Andrew H. Jheon

Characterization of a Novel KRAB/C2H2Zinc Finger Transcription Factor Involved in Bone Development

Osteogenic differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporo-spatial manner. Here we have used differential display to identify a novel zinc finger transcription factor (AJ18) that is induced during differentiation of bone cells in vitro and in vivo. The 64-kDa protein, encoded by a 7- kilobase mRNA, contains a Krüppel-associated box (KRAB) domain followed by 11 successive C2H2 zinc finger motifs. AJ18 mRNA, which is also expressed in kidney and brain, is developmentally regulated in embryonic tibiae and calvariae, with little expression in neonate and adult animals. During osteogenic differentiation in vitroAJ18 mRNA is expressed as cells approach confluence and declines as bone formation occurs. Using bacterially expressed, His-tagged AJ18 in a target detection assay, we identified a consensus binding sequence of 5′-CCACA-3′, which forms part of the consensus element forRunx2, a master gene for osteogenic differentiation. Overexpression of AJ18 suppressed Runx2-mediated transactivation of anosteocalcin promoter construct in transient transfection assays and reduced alkaline phosphatase activity in bone morphogenetic protein-induced C3H10T1/2 cells. These studies, therefore, have identified a novel zinc finger transcription factor in bone that can modulate Runx2 activity and osteogenic differentiation.

Zinc Finger Transcription Factors in Skeletal Development

Cellular and molecular processes that regulate the development of skeletal tissues resemble those required for regeneration. Given the prevalence of degenerative skeletal disorders in an increasingly aging population, the molecular mechanisms of skeletal development must be understood in detail if novel strategies are to be developed in regenerative medicine. Research in this area over the past decade has revealed that cell differentiation is largely controlled at the level of gene transcription, which in turn is regulated by transcription factors. Transcription factors usually recognize and bind to specific DNA sequences in the promoter of target genes via characteristic DNA-binding domains. Although the gene family containing C2H2 zinc fingers as DNA-binding motifs is the largest family of transciptional regulators, with several hundred individual members in mammals, only a small but increasing number of zinc finger genes have been implicated in bone, cartilage, or tooth development. These zinc finger proteins (ZFPs) contain multiple structural motifs that require zinc to maintain their structural integrity and function. Interestingly, zinc deficiency is known to result in skeletal growth retardation and has been identified as a risk factor in the pathogenesis of osteoporosis. This review attempts to summarize our current state of knowledge regarding the role of ZFPs in the molecular regulation of skeletogenesis.

The Pitx2:miR-200c/141:noggin pathway regulates Bmp signaling and ameloblast differentiation

The mouse incisor is a remarkable tooth that grows throughout the animal’s lifetime. This continuous renewal is fueled by adult epithelial stem cells that give rise to ameloblasts, which generate enamel, and little is known about the function of microRNAs in this process. Here, we describe the role of a novel Pitx2:miR-200c/141:noggin regulatory pathway in dental epithelial cell differentiation. miR-200c repressed noggin, an antagonist of Bmp signaling. Pitx2 expression caused an upregulation of miR-200c and chromatin immunoprecipitation assays revealed endogenous Pitx2 binding to the miR-200c/141 promoter. A positive-feedback loop was discovered between miR-200c and Bmp signaling. miR-200c/141 induced expression of E-cadherin and the dental epithelial cell differentiation marker amelogenin. In addition, miR-203 expression was activated by endogenous Pitx2 and targeted the Bmp antagonist Bmper to further regulate Bmp signaling. miR-200c/141 knockout mice showed defects in enamel formation, with decreased E-cadherin and amelogenin expression and increased noggin expression. Our in vivo and in vitro studies reveal a multistep transcriptional program involving the Pitx2:miR-200c/141:noggin regulatory pathway that is important in epithelial cell differentiation and tooth development.

