IGF1 treatment of developing mouse molar

When evolution leads to differences in body size, organs generally scale along. A well-known example of the tight relationship between organ and body size is the scaling of mammalian molar teeth. To investigate how teeth scale during development and evolution, we compared mouse and rat molar development from initiation through final size. Whereas the linear dimensions of the rat first lower molar are twice that of the mouse molar, their shapes are largely the same. We found that scaling of the molars starts early, and that the rat molar is patterned equally as fast but in a larger size than the mouse molar. Using transcriptomics, we discovered that a known regulator of body size, insulin-like growth factor 1 (Igf1), is more highly expressed in the rat molars compared to the mouse molars. Ex vivo and in vivo mouse models demonstrated that modulation of the IGF pathway reproduces several aspects of the observed scaling process. Furthermore, analysis of IGF1-treated mouse molars and computational modeling indicate that IGF signalling scales teeth by simultaneously inhibiting the cusp patterning programme and by enhancing growth, thereby providing a relatively simple mechanism for scaling teeth during development and evolution. Finally, comparative data from shrews to elephants suggest that this scaling of patterning mechanism regulates the minimum tooth size possible, as well as the patterning potential of large teeth. Overall design: Embryonic day 14 (E14) mouse molars were dissected and cultured in a hanging drop culture (Närhi & Thesleff, 2010) for 6 hours pairwise so that from each embryo one tooth was treated with IGF1-containing media (750ng/ml in 1x PBS + 0.1% BSA ) or control media (same volume of 1x PBS + 0.1% BSA as was added to treatment samples). Right and left sides were balanced, n = 5 for both the treatments and controls. Tissue was stored in RNAlater (Qiagen, Düsseldorf, Germany) at -80°C for RNAseq. For RNAseq, each tooth was handled individually. Samples were homogenized using Precellys 24 homogenizer (Bertin instruments). RNA was extracted using guanidium thiocyanate-phenol-chloroform method and purified using RNeasy Plus micro kit (Qiagen GmbH) following manufacturer's protocol. The putiry of RNA was measured using Nanodrop microvolume spectrophotometer (Thermo Fisher Scientific). The complementary DNA (cDNA) libraries were prepared using Ovation Mouse RNAseq System (Tecan).