Function and regulation of zebrafish nkx2.2a during development of pancreatic islet and ducts

In the mouse Nkx2.2 is expressed in the entire pancreatic anlage. Nevertheless, absence of Nkx2.2 only perturbs the development of endocrine cell types, notably beta-cells which are completely absent. In order to test the possibility that Nkx2.2 might fulfil additional functions during pancreas development we analysed its zebrafish homologue nkx2.2a using gene targeting and GFP-transgenic fish lines. Our results suggest similar roles for nkx2.2a and Nkx2.2 during the development of the endocrine pancreas. Morpholino-based knock-down of nkx2.2a leads to a reduction of alpha- and beta-cell number and an increase of ghrelin-producing cells but, as in mice, does not affect delta-cells. Moreover, like in the mouse, two spatially distinct promoters regulate expression of nkx2.2a in precursors and differentiated islet cells. In addition we found that in zebrafish nkx2.2a is also expressed in the anterior pancreatic bud and, later, in the differentiated pancreatic ducts. A nkx2.2a-transgenic line in which pancreatic GFP expression is restricted to the pancreatic ducts revealed that single GFP-positive cells leave the anterior pancreatic bud and move towards the islet where they form intercellular connections between each other. Subsequently, these cells generate the branched network of the larval pancreatic ducts. Morpholinos that block nkx2.2a function also lead to the absence of the pancreatic ducts. We observed the same phenotype in ptf1a-morphants that are additionally characterized by a reduced number of nkx2.2a-positive duct precursors. Whereas important details of the molecular program leading to the differentiation of endocrine cell types are conserved between mammals and zebrafish, our results reveal a new function for nkx2.2a in the development of the pancreatic ducts.     

Expression of Nkx2-5-GFP bacterial artificial chromosome transgenic mice closely resembles endogenous Nkx2-5 gene activity

Mouse Nkx2-5 gene is essential for early heart development and it is regulated by a complex array of regulatory modules. In order to establish an efficient in vivo system for mapping the Nkx2-5 genomic locus for regulatory regions, we developed improved homologous recombination technology for use in Escherichia coli and then knocked an IRES-hrGFP reporter gene into Nkx2-5 gene in a 120 kb Nkx2-5 bacterial artificial chromosome (BAC) clone. We employed the recombination genes redalpha and redbeta under the pBAD promoter, which was specifically induced by the addition of L-arabinose. Recombination was selected for by our universal targeting cassette which conferred kanamycin resistance in bacterial cells and neomycin resistance in mammalian cells. Transgenic mouse lines generated from this modified BAC clone closely resembled the endogenous Nkx2-5 expression in the heart, pylorus sphincter, and spleen, but expression was not detected in the tongue. Nkx2-5 BAC-GFP expression was copy number-dependent and locus site-independent. BAC transgenics using the GFP reporter offers an efficient model system to study gene expression and regulation.     

Two highly related homeodomain proteins, Nkx5-1 and Nkx5-2, display different DNA binding specificities.

The mouse Nkx5-1 and Nkx5-2 genes are related to NK genes in Drosophila and encode proteins with very similar homeodomains. In higher vertebrates Nkx5 genes are specifically expressed in the inner ear. Inactivation of the mouse Nkx5-1 gene by homologous recombination revealed a critical role for the formation of vestibular inner ear structures. Here, we investigated biochemical properties of the proteins encoded by the Nkx5 genes. A similar consensus binding sequence was isolated for both Nkx5 proteins using binding site selection. This sequence is related to target sequences previously identified for other Nkx proteins and contains the conserved homeodomain binding core TAAT. An additional, novel and unrelated high affinity binding sequence could be identified for the Nkx5-2 protein.     

单纯性先天性心脏病中TBX5基因表达异常的机制

为探讨人类单纯性先天性心脏病患者中TBX5基因表达下调的可能原因, 应用变性高效液相色谱(DHPLC)方法检测100例单纯性先天性心脏病患者中TBX5基因上游1 200 bp调控区的突变情况; 应用甲基化敏感性限制性内切酶(MS-RE)法检测50例单纯性先天性心脏病患者和5例非先天性心脏病患者心肌组织TBX5基因启动子区两个CpG岛(转录起始点上游-49~-188 bp和-247~-464 bp处)的甲基化情况; 应用P-match软件预测小鼠Tbx5基因上游转录因子Nkx2-5的结合位点, 构建Nkx2-5表达载体转染小鼠H9C2(2-1)心肌细胞, RT-PCR及Western blotting检测Tbx5基因表达, 凝胶阻滞实验(EMSA)验证Nkx2-5和Tbx5基因的作用。结果在100例单纯性先天性心脏病患者中, 未检测到TBX5基因上游1 200 bp调控区突变; 非先天性心脏病患者和单纯性先天性心脏病患者在两个CpG岛存在相同的甲基化; 小鼠Tbx5基因转录起始点上游-312~-315 bp可能存在Nkx2-5的结合位点, 转染Nkx2-5表达载体后Tbx5基因在mRNA及蛋白质水平均有表达增高趋势, Nkx2-5在体外可以与Tbx5基因上游-312~-315 bp序列相结合。以上结果提示TBX5基因调控区突变和两个CpG岛的甲基化不是单纯性先天性心脏病患者心肌组织中TBX5基因表达下调的原因, TBX5基因表达下调可能由于NKX2-5的表达异常引起。     

Expression pattern of novel chick T-box gene, Tbx20

Little is known about the molecular mechanisms involved with the initial specifications of the cardiac mesoderm. In order to identify potential regulatory factors that play important roles in early heart specification, we attempted to isolate the chick H15-related T-box gene and analyze its expression pattern during early development. The chick Tbx20 gene was found to be highly homologous to human, mouse, and zebrafish hrT/Tbx20. Its expression was initially detected in the posterior lateral mesoderm, after which it expanded to the anterior and was intensively co-expressed with a cardiogenic gene, Nkx2.5, in the anterior lateral mesoderm.