Gene Regulation And Function Of Cartilage

该项目不仅涵盖软骨，还涵盖牙齿和颅面发育。我们的目标是定义软骨细胞分化的机制并阐明软骨、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人类疾病来确定蛋白质因子在体内和体外对这些组织的功能。我们鉴定了与软骨、牙齿和颅面发育相关的新基因。 口腔和颅面基因 伴有口腔、颈部和头部异常的颅面出生缺陷是公众主要关注的问题。许多脊椎动物器官通过上皮和间质之间的诱导相互作用开始发育。牙齿发育是这一过程的典型例子，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，口腔外胚层内陷到下面的神经嵴衍生间充质中，随后分化为分泌牙釉质的成釉细胞和分泌牙本质的成牙本质细胞。 我们使用牙胚 cDNA 微阵列通过差异杂交鉴定了几个优先与牙胚 mRNA 杂交的新基因。其中一个克隆，我们将其命名为 Epiprofin，编码 KLF/Sp 锌指家族的成员以及 Sp6 的同源物。原位杂交表明，epiprofin mRNA 由增殖的牙上皮和毛囊基质上皮表达。 Epiprofin还在发育中的肢体和生殖器官的顶端外胚层嵴中短暂表达。表洛芬表达载体的转染表明，表洛芬促进牙上皮分化为成釉细胞表型，并增强釉质基质特异性成釉细胞蛋白基因的启动子活性。为了确定表洛芬在外胚层器官发育中的作用，我们创建了表洛芬基因敲除小鼠。我们的初步数据表明，基因敲除小鼠存活时生长迟缓，并出现牙釉质发育不全、牙齿数量过多（牙齿过多）、毛发形成严重缺陷、表皮增厚和手指融合。这些结果表明表洛芬具有多种功能，对于这些外胚层器官的发育至关重要。 在牙齿发育的早期阶段，基底膜将最终形成牙釉质和牙本质的两个关键组织分开，但基底膜的确切作用及其参与牙齿形态发生的潜在机制尚不清楚。层粘连蛋白α-5是层粘连蛋白-10/11的组成部分，是牙齿基底膜中主要的层粘连蛋白α链。我们使用层粘连蛋白 α-5 基因敲除小鼠研究了层粘连蛋白 α-5 在早期牙齿中的作用。突变小鼠发育出没有牙尖的小牙胚，其中内牙上皮没有极化，牙釉质结形成有缺陷。在正常小鼠中，层粘连蛋白α-5与与基底膜接触的上皮上的细胞表面受体整合素α-6/β-4相互作用，而在突变小鼠中，在缺乏层粘连蛋白α-5的情况下，牙齿上皮的细胞极性丢失，阻止与整合素受体的相互作用并导致牙齿发育畸形。我们发现层粘连蛋白α-5是细胞正常增殖、扩散和丝状伪足样微刺形成所必需的，这些都与细胞极性有关，并且我们阐明了参与这些过程的细胞信号分子。我们还发现层粘连蛋白 10/11 促进牙上皮的扩散和丝状伪足样微刺的形成，并且层粘连蛋白 α-5 和整合素 α-6/β-4 的相互作用通过 PI 3 激酶-Cdc42/Rac 信号传导介导这些细胞变化。这些结果表明，层粘连蛋白α-5对于牙上皮的增殖和分化至关重要，并表明基底膜中的层粘连蛋白α-5与上皮细胞表面上的整合素α-6/β-4相互作用，以调节牙胚的大小和形状。 软骨基因 软骨是一种高度专业化的结缔组织，含有广泛的细胞外基质，并提供机械强度以抵抗关节的压缩。在发育过程中，软骨充当大多数骨骼生长和发育的模板。在软骨细胞分化过程中，一组独特的 ECM 分子（包括 II 型、XI 型和 X 型胶原蛋白、连接蛋白和基底膜聚糖）以时间和位置特异性方式表达。软骨提供机械强度以抵抗关节的压缩，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结启动的，随后是一系列软骨细胞成熟过程，包括静息、增殖和肥大软骨细胞。 基底膜聚糖是一种硫酸乙酰肝素蛋白多糖，存在于所有基底膜以及没有基底膜的软骨中。我们之前表明，小鼠和人类的基底膜聚糖缺乏会导致围产期致命的软骨发育不良，这表明基底膜聚糖对于软骨发育至关重要。然而，基底膜蛋白聚糖在软骨发育中的功能尚不清楚。我们提出基底膜聚糖在正常软骨发育中至少有两个潜在功能：1.调节生长因子活性，例如FGF/FGFR3c，2.在肥厚区形成细胞外基质。 FGFR3c 是软骨细胞特异的 FGF 受体，通过抑制软骨细胞增殖和 Indian Hedgehog (Ihh) 的表达来调节软骨发育。 FGFR3c 的激活突变会导致致死性发育不良（最常见的人类致死性软骨发育不良），导致增殖区减少和生长板缩短，与基底膜蛋白缺失小鼠类似，而 Fgfr3 缺失小鼠则形成相反的表型，即增殖区和肥大区扩大并存活。为了测试基底膜聚糖对 Fgfr3c 活性的调节，我们使用 KO 小鼠的肢体器官培养物和 Fgfr 抑制剂。我们还创建了基底膜蛋白和 Fgfr3 双 KO 小鼠，以检查基底膜蛋白 KO 软骨的异常表型是否可以在 Fgfr3 缺失的情况下恢复。我们的数据表明，抑制 KO 小鼠生长板软骨中的 Fgfr 会增加软骨细胞增殖并恢复表达 Ihh 的肥大前软骨细胞区，但它们无法分化为肥大软骨细胞。这些结果表明，在正常软骨发育中，基底膜聚糖可能通过将 Fgf 捕获在基质中来抑制 Fgfr3c 活性。基底膜聚糖对 Fgfr3c 活性的调节使得生长板具有适当的尺寸和扩张。此外，基底膜聚糖在肥厚区的基质形成中起着关键作用。如果没有基质，细胞就无法形成有组织的柱状结构，并且无法很好地分化。由于基底膜聚糖主要位于肥大软骨细胞的细胞周间隙，因此基底膜聚糖可能与 ECM 和细胞表面受体相互作用，并稳定 ECM 和细胞的相互作用，从而在肥大区形成有组织的柱状细胞结构。

This project covers not only cartilage but also tooth and craniofacial development. Our objectives are to define the mechanisms of chondrocyte differentiation 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 animal models and cell culture, as well as in human disorders. We identify novel genes relevant to cartilage, 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 identified several new genes that were preferentially hybridized to tooth germ mRNA by differential hybridization using tooth germ cDNA micorroarrays. One of the