ROLE OF EXTRACELLULAR MATRIX REMODELING IN MANDIBULAR MORPHOGENESIS

发育生物学的一个主要问题是， 指令在胚胎发生期间被翻译成形式。 第一 鳃弓经历下颚结构的形态发生， 包括下颌骨软骨和骨骼 从胚胎第10天到第14天。 在E10组织中， 形态建成，并在随后的培养9天形成所有的 组织的下颌骨在无血清，化学成分确定的培养基。 因此，该系统是确定当地因素的理想选择， 调节骨和软骨的形态发生。 细胞外基质 重塑被认为在生长中起重要作用， 结缔组织的发育。 这项建议的目的是 确定细胞外基质重塑是否由 金属蛋白酶及其抑制剂，这是已知的主要 骨和软骨的组成部分，在形成和形成中起作用。 骨和软骨的细胞外基质成分在 下颌骨发育 为了验证这一假设，必须获得 金属蛋白酶表达的定量和定性数据 MMPs和金属蛋白酶抑制剂（TIMPs） 在体内和培养中的发育。 该方法将起诉RT-PCR， 蛋白酶和抑制剂mRNA转录本的初步鉴定， 然后通过酶和免疫组织化学分析这些基因的蛋白质产物， 免疫学手段。 一旦这些MMPs和TIMPs被鉴定出来， 他们的时间表达模式的定义，表达选定的 MMP和TIMP将通过以下方法定位于软骨形成和/或成骨病灶： 体内发育过程中原位杂交和免疫细胞化学 和文化。 那么这些MMPs和TIMPs的功能将是 研究了 一种方法将涉及添加纯化的或重组的 MMPs或TIMPs的下颌骨培养，看看这些干扰形态发生。 第二种方法将涉及产生亚纯型表型， MMPs或TIMPs的阻断抗体或反义寡核苷酸， 如果这些影响形态发生。 第三种方法将涉及调制 下颌骨的生长和形态发生 或通过抗体消除这些因子 或反义寡核苷酸，然后测定它们的作用 对MMP和TIMP表达的影响，以及对MMP和TIMP表达的时间、位置和表达的影响。 软骨形成和骨形成。 这些实验应该会导致 了解参与细胞外 骨和软骨形态发生的基质方面，一般而言， 特别是下颌骨，并导致新的治疗策略， 下颌骨畸形和骨软骨修复。

A major issue in developmental biology is how the temporal and spatial instructions are translated to form during embryogenesis. The first branchial arch undergoes morphogenesis to structures of the lower jaw, including the mandibular cartilage and bone from an amorphous mesenchyme from embryonic day (E)10 to 14 in vivo. In E10 tissue the program of morphogenesis is set, and in subsequent culture for 9 days forms all the tissues of the mandible in serum-less, chemically defined culture medium. Thus, this system is ideal for determining the local factors that regulate the morphogenesis of bone and cartilage. Extracellular matrix remodeling is believed to play a significant role in growth and development of connective tissues. The aim of this proposal is to determine whether extracellular matrix remodeling mediated by metalloproteinases and their inhibitors, which are known to be major components of bone and cartilage, plays a role in the formation and form of the extracellular matrix constituents of bone and cartilage during mandible development. To test this hypothesis it is essential to obtain quantitative and qualitative data on the expression of metalloproteinases (MMPs) and metalloproteinase inhibitors (TIMPs) during mandibular development in vivo and in culture. The approach will sue RT-PCR for initial identification of proteinase and inhibitor mRNA transcripts, followed by analysis of protein products of these genes by enzymatic and immunological means. Once these MMPs and TIMPs have been identified and their temporal patterns of expression defined, the expression of selected MMPs and TIMPs will be mapped to chondrogenic and/or osteogenic foci by in situ hybridization and immunocytochemistry during development in vivo and in culture. Then the function of these MMPs and TIMPs will be studied. One approach will involve addition of purified or recombinant MMPs or TIMPs to mandible cultures to see if these perturb morphogenesis. A second approach will involve production of a hypomorphic phenotype with blocking antibodies or antisense oligonucleotides to MMPs or TIMPs to see if these affect morphogenesis. A third approach will involve modulation of mandibular growth and morphogenesis by addition of exogenous growth and differentiation factors or by ablation of these factors by antibodies or antisense oligonucleotides followed by determination of their effects on MMP and TIMP expression, and on timing, position, and expression of chondrogenesis and osteogenesis. These experiments should lead to an understanding of the molecular factors involved in the extracellular matrix aspects of bone and cartilage morphogenesis, in general, and in the mandible, in particular, and lead to novel therapeutic strategies for mandibular malformations and bone and cartilage repair.