Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Spatially restricted dental regeneration drives pufferfish beak development

doi: 10.1073/pnas.1702909114

Figure Lengend Snippet: Localization of a dental progenitor cell niche within the pufferfish dental lamina. (A–C) P. baileyi PCNA immunohistochemistry reveals high levels of cellular proliferation within the oral epithelium. As replacement teeth progress from late-initiation (A) to morphogenesis (C), the new tooth generation (R1) buds from the dental lamina (B). Successive rounds of replacement show that the dental generations stack on one another within an enameloid outer casing (black line) (C). (D) Sox2 immunohistochemical labeling during dental replacement initiation depicts high levels of Sox2 within both the developing taste buds (TB) and the dental progenitor site located within the labial oral epithelium (dental lamina) (black arrowhead). (E) Double immunofluorescence treatment for Sox2/PCNA in T. niphobles shows low levels of PCNA expression within the Sox2+ cells of the presumptive dental progenitor niche and cells within the aboral dental lamina exhibiting high levels of PCNA. The horizontal dashed line depicts image stitching of two adjacent images. White arrowhead marks region of overlapping PCNA/Sox2 expression. (F) BrdU pulse/chase experiments (0.2 mM) show the incorporation of BrdU into dividing cells after 6 wk of treatment, with high levels of incorporation noted in the distal dental lamina next to the base of the beak (white arrowhead). (G) After a further 8-wk chase, label-retaining cells were found in the most superficial dental lamina cells (open arrowhead) but not in the distal dental lamina (white arrowhead). Label-retaining cells found in the dental epithelium of the developing tooth are indicated by a white arrow. Images in F and G are composites of multiple images taken at high magnification and stitched together. (H) DiI labeling of the labial oral epithelium in P. suvattii highlighted this region as a presumptive source of dental progenitor cells. DiI was detected within the outer dental epithelium of the tooth (white arrowhead) 72 h post DiI treatment. (I) As summarized in a schematic representation, we observed a continuous field of Sox2+ cells between the labial taste bud and the dental progenitor site, with cells from the latter migrating and contributing to the new dental generations. Black arrows represent the direction of cell movement. (J) Sox2/ABC double immunohistochemical labeling on adult C. travancoricus highlights epithelial Sox2+/ABC− (a′, white filled arrow), Sox2+/ABC+ (b′, white arrow), and Sox2−/ABC+ (c′, white arrowhead) regions within the dental lamina. Coexpression of these markers marks the site of activation of putative dental progenitors within the oral epithelium. Dashed line across (J) depicts image stitching of two adjacent images. Images are orientated with labial to the left and oral to the top. The dotted line in all images depicts the boundary of the oral epithelium and the end of the dental lamina. DM, dental mesenchyme; ODE, outer dental epithelium; R1–3, replacement tooth generations; RT, regenerating tooth; TB, labial taste bud. (Scale bars: 25 µm in A–E; 20 µm in F, a′–F, c′; 50 µm in G and H; 15 µm in I.)

Article Snippet: Rabbit anti-Sox2/mouse anti-PCNA and rabbit anti-Sox2/mouse anti-active β-catenin (1:500) (05-665; Merck) double immunofluorescence was carried out as described by Martin et al. ( 17 ).

Techniques: Immunohistochemistry, Immunohistochemical staining, Labeling, Immunofluorescence, Expressing, Pulse Chase, Activation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Spatially restricted dental regeneration drives pufferfish beak development

doi: 10.1073/pnas.1702909114

Figure Lengend Snippet: Conserved odontogenic signaling regulates dental regeneration in pufferfish. (A–I) Expression of well-documented odontogenic markers belonging to Sox (sox2, A); canonical Wnt signaling (β-catenin, B; lef1 C); Pitx (pitx2, D); Shh (E), Notch (hes1, F; notch3 G); Bmp (bmp2, H); and Fgf (fgf3, I) gene families in T. niphobles embryos. The thin arrow marks the site of presumptive dental progenitors, with expression of pitx2 (D), lef1 (C), and sox2 (A) within this region. The thick arrow marks the distal end of the dental lamina. The filled arrowhead highlights an opening within the osteodentine beak casing through which new odontogenic cells bud from the dental lamina. β-cat (B), shh (E), hes1 (F), notch3 (G), bmp2 (H), and fgf3 (I) are all expressed within the epithelium of the latest developing teeth. (J) A diagrammatic illustration of odontogenetically similar structures in various polyphyodonts [pufferfish, alligator (7, 16), cichlid (5), and catshark (43)]. Four main developmental regions are highlighted: presumptive dental progenitors, progenitor cell activation marked by the coexpression of Sox and Wnt signals, dental epithelium differentiation marked by the up-regulation of various developmental genes at the distal tip of the dental lamina and the growth of a tooth bud, and dental morphogenesis. The dotted line depicts the boundary of the oral epithelium and the end of the dental lamina. All images were taken from 14-µm sagittal paraffin-embedded sections. A, B, E, F, H, and I are from T. niphobles embryos at 50 dpf. C, D, and G are from embryos at 32 dpf. R1-2, replacement tooth generations; S, suture; TB, labial taste bud. (Scale bars: 50 µm in B–D, F, and G; 35 µm in A, E, H, and I.)

Article Snippet: Rabbit anti-Sox2/mouse anti-PCNA and rabbit anti-Sox2/mouse anti-active β-catenin (1:500) (05-665; Merck) double immunofluorescence was carried out as described by Martin et al. ( 17 ).

Techniques: Expressing, Activation Assay