(a) In situ hybridisation for Shh in the developing palatal shelves from E12.0 to E14.5 (anterior right, medial up) illustrating the sequential addition of new rugae (white arrowheads) anterior to r8 (black arrowhead), as described ^17,18. Scale bar = 500 μm. (b) Schematic illustrating this sequential addition of rugae with growth. (c) Inter-rugal intervals measured at E13.5 and E14.5, along an axis defined as that of a line from the base of the anterior of the palatal shelf, parallel to the midline of the head. Colours for different interrugal intervals correspond to those used in histogram in panel (d). Scale bar = 200 μm. (d) Ratios of the E14.5 to the E13.5 lengths of the inter-rugal intervals, indicating high levels of growth between r8 and r5, and also elevated growth between r5 and r4, with little growth anterior to r4. Error bars indicate standard deviations. (e) Growth anterior to r2. Coloured dotted lines illustrate the orthogonal distance from r2 Shh expression to the anterior shelf edge (black dotted line) at the base of the shelf, medial edge of the Shh stripe, and midway between (blue, red and yellow respectively). Growth in more medial regions correlates with the appearance of r1 Shh expression at the anterior edge. Scale bar = 200 μm.

(a) Schematic of a lateral inhibition hypothesis for rugal spacing. Curves represent levels of inhibitor produced by rugae and dashed line represents inhibitory threshold. Growth between rugae would allow the level of inhibition to fall below threshold (asterisk) allowing the formation of a new ruga (dashed rectangle). (b,c) Rugal Shh stripes of palatal shelves of littermates cultured for 0, 24 and 48 hours explanted at (b) E12.5 and (c) E13.5 demonstrating the addition of rugae without AP growth at closer spacing than the equivalent stripes in vivo. (d) Schematic representing the predicted effect of removing a ruga under a lateral inhibition model. Removing the anterior of the palatal shelf by cutting posterior to the second ruga (vertical dashed line), removes inhibition from second ruga allowing inhibition to fall below threshold level at the cut edge (asterisk) leading to the formation of a new ruga (dashed rectangle). (e,f,g) Experimental result differs dramatically from the prediction: posterior palatal shelves cut adjacent to ruga 2 and cultured for 48 hours (two examples, f and g, with immediately fixed anterior, uncultured anterior pieces to the right) analysed by Shh in situ hybridisation show branches to ruga 3 at curves in the ruga (black arrowheads), not seen in uncut controls (e). (Dashed line in (e) represents where cut is in cut shelves.) (h, i) Branches to stripes are readily replicated in reaction-diffusion simulations generated using Turing equations as in ref ² – compare pattern in circles in h and i with those at arrowheads in f, g. All specimens oriented with anterior to right, medial up.

Palates of P0 mice viewed from the oral side, anterior up. (a-d) Increased FGF signaling in Spry1^−/−;Spry2^−/− mutant mice results in disorganized and compacted rugae (b, detail in d) compared to wildtype (a, detail in c). Rugal phenotype can be distinguished despite cleft palate in these mutants. (e-h) Down-regulation of Shh in K14-Cre/Shh^fl/fl mice results in a similar phenotype of disorganized, compacted rugae (f, detail in h) compared to wildtype controls (e, detail in g). Scale bar in panel a = 1 mm (for a, b, e, f); scale bar in panel c = 0.3 mm (for c, d, g, h).

Pattern of rugae, visualized by Shh in situ hybridization in palatal shelf explants cut posterior to ruga 2 and cultured for 24 (a-c, g-i) and 48 (d-f, i-l) hours. (a-f) Culture with the FGF inhibitor SU5402 shows reduced levels of Shh expression in treated explants (c,f) relative to controls (a,b,d,e). (g-l) Culture with the Hedgehog inhibitor cyclopamine shows increased levels of Shh expression in treated explants after 24 hours (i) relative to controls (g, h). Levels of Shh expression then fall after 48 hours culture with Cyclopamine (l) compared to controls (j,k). All specimens oriented with anterior to right, medial up. Scale bar = 200 μm.