Regulation of tooth number by fine-tuning levels of receptor-tyrosine kinase signaling

Much of our knowledge about mammalian evolution comes from examination of dental fossils, because the highly calcified enamel that covers teeth causes them to be among the best-preserved organs. As mammals entered new ecological niches, many changes in tooth number occurred, presumably as adaptations to new diets. For example, in contrast to humans, who have two incisors in each dental quadrant, rodents only have one incisor per quadrant. The rodent incisor, because of its unusual morphogenesis and remarkable stem cell-based continuous growth, presents a quandary for evolutionary biologists, as its origin in the fossil record is difficult to trace, and the genetic regulation of incisor number remains a largely open question. Here, we studied a series of mice carrying mutations in sprouty genes, the protein products of which are antagonists of receptor-tyrosine kinase signaling. In sprouty loss-of-function mutants, splitting of gene expression domains and reduced apoptosis was associated with subdivision of the incisor primordium and a multiplication of its stem cell-containing regions. Interestingly, changes in sprouty gene dosage led to a graded change in incisor number, with progressive decreases in sprouty dosage leading to increasing numbers of teeth. Moreover, the independent development of two incisors in mutants with large decreases in sprouty dosage mimicked the likely condition of rodent ancestors. Together, our findings indicate that altering genetic dosage of an antagonist can recapitulate ancestral dental characters, and that tooth number can be progressively regulated by changing levels of activity of a single signal transduction pathway.

From molecules to mastication: the development and evolution of teeth

Teeth are unique to vertebrates and have played a central role in their evolution. The molecular pathways and morphogenetic processes involved in tooth development have been the focus of intense investigation over the past few decades, and the tooth is an important model system for many areas of research. Developmental biologists have exploited the clear distinction between the epithelium and the underlying mesenchyme during tooth development to elucidate reciprocal epithelial/mesenchymal interactions during organogenesis. The preservation of teeth in the fossil record makes these organs invaluable for the work of paleontologists, anthropologists, and evolutionary biologists. In addition, with the recent identification and characterization of dental stem cells, teeth have become of interest to the field of regenerative medicine. Here, we review the major research areas and studies in the development and evolution of teeth, including morphogenesis, genetics and signaling, evolution of tooth development, and dental stem cells. WIREs Dev Biol 2013, 2:165–182. doi: 10.1002/wdev.63

Stem Cell and Biomaterials Research in Dental Tissue Engineering and Regeneration

This review summarizes approaches used in tissue engineering and regenerative medicine, with a focus on dental applications. Dental caries and periodontal disease are the most common diseases resulting in tissue loss. To replace or regenerate new tissues, various sources of stem cells have been identified such as somatic stem cells from teeth and peridontium. Advances in biomaterial sciences including microfabrication, self-assembled biomimetic peptides, and 3-dimensional printing hold great promise for whole-organ or partial tissue regeneration to replace teeth and periodontium.

FGF Signaling Regulates the Number of Posterior Taste Papillae by Controlling Progenitor Field Size

The sense of taste is fundamental to our ability to ingest nutritious substances and to detect and avoid potentially toxic ones. Sensory taste buds are housed in papillae that develop from epithelial placodes. Three distinct types of gustatory papillae reside on the rodent tongue: small fungiform papillae are found in the anterior tongue, whereas the posterior tongue contains the larger foliate papillae and a single midline circumvallate papilla (CVP). Despite the great variation in the number of CVPs in mammals, its importance in taste function, and its status as the largest of the taste papillae, very little is known about the development of this structure. Here, we report that a balance between Sprouty (Spry) genes and Fgf10, which respectively antagonize and activate receptor tyrosine kinase (RTK) signaling, regulates the number of CVPs. Deletion of Spry2 alone resulted in duplication of the CVP as a result of an increase in the size of the placode progenitor field, and Spry1−/−;Spry2−/− embryos had multiple CVPs, demonstrating the redundancy of Sprouty genes in regulating the progenitor field size. By contrast, deletion of Fgf10 led to absence of the CVP, identifying FGF10 as the first inductive, mesenchyme-derived factor for taste papillae. Our results provide the first demonstration of the role of epithelial-mesenchymal FGF signaling in taste papilla development, indicate that regulation of the progenitor field size by FGF signaling is a critical determinant of papilla number, and suggest that the great variation in CVP number among mammalian species may be linked to levels of signaling by the FGF pathway.

