Gene Regulation /Function Of Cartilage

软骨的基因调控/功能

• 批准号: 7146108
• 负责人: Yoshihiko Yamada
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7146108
• 关键词: bone development; cartilage; cell differentiation; chondrocytes; chromatin immunoprecipitation; cleft palate; craniofacial; dental development; developmental genetics; ectoderm; gene induction /repression; genetic regulation; genetically modified animals; heparan sulfate; histogenesis; molecular cloning; proteoglycan; tissue /cell culture

This project covers not only cartilage but also tooth and craniofacial development. Our objectives are to define the mechanisms for activating chondrocyte-specific genes and to elucidate the molecular basis of cartilage, tooth, and craniofacial development. We determine the function of protein factors in vivo and in vitro for these tissues using an animal model and cell culture as well as in human disorders. We identify novel genes relevant to tooth and craniofacial development. Oral and Craniofacial Genes Craniofacial birth defects with anomalies of the mouth, neck, and head are of major public concern. Many vertebrate organs begin their development by inductive interactions between an epithelium and a mesenchyme. Tooth development is a classic example of this process and provides a useful experimental system for understanding the molecular mechanisms of organogenesis. The early morphogenetic event of mouse tooth development occurs in the embryo by invagination of the oral ectoderm into the underlying neural crest-derived mesenchyme, which later differentiates into enamel-secreting ameloblasts and dentin secreting odontoblasts. We previously identified a new enamel matrix protein, which we named ameloblastin, by cloning from an incisor cDNA library. To identify in vivo functions of ameloblastin, we created gene knockout mice for ameloblastin. Ameloblastin-deficient mice showed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of differentiation marker factors such as Msx2, p27, and p75 was deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited dental epithelial cell proliferation. In addition, the mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation. We also identified epiprofin, a Kruppel-like-factor containing three characteristic C2H2-type zinc-finger motifs by differential hybridization. Epiprofin is highly cell-type specific, primarily expressed by epithelium of developing tooth, hair follicles and limb bud. To examine the in vivo function of epiprofin, we have created epiprofin knockout mice. Cartilage Genes Cartilage provides mechanical strength to resist compression in joints and also serves as the template for the growth and development of most bones. Cartilage development is initiated by mesenchymal cell condensation followed by a series of chondrocyte maturation processes including resting, proliferative, and hypertrophic chondrocytes. Cartilage contains an extensive extracellular matrix that includes type II, IX, and XI collagens. Type II collagen, a major collagen in cartilage, forms collagen fibrils and provides a structural framework for cartilage matrix. Type XI collagen co-assembles stoichiometrically with type II collagen in the fibrils, whereas type IX collagen is associated with the surface of the fibrils. Type XI collagen is composed of three chains, alpha-1(XI), alpha-2(XI) and alpha-3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. We previously reported that the promoter segment of the alpha-2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. We also identified a protein factor, NT2, that binds the 24-bp sequence of the Col11a2 promoter. NT2 is a zinc-finger protein containing a Kruppel-associated box (KRAB). We found that NT2 is expressed in the mesenchyme cells and hypertrophic chondrocytes, and we demonstrate that NT2 functions as a negative regulator of Col11a2. In collaboration with Dr. Rausher, we demonstrated by chromatin immunoprecipitation experiments that the Col11a2 silencing site is the target site for NT2 and other chromatin-associated repressor proteins, suggesting that NT2 suppresses Col11a2 transcription by binding the Col11a2-promoter via its zinc-finger motif and recruiting the suppressor protein via its KRAB domain. To study the role of NT2 in cartilage development, we prepared a Cre-inducible NT2 activation vector to create conditional ectopic NT2 expression in mice and also a Cre-mediated NT2 targeted vector to create conditional knockout mice. Perlecan, a large heparan sulfate proteoglycan, is present in all basement membranes and some other tissues such as cartilage. We previously created perlecan knockout (perl-/-) mice, which developed severe chondrodysplasia, and the majority of them died immediately after birth due to respiratory failure. We have created transgenic mice expressing recombinant perlecan under the control of the Col2a1 promoter and enhancer and crossed them with perl+/- mice. These mice show normal cartilage development and survive. To identify domains and sequences of perlecan important for cartilage development, we have created several Col2a1-mutant perlecan constructs and created transgenic mice expressing truncated perlecan molecules.

