Gene expression detection in developing mouse tissue using in situ hybridization and µCT imaging.

High resolution and noninvasiveness have made soft-tissue X-ray microtomography (µCT) a widely applicable three-dimensional (3D) imaging method in studies of morphology and development. However, scarcity of molecular probes to visualize gene activity with µCT has remained a challenge. Here, we apply horseradish peroxidase-assisted reduction of silver and catalytic gold enhancement of the silver deposit to in situ hybridization in order to detect gene expression in developing tissues with µCT (here called GECT, gene expression CT). We show that GECT detects expression patterns of collagen type II alpha 1 and sonic hedgehog in developing mouse tissues comparably with an alkaline phosphatase-based detection method. After detection, expression patterns are visualized with laboratory µCT, demonstrating that GECT is compatible with varying levels of gene expression and varying sizes of expression regions. Additionally, we show that the method is compatible with prior phosphotungstic acid staining, a conventional contrast staining approach in µCT imaging of soft tissues. Overall, GECT is a method that can be integrated with existing laboratory routines to obtain spatially accurate 3D detection of gene expression.

The developmental basis for scaling of mammalian tooth size.

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 molar development from initiation through final size in the mouse and the rat. Whereas the linear dimensions of the rat molars are twice that of the mouse molars, their shapes are largely the same. Here, we focus on the first lower molars that are considered the most reliable dental proxy for size-related patterns due to their low within-species variability. 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 signaling scales teeth by simultaneously enhancing growth and by inhibiting the cusp-patterning program, thereby providing a relatively simple mechanism for scaling teeth during development and evolution. Finally, comparative data from shrews to elephants suggest that this scaling mechanism regulates the minimum tooth size possible, as well as the patterning potential of large teeth.

Dicer1 ablation in osterix positive bone forming cells affects cortical bone homeostasis.

The RNAse III enzyme Dicer plays a major role in the processing of microRNAs from large pre-miRNAs. Dicer1 processed microRNAs are known to play a comprehensive role in osteoblast differentiation, bone remodeling and skeletal disorders. Targeted deletion of Dicer1 in osteo-progenitor cells is deleterious to fetal survival whereas targeted deletion in mature osteoblasts leads to an increase in bone mass. To address the role of Dicer1 in post-natal skeletal homeostasis, we generated a pre-osteoblast specific Dicer1 knockout model employing Tamoxifen controllable Cre allele, enabling us, via tamoxifen administration, to time-controllably ablate Dicer1 gene expression in osterix expressing bone forming cells in post-natal mice. Inactivation of Dicer1 in osterix positive bone forming cells led to striking dysregulation of cortical bone formation in pre-pubertal as well as adult mice. Cortical bone thickness was found to be significantly decreased in the Cre+ femora of both young and adult mice. Further, biomechanical testing experiments showed increased ductility, reduced stiffness and altered load at upper yield among the Cre+ tibiae. Our results suggest that Dicer1 processed microRNAs might play an important role in the regulation of post-natal cortical bone formation.