Gene Regulation /Function Of Cartilage

这个项目不仅包括软骨，还包括牙齿和颅面发育。我们的目标是确定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。我们使用动物模型和细胞培养以及在人类疾病中确定这些组织的蛋白质因子在体内和体外的功能。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 颅面出生缺陷与口腔、颈部和头部的异常是公众关注的主要问题。许多脊椎动物器官开始它们的发育是通过上皮细胞和间充质细胞之间的诱导相互作用。牙齿发育是这一过程的一个典型例子，并为了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，通过口腔外胚层内陷到下面的神经嵴来源的间充质中，其随后分化成分泌釉质的成釉细胞和分泌牙本质的成牙本质细胞。我们以前确定了一个新的釉基质蛋白，我们命名为成釉蛋白，从切牙cDNA文库克隆。为了鉴定成釉蛋白的体内功能，我们建立了成釉蛋白基因敲除小鼠。成釉细胞蛋白缺陷小鼠表现出严重的釉质发育不全。在突变牙中，牙上皮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，并形成多细胞层。在突变成釉细胞中，分化标记因子如Msx2、p27和p75的表达失调，其表型逆转为未分化上皮。我们发现，重组成釉蛋白特异性地粘附于成釉细胞，并抑制牙上皮细胞增殖。此外，突变小鼠发展成牙上皮起源的牙源性肿瘤。因此，成釉蛋白是成釉细胞形成所必需的细胞粘附分子，其通过与成釉细胞结合并抑制增殖而在维持分泌期成釉细胞的分化状态中起作用。我们还确定epiprofin，Kruppel样因子含有三个特征C2H2型锌指基序的差异杂交。Epiprofin具有高度的细胞类型特异性，主要在发育中的牙齿、毛囊和肢芽的上皮中表达。为了检查表普罗芬的体内功能，我们创建了表普罗芬敲除小鼠。 软骨素基因 软骨提供机械强度以抵抗关节中的压缩，并且还充当大多数骨骼生长和发育的模板。软骨发育是由间充质细胞凝聚，随后是一系列软骨细胞成熟过程，包括静止，增殖和肥大的软骨细胞。软骨含有大量的细胞外基质，包括II、IX和Xi型胶原。II型胶原是软骨中的主要胶原，形成胶原原纤维并为软骨基质提供结构框架。Xi型胶原与原纤维中的II型胶原化学计量地共组装，而IX型胶原与原纤维的表面缔合。 Xi型胶原蛋白由三条链α-1（Xi）、α-2（Xi）和α-3（Xi）组成，并且在软骨胶原原纤维的形成和骨骼形态发生中起关键作用。我们以前报道，α-2（Xi）胶原基因（Col11a2）的启动子片段足以用于软骨特异性表达，并且当将该序列置于异源神经丝轻基因（NFL）启动子中时，来自该片段的24 bp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。我们还确定了一个蛋白质因子，NT 2，它结合了Col11a2启动子的24 bp序列。NT2是一种含有Kruppel相关盒（KRAB）的锌指蛋白。我们发现，NT 2在间充质细胞和肥大软骨细胞中表达，我们证明NT 2作为Col11a2的负调节剂发挥作用。与Rausher博士合作，我们通过染色质免疫沉淀实验证明了Col11a2沉默位点是NT 2和其他染色质相关阻遏蛋白的靶位点，这表明NT 2通过锌指基序结合Col11a2启动子并通过其KRAB结构域招募抑制蛋白来抑制Col11a2转录。为了研究NT 2在软骨发育中的作用，我们制备了Cre诱导的NT 2激活载体以在小鼠中产生条件性异位NT 2表达，并且还制备了Cre介导的NT 2靶向载体以产生条件性敲除小鼠。 串珠素是一种大的硫酸乙酰肝素蛋白多糖，存在于所有基底膜和一些其他组织如软骨中。我们以前创造了串珠素基因敲除（perl-/-）小鼠，这些小鼠出现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。我们已经创建了在Col2a1启动子和增强子的控制下表达重组串珠素的转基因小鼠，并将它们与perl +/-小鼠杂交。这些小鼠显示正常的软骨发育并存活。为了鉴定对软骨发育重要的串珠素的结构域和序列，我们已经创建了几个Col2a1突变串珠素构建体，并创建了表达截短串珠素分子的转基因小鼠。

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.