该项目不仅包括软骨，还包括牙齿和颅面发育。我们的目标是明确激活软骨细胞特异性基因的机制，并阐明软骨、牙齿和颅面发育的分子基础。我们通过动物模型和细胞培养，以及在人类疾病中，确定了蛋白质因子在体内和体外对这些组织的作用。我们发现了与牙齿和颅面发育相关的新基因。 口腔和颅面基因 有口腔、颈部和头部异常的头面部出生缺陷是公众主要关注的问题。许多脊椎动物的器官是通过上皮和间质之间的诱导相互作用开始发育的。牙齿发育是这一过程的一个经典例子，为理解器官发生的分子机制提供了一个有用的实验系统。小鼠牙齿发育的早期形态发生事件发生在胚胎中，通过将口腔外胚层内陷到下面的神经沟来源的间质中，该间质后来分化为分泌成釉细胞和分泌牙本质的成牙本质细胞。我们先前通过从切牙cDNA文库中克隆出一种新的釉质基质蛋白，我们将其命名为成釉蛋白。为了鉴定成釉蛋白在体内的功能，我们建立了成釉蛋白基因敲除小鼠。成釉蛋白缺陷小鼠表现出严重的釉质发育不良。在突变牙中，牙上皮细胞分化为分泌釉质的成釉细胞，但细胞从基质中分离，随后失去细胞极性，恢复增殖，形成多细胞层。在突变的成釉细胞中，分化标志因子MSX2、p27和p75的表达被解除调控，其表型逆转为未分化上皮。我们发现重组成釉蛋白能特异性地与成釉细胞黏附，抑制牙上皮细胞的增殖。此外，突变的小鼠还患上了牙源性上皮源性肿瘤。因此，成釉蛋白是成釉细胞发生所必需的一种细胞黏附分子，它通过与成釉细胞结合、抑制细胞增殖来维持分泌期成釉细胞的分化状态。我们还通过差异杂交鉴定了一种含有三个特征C2H2型锌指基序的Kruppel样因子--表普洛林。Epiproin是高度细胞特异性的，主要表达于发育中的牙齿、毛囊和肢芽的上皮细胞。为了检测表普洛林在体内的功能，我们创造了表普洛林基因敲除小鼠。 软骨基因 软骨提供抵抗关节压缩的机械强度，也是大多数骨骼生长和发育的模板。软骨的发育始于间充质细胞的凝聚，随后是一系列软骨细胞的成熟过程，包括静止、增殖和肥大的软骨细胞。软骨含有广泛的细胞外基质，包括II型、IX型和XI型胶原。II型胶原是软骨中的一种主要胶原，形成胶原纤维，为软骨基质提供结构框架。XI型胶原与II型胶原在纤维中以化学计量的方式共同组装，而IX型胶原与纤维表面相关。 XI型胶原由α-1(XI)、α-2(XI)和α-3(XI)三条链组成，在软骨胶原纤维的形成和骨骼形态发生中起关键作用。我们先前报道，α-2(XI)胶原基因的启动子片段(Col11a2)足以进行软骨特异性表达，并且当该序列被置于异源神经丝轻链基因(NFL)启动子中时，该片段的24bp序列能够将启动子活性从神经组织切换到转基因小鼠的软骨。我们还鉴定了一个蛋白因子NT2，它与Col11a2启动子的24个碱基序列结合。NT2是一个含有Kruppel相关框(KRAb)的锌指蛋白。我们发现NT2在间充质细胞和肥大的软骨细胞中都有表达，并且我们证明了NT2是CO11a2的负调控因子。我们与Rausher博士合作，通过染色质免疫沉淀实验证明了Col11a2沉默位点是NT2和其他染色质相关抑制蛋白的靶点，表明NT2通过其锌指基序与Col11a2启动子结合并通过其KRAB结构域招募抑制蛋白来抑制Col11a2的转录。为了研究NT2在软骨发育中的作用，我们制备了Cre诱导的NT2激活载体，用于在小鼠中建立条件性异位表达NT2，并制备了Cre介导的NT2靶向载体，以建立条件性基因敲除小鼠。 Perlecan是一种大的硫酸乙酰肝素蛋白多糖，存在于所有的基底膜和其他一些组织中，如软骨。我们之前创造了Perlecan基因敲除(Perl-/-)小鼠，这种小鼠患上了严重的软骨发育不良，其中大多数在出生后立即死于呼吸衰竭。我们在Col2a1启动子和增强子的控制下建立了表达重组Perlecan的转基因小鼠，并将它们与Perl+/-小鼠杂交。这些小鼠表现出正常的软骨发育和存活。为了确定对软骨发育重要的Perlecan结构域和序列，我们创建了几个Col2a1突变的Perlecan构建物，并创建了表达截短Perlecan分子的转基因小鼠。