The turtle shell is an evolutionary novelty composed of two main parts, the upper carapace and the lower plastron. What "makes a turtle a turtle" is the migration of the ribs into the dermis of the back skin rather than their migrating downward to form a rib cage. This study will use cell transplantation and gene labeling techniques to identify the molecules secreted by the ribs which cause the skin to turn into bone. We expect that the ribs are secreting proteins called Bone Morphogenetic Proteins (BMPs) and that the timing of bone formation is regulated by the interplay between these BMPs and inhibitors of BMPs that exist in the dermis.
Cells forming the plastron originate in an embryonic structure called the trunk neural crest. The trunk neural crest has not been seen to produce bones on any other extant species. The cranial neural crest, however, usually makes facial bones. If trunk neural crest cells lose the expression of Hox genes, they simultaneously gain the ability to form skeleton. Hox gene expression appears to inhibit the ability of neural crest cells to form bone. So another major objective of this project is to test the hypothesis that in turtle embryos, a population of neural crest cells is produced late, loses its Hox expression, and migrates to form the plastron bones.
If these hypotheses are validated, this research goes far in explaining the "age-old" question of how the turtle got its shell. It would provide a solution to the question of how a novel anatomical structure can evolve so quickly in the fossil record, and it would also provide a readily understood example how evolution works to make new structures. It will be done by undergraduates and should acquaint them with the basic techniques of evolutionary developmental genetics.
How can the anatomy of an existing group of animals be altered to create a new type of animals? We looked at the origin of turtles, asking the question, "How did the turtle get its shell?" Or, rather, we rephrased the question, "How does changing the timing, placement, or amount of gene expression enable the formation of over 50 bones that no other vertebrate possesses?" Creationists have claimed that the turtle could not have arisen by evolution, as it appeared suddenly in the fossil record. We sought to discover how this anatomical structure--the turtle shell--develops from the basic body plan of a usual land-dwelling vertebrate. We first found that the carapace--the upper shell--forms by three major steps. First, a particular protein, Fgf10, is made in a small portion of the turtle dermis. This protein appears to be responsible for causing the ribs to migrate into the back skin. (A turtle, therefore, has no rib cage.) If we block Fgf10 from functioning, the ribs do not enter the skin. Second, once the ribs are in the skin's dermis, they do what ribs normally do, transforming themselves from cartilage cells into bone cells. To do this, a protein called bone morphogenetic protein-4 (BMP4) is secreted from the rib. BMP4 not only turns the rib cartilage into bone, we found that it also turns the surrounding dermis into bone. So each rib acts as a signaling center to form bone around it. The third step involves the formation of the nuchal bone, the most anterior bone of the turtle shell. We believe this is formed from neural crest cells (like the face is), since it differs from the other carapacial bones by staining positivly for HNK-1 and PDGFR-alpha, two markers of the skeletogenic (cranial) neural crest. The bottom shell--the plastron--forms in a totally different manner. We have evidence that the bones of the plastron derive from the trunk neural crest. This would be remarkable, since trunk neural crest cells do not make bone in any other species of vertebrate. Rather, the cranial neural crest, above the neck, midbrain, and hindbrain, make the bones of the face and part of the skull. Our first evidence for the plastron bones being derived from the neural crest was that the cells forming the nine plastron bones are each staining for HNK-1 and PDGFR-alpha. We then looked at the neural crest cells and discovered that in turtles there is a second wave of neural crest cell emigration from the neural tube. This had not been seen in any other animal. This second wave of emigrating cells forms a thick band of cells in the carapacial dermis, and these cells stain positively for neural crest markers. Moreover, we can see these cells migrating toward the plastron region and forming bone there. These bone forming cells stained positively for HNK-1, PDGFR-alpha, p75, and other markers of skeletogenic neural crest. When we labeled the inside of the turtle neural tube with DiI, a fluorescent dye that labels cell membranes, we saw DiI-labeled cells in the ventral regions of the turtle embryo after four days. Thus, cells that used to be in the neural tube have migrated to the region where plastron bones form. We also allowed these cells to migrate from young and old neural tubes in culture. The neural crest cells from young embryos stained positively for HNK-1; but they did not stain positively for PDGFR-alpha. The neural crest cells coming from older embryos stained for both HNK-1 (neural crest) and PDGFR-alpha (cranial, skeletogenic). Further analysis showed that nearly all the cells of the plastron dermis stain positively for the markers of cranial neural crest. We therefore propose that the bones of the turtle plastron arose by a re-specification of the trunk neural crest into cranial-like neural crest.We have established an embryonic turtle transcriptome and are now testing to see if Hox gene expression has been downregulated. (Hox gene expression inhibits bone formation and is seenin the trunk but not the cranial neural crest cells.) The mechanisms by which certain cells are told to initiate craniofacial bone formation are as yet unknown,and the turtle plastron may be an excellent model in which to look at the mechanisms by which bones form in the face and skull. By altering the placement of gene expression (Fgf10 in the dermis), the carapace can be formed. By changing the timing of gene expression (enabling a second wave of neural crest cell emigration from the neural tube), the plastron can be formed. This grant has also enabled the training of numerous undergraduates to master the basic techniques (DNA cloning, in situ hybridization, immunohistochemistry, cell culture, organ culture, histology) that will be useful in any laboratory studying development, cancer, or gene regulation.
