RUI: Roles of Paracrine Factors in Formation of the Turtle Shell

吉尔伯特龟壳是一种非凡的进化新奇事物。没有其他现存的脊椎动物的外壳几乎完全由骨头制成，也没有其他脊椎动物的四肢是在肋骨内形成的。甲鱼的甲壳由背甲和腹板两个主要部分组成。在它们之间，在侧面，是一座桥。甲壳有50块骨头，由肋骨支撑，肋骨生长在背侧，而不是腹侧。该项目的主要工作将扩大早期对甲壳发育的研究。这些早期的研究发现(1)形成甲壳骨板的肋骨从肋骨向两个方向生长，(2)肋骨前面的软骨内骨化经过真皮的那个区域后开始形成，(3)肋骨周围的骨骼形成早于肋骨的后部。目前的项目旨在测试肋骨是否会导致甲壳真皮的骨化和甲壳骨板的形成。具体地说，它测试了一种假设，即海龟真皮的骨化是肋骨在软骨内骨化时分泌的旁分泌因子的副产品。这一发育方案的第一部分认为，随着肋骨经历正常的软骨内成骨，在此过程中分泌的印度刺猬将诱导周围的真皮细胞表达和分泌BMP2/4/7。这一方案的第二部分是骨形态发生蛋白将以旁分泌和自分泌方式作用于真皮，使发育中的甲鱼背部真皮发生膜内成骨的进行性前沿。为了验证这一点，我们将克隆海龟的MSX2、IHH、BMP2、Bmp4、Bmp7、Fgf8和noggin基因的同源物，并通过原位杂交分析它们的表达。其次，将分泌BMP抑制蛋白Noggin的珠子或细胞植入孵化的甲壳中，看看这种抑制是否能阻止龟壳的形成。相反，含有BMP的珠子将被植入背部真皮，看看它们是否能在甲壳真皮的其他部分诱导早熟骨骼发育。如果这一假设得到证实，这项研究将产生广泛的影响。首先，它可以很好地解释“乌龟是如何长出壳来的”这个“古老”的问题。其次，这项研究将表明，形成海龟甲壳的发育程序是将肋骨软骨放入体内新位置的结果。第三，这项研究的结果将有助于重建导致海龟在晚三叠世突然出现的系统发育路径。这些数据将为甲壳的快速发展提供一种机制(根据化石记录推断)。在教学上，如果这一假设是正确的，这项研究将为脊椎动物的发育选择性提供一个极好的、容易理解的例子。这项工作对生物专业学生的培养和教学科研一体化具有重要意义。在过去的两年里，该项目受益于10名本科生、1名研究生和3名博士后研究员的才华。其中包括几名妇女以及残疾人和代表性不足的少数群体的人。该项目的第一年将在托马斯·杰斐逊大学医学院进行，骨科研究小组正在研究刺猬和骨形成蛋白在颌骨发育中的作用。在第二个两年中，这项研究将在斯沃斯莫尔学院(学年)和托马斯·杰斐逊大学(夏季)进行。。

0316025GilbertThe turtle shell is a remarkable evolutionary novelty. No other extant vertebrate has a casing made almost exclusively of bone, and no other vertebrate has its limbs forming within its ribs. The turtle shell is composed of two main parts, the dorsal carapace and the ventral plastron. Between them, on the lateral sides, is a bridge. The carapace contains fifty bones and is supported by the ribs, which grow dorsolaterally rather than ventrally. The major effort of this project will expand earlier studies of carapace development. These earlier studies found (1) that the costal bones that form the bony plate of the carapace grow out from the ribs in both directions, (2) that the costal bones begin to form after the endochondral ossification front of the rib has passed that region of the dermis, and (3) that the bones around the anterior ribs form earlier than those of the posterior ribs. The current project seeks to test whether the ribs induce the ossification of the carapacial dermis and the formation of the bony plate of the carapace. Specifically, it tests the hypothesis that the ossification of the turtle dermis is a by-product of paracrine factors secreted by the ribs as they undergo endochondral ossification. The first part of this developmental scenario contends that as the ribs undergo their normal endochondral ossification, the Indian hedgehog that they secrete during this process would induce the dermal cells around them to express and secrete BMP2/4/7. The second part of this scheme is that the Bone Morphogenetic Proteins would act in both paracrine and autocrine fashions to ossify the dermis, causing a progressive front of intramembranous ossification in the dorsal dermis of the developing turtle. To test this, the turtle homologues for the msx2, ihh, bmp2, bmp4, bmp7, fgf8, and noggin genes would be cloned and their expression analyzed by in situ hybridization. Second, beads or cells secreting the BMP-inhibitory protein, Noggin, would be implanted into hatchling carapaces to see if this inhibition would prevent turtle shell formation. Conversely, BMP-containing beads would be implanted into the dorsal dermis to see if they could induce precocious bone development in other parts of the carapacial dermis. If this hypothesis is validated, this research has wide-ranging implications. First, it would go very far to explaining the "age-old" question of "how the turtle got its shell." Second, this research would show that the developmental program used to form the turtle carapace is a consequence of putting rib cartilage into new positions in the body. Third, the results of this research will aid in the reconstruction of the phylogenetic path leading to the "sudden" emergence of turtles in the late Triassic. The data would provide a mechanism for the rapid development of the carapace (as inferred by the fossil record). Pedagogically, if this hypothesis were correct, this research would provide an excellent and readily understood example of developmental co-option in vertebrates. This work is especially relevant for the training of biology students and the integration of teaching and research. During the past two years, the project has benefited from the talents of ten undergraduates, one graduate student, and three postdoctoral fellows. These have included several women, as well as persons from disabled and under-represented minority groups. The first year of this program will be undertaken at the Thomas Jefferson University School of Medicine, where the Orthopedics Research group is studying the role of hedgehog and BMPs in jaw development. During the second two years this research will be performed at Swarthmore College (during the academic year) and Thomas Jefferson University (during the summers). .