Application of lentivirus‐mediated RNAi in studying gene function in mammalian tooth development

Abstract

RNA interference (RNAi) has recently become a powerful tool to silence gene expression in mammalian cells, but its application in assessing gene function in mammalian developing organs remains highly limited. Here we describe several unique developmental properties of the mouse molar germ. Embryonic molar mesenchyme, but not the incisor mesenchyme, once dissociated into single cell suspension and re-aggregated, retains its odontogenic potential, the capability of a tissue to instruct an adjacent tissue to initiate tooth formation. Dissociated molar mesenchymal cells, even after being plated in cell culture, retain odontogenic competence, the capability of a tissue to respond to odontogenic signals and to support tooth formation. Most interestingly, while dissociated epithelial and mesenchymal cells of molar tooth germ are mixed and re-aggregated, the epithelial cells are able to sort out from the mesenchymal cells and organize into a well-defined dental epithelial structure, leading to the formation of a well-differentiated tooth organ after sub-renal culture. These unique molar developmental properties allow us to develop a strategy using a lentivirus-mediated RNAi approach to silence gene expression in dental mesenchymal cells and assess gene function in tooth development. We show that knockdown of Msx1 or Dlx2 expression in the dental mesenchyme faithfully recapitulates the tooth phenotype of their targeted mutant mice. Silencing of Barx1 expression in the dental mesenchyme causes an arrest of tooth development at the bud stage, demonstrating a crucial role for Barx1 in tooth formation. Our studies have established a reliable and rapid assay that would permit large-scale analysis of gene function in mammalian tooth development.

FGF signaling sustains the odontogenic fate of dental mesenchyme by suppressing β-catenin signaling

“…We reported previously that E13.5 mouse molar germ, after dissociation and reaggregation, is able to form a well differentiated tooth organ, whereas incisor germ fails (Song et al, 2006) (Fig. 1A,B).…”

“…About 1×10 6 cells from either the incisor or molar pool were added to a 1.5-ml Eppendorf tube, centrifuged at 3000 rpm (550 g) for 5 minutes, and incubated at 37°C and 5% CO 2 for 1 hour to allow the formation of a firm cell pellet. Cell pellets were removed from Eppendorf tubes, placed in Trowell type organ culture in DMEM supplemented with 20% FBS overnight prior to being subjected to subrenal culture as described previously (Zhang et al, 2003;Song et al, 2006).…”

“…In this study, we investigated the mechanisms underlying our previous finding that early molar and incisor tooth germs exhibit distinct tooth-forming capability after dissociation and reaggregation in vitro (Song et al, 2006).…”

Developmental stalling and organ-autonomous regulation of morphogenesis

“…The fact that following stalling of the intrinsic developmental clock, development then accelerates to be back in synchrony with the general timing of embryonic development, illustrates the importance of temporal coupling of developmental processes. Surprisingly, in vitro knockdown of Barx1 using lentiviruses expressing Barx1 siRNA led to a complete arrest of tooth development at the bud stage (16), suggesting that the ability to restart development is lost in this system. The subrenal culture of tooth rudiments is unlikely to be the cause of this definitive arrest of development, because Barx1 −/− molar tooth rudiments grafted under a kidney capsule do form normal mineralized molars.…”

Optimization of Feline Immunodeficiency Virus Vectors for RNA Interference

“…RNAi can be induced in cells by naturally occurring endogenous microRNAs (miRNAs) or through artificial expression of inhibitory RNAs (e.g., small inhibitory RNAs [siRNAs], short hairpin RNAs [shRNAs], and synthetic microRNAs) designed to specifically reduce expression of a gene of interest (4,11,29,54). Over the past several years, inhibitory RNAs have been used in basic research to elucidate gene function or as tools in gene therapy to develop preclinical antivirals (5,24,31,40,50) and treatments for cancer (36,44,46) and dominant genetic diseases, such as Huntington's disease and spinocerebellar ataxia type 1 (13,32,33,35,52). Numerous methods have been developed to introduce inhibitory RNAs into cells, including lipid-based transfection of in vitro-synthesized siRNAs and gene transfer using viral vectors containing promoters that constitutively transcribe shRNAs or microRNAs (10,13,32,33,49,52,53).…”