Tooth replacement in an evo-devo context : the dental lamina as a possible source of stem cells

The enthralling ability to continuously replace teeth throughout life has fascinated scientists for decades. This ability (polyphyodonty) is maintained in almost all tooth-bearing non-mammalian vertebrates. Teeth are organised in tooth families, i.e. a functional tooth and all of its successors (Reif, 1982). In general, first and later generation teeth develop within an epithelial dental lamina as result of interaction of the epithelial cells with the underlying neural crest derived mesenchyme. Many aspects of tooth development and tooth evolution have been intensively studied over the past century; however the mechanism that drives lifelong tooth renewal remains largely unknown. Huysseune and Thesleff (2004) were the first to hypothesise that epithelial stem cells might be involved in this process, and suggested the dental lamina as a putative stem cell niche. In this thesis, we wished to test this hypothesis using a selection of polyphyodont species. In each of the species examined, we focussed on the lower jaw, and addressed three key issues: (1) the morphology and architecture of the dental tissues, with a particular interest for the dental lamina, (2) the spatiotemporal pattern of cell proliferation within the different layers of the dental lamina, and (3) the potential presence of epithelial stem cells. We selected three polyphyodont species that are interesting from a comparative, evo-devo perspective: a chondrichthyan (Scyliorhinus canicula) and two actinopterygian osteichthyans (Polypterus senegalus as a member of a basal clade within the Actinopterygii and Salmo salar as a basal protacanthopterygian). (1) Scyliorhinus canicula has many successor teeth, which are prefabricated before the functional tooth is shed (CHAPTER 3). This results in tooth family sizes of seven to eight members. All tooth families are interconnected by a continuous and permanent dental lamina and are organised in an alternating pattern of developmental stages along the jaw margin. Three to four members of each family reside in the dental lamina. Polypterus senegalus and Salmo salar have their teeth organised in tooth families with two to three members; however, neither for the establishment of the first-generation tooth, nor for the development of its successors, a distinct dental lamina could be determined (CHAPTER 5,7). The maturation stage of functional teeth in Polypterus senegalus showed an alternating pattern, similar on both jaw halves (CHAPTER 4), while in Salmo salar a pattern with tooth families in similar developmental stages occurred every third position (CHAPTER 6). We propose to define the dental lamina in broad terms as ‘all epithelial cells that enclose a tooth family’. This allows us to interpret the dental lamina in Polypterus senegalus (CHAPTER 5) and Salmo salar (CHAPTER 7) as being extremely small or spatially compressed rather than being absent, and morphologically indistinguishable from the superficial epithelium. In conclusion of our morphological observations, we have constructed a hypothetical evolutionary model that generalises the dental lamina as a homologous modular structure in dental development (CHAPTER 8). (2) BrdU proliferation studies showed dividing cells during the development of the youngest tooth germs in all three species (CHAPTERS 3,5,7). These tooth germs arise at the distal end of the dental lamina in Scyliorhinus canicula and the lingual side of the dental lamina in Polypterus senegalus and Salmo salar. In all three species, the middle dental epithelium (MDE) and outer dental epithelium (ODE) in the lingualmost part (i.e. most distal) of the dental lamina showed intensive proliferation, while the transition zone of the outer dental epithelium towards the oral epithelium was not proliferating in any of the three species. Further similarities were obvious from BrdU chase experiments (CHAPTERS 3,5,7). All three species showed a shift in position of proliferative areas in the epithelium from the lingual side of the dental lamina towards a labial and oral position. This is consistent with a shift that developing successor teeth undergo towards the superficial epithelium, i.e. the location where they replace functional teeth. (3) BrdU pulse-chase experiments were conducted in all three species, with the intention to detect label-retaining cells (LRCs) in the dental lamina as a possible indication for the presence of stem cells. In Scyliorhinus canicula, LRCs were found in the lingualmost part of the dental lamina both within the dental and interdental region (CHAPTER 3). Our experiments on Salmo salar and Polypterus senegalus did not reveal any of such cells in the epithelial tissues surrounding a tooth family (CHAPTERS 5,7). Immunohistological staining for Sox2, used on all three species as proxy for the potential presence of stem cells, yielded consistent results, with positive staining in the oral epithelium, taste buds and ODE transition zone but absent in the dental lamina (CHAPTERS 3,7). The Sox2+ cells in Scyliorhinus canicula did not coincide with the LRCs in the MDE. The combination of these conflicting results obtained for the three species studied, provided arguments for either of two scenarios: one supporting the presence, and one, conversely, in support of the absence of stem cells in the dental lamina. While only limited support for the presence of epithelial stem cells could be collected using the commonly proposed character of label retention, we consider the presence of cells with stem cell like characteristics likely. At the same time we emphasise that the concept of what ‘true’ stem cells are, needs to be critically re-evaluated. In the species examined, the lingualmost part of the MDE could serve as a site within the dental lamina to maintain a population of such potential stem cells. Continued experimental research on the proposed candidate area for stem cells should be pursued