clones, which we named epiprofin, encodes a member of the KLF/Sp zinc-finger family and homologue to Sp6. In situ hybridization revealed that epiprofin mRNA is expressed by proliferating dental epithelium and also hair follicle matrix epithelium. Epiprofin is also transiently expressed in the apical ectodermal ridge in developing limb and genital organ. Transfection of an epiprofin expression vector showed that epiprofin promotes differentiation of dental epithelium into an ameloblast phenotype and enhances promoter activity of the enamel matrix-specific ameloblastin gene. To determine the role of epiprofin in ectodermal organ development, we have created gene knockout mice for epiprofin. Our preliminary data indicate that knockout mice survive with growth retardation and develop with enamel hypoplasia, an excess number of teeth (hyperdontia), severe defects in hair formation, thickening of epidermis, and digit fusions. These results suggest diverse functions of epiprofin that are essential for the development of these ectodermal organs. At the early stage of tooth development, the basement membrane separates two key tissues that ultimately form enamel and dentin, but the precise role of the basement membrane and the underlying mechanism of its involvement in tooth morphogenesis are not clear. Laminin alpha-5, a component of laminin-10/11, is the major laminin alpha chain in tooth basement membrane. We examined the role of laminin alpha-5 in early tooth using gene knockout mice for laminin alpha-5. Mutant mice develop a small tooth germ with no cusps, in which the inner dental epithelium is not polarized and enamel knot formation is defective. In normal mice, laminin alpha-5 interacts with a cell surface receptor integrin alpha-6/beta-4 on the epithelium that is in contact with the basement membrane, whereas in mutant mice, cell polarity of the dental epithelium is lost in the absence of laminin alpha-5, preventing the interaction with integrin receptors and leading to deformities in tooth development. We found that laminin alpha-5 is required for proper cell proliferation, spreading, and filopodia-like microspike formation, which are involved in cell polarity, and we elucidated the cell signaling molecules involved in these processes. We also found that laminin 10/11 promotes spreading and filopodia-like microspike formation of dental epithelium and that the interaction of laminin alpha-5 and integrin alpha-6/beta-4 mediates these cellular changes through PI 3 kinase-Cdc42/Rac signaling. These results demonstrate that laminin alpha-5 is critical for the proliferation and differentiation of the dental epithelium and suggest that laminin alpha-5 in the basement membrane interacts with integrin alpha-6/beta-4 on the epithelial-cell surface to regulate the size and shape of the tooth germ. Cartilage Genes Cartilage, a