A periodic pattern generator for diversity in Malawi cichlid dentitions. (A) A cross-section through three thickened tooth germs showing planar signalling in the zone of inhibition (ZOI)/inter-germ space (grey, wnt7b) demarcating the location of teeth (green, shh and edar), with contribution from dental mesenchyme (blue, eda, bmp4, pax9 and runx2). Non-dental mesenchyme, purple. (B) The proposed interactions of wnt7b and eda (around the thickened tooth germs) with shh and edar within the thickened epithelial germ [9, 30, 44–51] in the context of our observations. wnt7b acts as an inhibitor of shh during tooth patterning and eda from the mesenchyme acts as an activator maintaining shh, perhaps via the eda-receptor edar, within the tooth germ. Wnt signals (possibly including wnt7b) may induce eda in the mesenchyme. DE, dental epithelium; DM, dental mesenchyme; OE, oral epithelium; OM, oral mesenchyme. (C)-(K) the induction of each tooth row acts in a 'copy-and-paste' mechanism utilizing the same genes for the pattern and organization of teeth and new tooth rows. Differential regulation of the genes during periodic patterning lead to the species diversityRedeployment of genes from OB1 establishes new tooth rows periodically in a lingual progression until the 'copy-and-paste' mechanism fails, due lack of combinatorial pitx2 and shh, essential for sequential row addition. Upper panel (C)-(E) around 5–6 days post-fertilization (dpf); middle panel (F)-(H) around 6–7 dpf; lower panel (I)-(K) around 12 dpf; each panel represents the left half of the dentary in dorsal view. (C)-(E the first tooth initiates from OB, a combinatorial expression of pitx2 (white) and shh (green). The initial pitx2 positive region is greatest in LF, with more rows. Up-regulation of shh and edar in the epithelial germs plus the establishment of a ZOI (eda and wnt7b) set the size and space restriction for the epithelial tooth germs. Local eda surrounds the first germ for each species (darker blue denotes stronger local expression of eda, which later becomes a broader domain and less intense, lighter blue). (F)-(H) eda remains strong and local in CA and at this stage (3–4-teeth stage) is broader and less intense in LF. CA does not initiate more than two tooth rows because pitx2 is constrained along the oral-aboral axis (lingual to the first teeth). The size, spacing and positioning of teeth and tooth rows is regulated by the interactions of wnt7b (grey), eda (blue) and shh (green) within the pitx2-positive field (white; see the main text and Figure 5). Future tooth row initiation in (J) MZ (OB3) and (K) LF (OB4) depends on the lingual co-expression of pitx2 and shh. (I) CA does not initiate a third row of teeth, and lacks co-expression of shh and pitx2 in a third OB (see Figure 4). Arrows mark the direction of tooth initiation for continued tooth addition on older rows. (I)-(K) eda and wnt7b expression domains are predicted from earlier stages. A more diffuse expression of eda (light blue) will continue to regulate size, spacing and row initiation of first-generation teeth. Numbers indicate the order of appearance for tooth rows. S, jaw symphysis.