable and rapid assay that would permit large-scale analysis of gene function in mammalian tooth development.