highly specialized connective tissue, contains an extensive extracellular matrix, and provides mechanical strength to resist compression in joints. In development, cartilage serves as the template for the growth and development of most bones. During chondrocyte differentiation, a unique set of ECM molecules including type II, XI and X collagens, link protein, and perlecan, are expressed in a temporal and location-specific manner. 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. Perlecan, a heparan sulfate proteoglycan, is present in all basement membranes and also in cartilage where there is no basement membrane. We previously showed that perlecan deficiency in mice and humans causes perinatal lethal chondrodysplasia, indicating that perlecan is essential for cartilage development. However, the functions of perlecan in cartilage development are unknown. We propose at least two potential functions of perlecan in normal cartilage development: 1. modulation of growth factor activity, such as FGF/FGFR3c, and 2. formation of the extracellular matrix in the hypertrophic zone. FGFR3c, the FGF receptor specific to chondrocytes, regulates cartilage development by inhibiting chondrocyte proliferation and expression of Indian hedgehog (Ihh). Activating mutations of FGFR3c, which cause Thanatophoric dysplasia, the most common human lethal chondrodysplasia, result in a reduced proliferative zone and a shortened growth plate, similar to perlecan-null mice, whereas Fgfr3-null mice develop an opposite phenotype, i.e., expansion of the proliferative and hypertrophic zones and survival. To test the modulation of Fgfr3c activity by perlecan, we used limb organ cultures from KO mice and Fgfr inhibitors. We also created double-KO mice for perlecan and Fgfr3 to examine if the abnormal phenotypes of perlecan KO cartilage can be restored in the absence of Fgfr3. Our data indicated that the inhibition of Fgfr in the growth plate cartilage of KO mice increased chondrocyte proliferation and restored the Ihh-expressing prehypertrophic chondrocyte zone, but they failed to differentiate into hypertrophic chondrocytes. These results suggest that in normal cartilage development, perlecan inhibits Fgfr3c activity, probably by trapping Fgf in the matrix. The modulation of Fgfr3c activity by perlecan allows the appropriate size and expansion of the growth plate. Further, perlecan plays a critical role in matrix formation in the hypertrophic zone. Without the matrix, cells fail to form an organized columnar structure and cannot differentiate well. Since perlecan is predominantly located in the pericellular space of hypertrophic chondrocytes, it is possible that perlecan interacts with both ECM and a cell surface receptor and stabilizes the interaction of ECM and cells to form organized columnar cell structure in the hypertrophic zone.