Distinct tooth regeneration systems deploy a conserved battery of genes

Abstract

Background Vertebrate teeth exhibit a wide range of regenerative systems. Many species, including most mammals, reptiles, and amphibians, form replacement teeth at a histologically distinct location called the successional dental lamina, while other species do not employ such a system. Notably, a ‘lamina-less’ tooth replacement condition is found in a paraphyletic array of ray-finned fishes, such as stickleback, trout, cod, medaka, and bichir. Furthermore, the position, renewal potential, and latency times appear to vary drastically across different vertebrate tooth regeneration systems. The progenitor cells underlying tooth regeneration thus present highly divergent arrangements and potentials. Given the spectrum of regeneration systems present in vertebrates, it is unclear if morphologically divergent tooth regeneration systems deploy an overlapping battery of genes in their naïve dental tissues. Results In the present work, we aimed to determine whether or not tooth progenitor epithelia could be composed of a conserved cell type between vertebrate dentitions with divergent regeneration systems. To address this question, we compared the pharyngeal tooth regeneration processes in two ray-finned fishes: zebrafish (Danio rerio) and threespine stickleback (Gasterosteus aculeatus). These two teleost species diverged approximately 250 million years ago and demonstrate some stark differences in dental morphology and regeneration. Here, we find that the naïve successional dental lamina in zebrafish expresses a battery of nine genes (bmpr1aa, bmp6, cd34, gli1, igfbp5a, lgr4, lgr6, nfatc1, and pitx2), while active Wnt signaling and Lef1 expression occur during early morphogenesis stages of tooth development. We also find that, despite the absence of a histologically distinct successional dental lamina in stickleback tooth fields, the same battery of nine genes (Bmpr1a, Bmp6, CD34, Gli1, Igfbp5a, Lgr4, Lgr6, Nfatc1, and Pitx2) are expressed in the basalmost endodermal cell layer, which is the region most closely associated with replacement tooth germs. Like zebrafish, stickleback replacement tooth germs additionally express Lef1 and exhibit active Wnt signaling. Thus, two fish systems that either have an organized successional dental lamina (zebrafish) or lack a morphologically distinct successional dental lamina (sticklebacks) deploy similar genetic programs during tooth regeneration. Conclusions We propose that the expression domains described here delineate a highly conserved “successional dental epithelium” (SDE). Furthermore, a set of orthologous genes is known to mark hair follicle epithelial stem cells in mice, suggesting that regenerative systems in other epithelial appendages may utilize a related epithelial progenitor cell type, despite the highly derived nature of the resulting functional organs.

EvolvedBmp6enhancer alleles drive spatial shifts in gene expression during tooth development in sticklebacks

“…NFATc1 was shown to have importance in the balancing of quiescent and actively dividing stem cells in hair follicles (Horsley et al, 2008) which share homology with teeth (Ahn, 2015;Biggs & Mikkola, 2014;Pispa & Thesleff, 2003), and so a difference in NFATc1 binding may potentially play a role in the Bmp6 allele specific expression and enhancer activity differences observed previously and here. Supporting this hypothesis, Nfatc1b expression was recently shown to be present in stickleback tooth germs and functional tooth mesenchyme (Square et al, 2021).…”

“…The in situ hybridization (ISH) for Bmp6 was carried out as described previously (Square et al, 2021), with some modifications to ensure maximally comparable assays were carried out on marine and freshwater samples in parallel. A previously published Bmp6 riboprobe was used in this study Square et al, 2021).…”

“…The in situ hybridization (ISH) for Bmp6 was carried out as described previously (Square et al, 2021), with some modifications to ensure maximally comparable assays were carried out on marine and freshwater samples in parallel. A previously published Bmp6 riboprobe was used in this study Square et al, 2021). The Bmp6 riboprobe was synthesized with digoxygenin-labeled UTP and added at a concentration of ~300 ng/mL in 20 mL of hybridization buffer, split between 2 different LockMailer slide containers (Sigma-Aldrich), and agitated overnight in a rotating hybridization oven at 67° C. Slides from marine and freshwater fish were cohoused in the hybridization buffers to ensure equal exposure to the riboprobe between marine and freshwater samples.…”

“…The Bmp6 riboprobe was synthesized with digoxygenin-labeled UTP and added at a concentration of ~300 ng/mL in 20 mL of hybridization buffer, split between 2 different LockMailer slide containers (Sigma-Aldrich), and agitated overnight in a rotating hybridization oven at 67° C. Slides from marine and freshwater fish were cohoused in the hybridization buffers to ensure equal exposure to the riboprobe between marine and freshwater samples. Hybridization buffer washes, blocking, and antibody incubation steps were as previously described (Square et al, 2021). Signal development was carried out for 2, 3, or 7 days to visualize mRNA localization.…”

EvolvedBmp6enhancer alleles drive spatial shifts in gene expression during tooth development in sticklebacks

“…Marine and freshwater fish were always collected and prepared in parallel such that all storage and preparation intervals were equivalent. The ISH for Bmp6 was carried out as described previously ( Square et al 2021 ), with some modifications to ensure maximally comparable assays were performed on marine and freshwater samples in parallel. A previously published Bmp6 riboprobe was used in this study ( Cleves et al 2014 ; Square et al 2021 ).…”