The continuously growing mouse incisor provides a valuable model system to study adult stem cells. A single incisor contains two different epithelial stem cell niches, called the laCL and liCL, which are both derived from oral epithelium and surrounded by dental mesenchyme. Of these two niches, only the laCL generates enamel-forming ameloblasts. Therefore, we hypothesized that molecular profiling of the laCL and liCL, as well as the ameloblast region, would shed light on the renewal and differentiation of ameloblast stem cells.
miRNAs are important regulators of signaling pathways during morphogenesis and organogenesis, as well as in the control of embryonic and adult stem cells 
. The importance of Dicer
and miRNAs during tooth development has been demonstrated 
, but there has been relatively little progress in the identification and characterization of the roles of specific miRNAs. Such molecular characterization of dental stem cells may one day help us to repair and regenerate human teeth.
The miRNA microarray analysis from laCL, liCL, and ameloblasts yielded numerous differentially expressed miRNA transcripts (, ). All 10 miRNAs that we chose for validation by qPCR were indeed differentially expressed in the laCL/liCL and ameloblast/laCL comparisons at levels consistent with the microarray data (). Because the laCL, liCL, and ameloblast regions isolated from the incisor included mesenchyme-derived tissues, we selected two of the highest differentially expressed genes (i.e. miR-31, -138) for in situ hybridization analysis. Our results showed high expression of miR-31 in the laCL and miR-138 in ameloblasts, consistent with the microarray and qPCR analyses.
miR-31 was previously reported to be involved in the cycling of ectoderm-derived hair cells 
. Because the hair stem cell niche shares many characteristics with the laCL 
, it is likely that miR-31 will also play a role in the renewal of stem cells during continuous incisor growth. Interestingly, Fgf10
was previously identified as a direct target of antagonism by miR-31 
is expressed largely in the dental papilla adjacent to the laCL 
is required for tooth stem cell survival 
, and its inactivation leads to the formation of smaller teeth 
, whereas upregulation of Fgf10
is associated with the generation of supernumerary teeth and ectopic enamel 
. Taking into account the high expression of miR-31 in the ectoderm-derived laCL, it is possible that miR-31 may play a role in fine-tuning Fgf10
levels in the dental papilla. Further experiments will be required to study the interaction between miR-31 and Fgf10
during continuous tooth growth.
miR-138, whose expression is correlated with many different types of cancers and diseases, is also involved in cardiac patterning, specifically through the regulation of expression of genes such as aldehyde dehydrogenase (Aldh
) -1a2 and versican 
. Because Aldh1
is a marker of stem cells in certain contexts 
and versican is a secreted extracellular matrix protein that is present in many mineralized tissues including the dental epithelium of developing tooth germs 
, miR-138 may be involved in the differentiation of stem cells towards enamel matrix-secreting ameloblasts.
Members of the miR-200 family (i.e. miR-200a, -200b, -200c, -141, -429) were differentially expressed in both laCL/liCL and laCL/ameloblast comparisons. These transcripts along with miR-199b* were the only miRNAs that were differentially expressed in both comparisons, demonstrating the potential importance of specific subsets of miRNAs in the renewal vs. differentiation of tooth stem cells.
Interestingly, many of the microarray-identified, differentially expressed miRNAs were miRNA* (i.e. star strand) species. The ~21-nucleotide, obligate intermediate miRNA:miRNA* duplex associates with the Argonaute protein in miRISCs, such that the miRNA strand is usually the one that becomes stably incorporated, whereas the miRNA* strand dissociates and is thought to be degraded 
. Thus, by convention, the mature miRNA is defined as the duplex strand that is present at the higher steady-state level than its miRNA* partner strand. However, Yang et al. 
recently demonstrated that miRNA* species, rather than simply being the by-product of non-incorporation into miRISCs, also could directly repress translation and/or mRNA stability by specific binding to the 3′-UTR of genes. Further, the target seed sequence, which is a conserved heptametrical sequence that is essential for the binding of the miRNA to the mRNA, was evolutionarily conserved in miRNA* species 
. Thus, it will be of interest to determine the function of the identified miRNA* species during stem cell renewal and differentiation.
Together, our analyses utilizing microarray technology, qPCR, in situ hybridization, and target prediction tools have uncovered miRNAs that may play important roles in stem cell-based renewal of teeth.