G1 cell population domain coincides with Shh expression in placodes and buds. Fucci G1 cell cycle transgene expression in control or K17-GFP background was analyzed with fluorescence microscopy. The same specimens were then analyzed by whole-mount in situ hybridization with probes specific for Foxi3, Sox2, Dkk4, or Shh. (A) The early G1 population coincided with, but was more restricted than, the Foxi3 expression domain in the dental lamina at E11.5 and later in the forming placode and bud. Foxi3 expression colocalized with K17-GFP positivity. (B) Early G1 population and K17-GFP–positive cells partially colocalized, but mostly resided buccally to Sox2-expressing cells. (C) Dkk4 colocalized with the G1 population; however, by E13.0 Dkk4 expression was already down-regulated, whereas the G1 focus persisted in the anterior part of the incisor bud. (D) Shh expression colocalized with the G1 cell population at all observed time points (E11.5, 12.5, 12.0, and 13.5; emerging enamel knot marked with an asterisk). This identifies the early G1 cell population as an early signaling center characterized by cell cycle cessation and signaling center marker expression. Bars, 100 µm.