The Cells that Fill the Bill: Neural Crest and the Evolution of Craniofacial Development

Avian embryos, which have been studied scientifically since Aristotle, continue to persevere as invaluable research tools, especially for our understanding of the development and evolution of the craniofacial skeleton. Whether the topic is beak shape in Darwin’s finches or signaling interactions that underlie bone and tooth formation, birds offer advantages for craniofacial biology that uniquely complement the strengths of other vertebrate model systems, such as fish, frogs, and mice. Several papers published during the past few years have helped pinpoint molecular and cellular mechanisms that pattern the face and jaws through experiments that could only have been done together with our feathered friends. Ultimately, such knowledge will be essential for devising novel clinical approaches to treat and/or prevent diseases, injuries, and birth defects that affect the human craniofacial skeleton. Here we review recent insights plucked from avians on key developmental processes that generate craniofacial diversity.

An L1 element disrupts human bone sialoprotein promoter: lack of tissue-specific regulation by distalless5 (Dlx5) and runt homeodomain protein2 (Runx2)/core binding factor a1 (Cbfa1) elements.

Bone sialoprotein (BSP) is a phosphorylated and sulphated glycoprotein with hydroxyapatite nucleating properties that is specifically expressed in association with physiological and pathological mineralization. Although previous studies have indicated that tissue-specific expression of murine BSP is regulated through a proximal homeodomain element that binds distalless5 (Dlx5) transcription analysis of the homologous human promoter revealed modest enhancement in osteogenic cells. Moreover, whereas forced expression of an antisense Dlx5 vector increased transcription, Dlx5 expression did not alter transcription significantly. Since extended promoter sequences are required to confer absolute tissue-specific expression of BSP in vivo, we characterized the upstream region of the human BSP gene. In contrast to the rat and mouse promoters, which show conserved sequences extending several kbs upstream, analysis of approximately 3 kb of the human promoter showed no sequence conservation beyond -0.99 kb. Southern blot analysis of genomic DNA from four different BAC clones showed that this sequence was not an aberration in the human genomic library used to isolate the BSP gene. Using clone BAC H-NH0811I08, the human BSP promoter sequence was extended approximately 8 kb upstream from which the non-homologous region was characterized as a 3.48 kb insert coding for an L1 retrotransposon element. Transcriptional analyses of chimeric promoter constructs revealed that the retrotransposon element suppresses transcription <80%. Upstream of the inserted DNA several regions, varying in length from 26 to 161 bps, were conserved within the mouse and human promoters. One of these conserved regions included a runt homeodomain protein2 (Runx2)/core binding factor a1 (Cbfa1) elements consensus element in reverse orientation. Whereas a multimeric form of the element was transcriptionally active in response to Runx2/Cbfa1 when ligated to the BSP basal promoter, the single element in the context of the extended promoter was unresponsive. These studies have characterized the upstream promoter of the human BSP gene, which is interrupted by a unique high-frequency DNA insert that suppresses BSP gene transcription.

Expression of MicroRNAs in the Stem Cell Niche of the Adult Mouse Incisor

The mouse incisor is a valuable but under-utilized model organ for studying the behavior of adult stem cells. This remarkable tooth grows continuously throughout the animals lifetime and houses two distinct epithelial stem cell niches called the labial and lingual cervical loop (laCL and liCL, respectively). These stem cells produce progeny that undergo a series of well-defined differentiation events en route to becoming enamel-producing ameloblasts. During this differentiation process, the progeny move out of the stem cell niche and migrate toward the distal tip of the tooth. Although the molecular pathways involved in tooth development are well documented, little is known about the roles of miRNAs in this process. We used microarray technology to compare the expression of miRNAs in three regions of the adult mouse incisor: the laCL, liCL, and ameloblasts. We identified 26 and 35 differentially expressed miRNAs from laCL/liCL and laCL/ameloblast comparisons, respectively. Out of 10 miRNAs selected for validation by qPCR, all transcripts were confirmed to be differentially expressed. In situ hybridization and target prediction analyses further supported the reliability of our microarray results. These studies point to miRNAs that likely play a role in the renewal and differentiation of adult stem cells during stem cell-fueled incisor growth.