Frederic Michon

Tooth morphogenesis and ameloblast differentiation are regulated by micro-RNAs

Teeth form as appendages of the ectoderm and their morphogenesis is regulated by tissue interactions mediated by networks of conserved signal pathways. Micro-RNA (miRNA) pathway has emerged as important regulator of various aspects of embryonic development, but its function in odontogenesis has not been elucidated. We show that the expression of RNAi pathway effectors is dynamic during tooth morphogenesis and differentiation of dental cells. Based on microarray profiling we selected 8 miRNAs expressed during morphogenesis and 7 miRNAs in the incisor cervical loop containing the stem cell niche. These miRNAs were mainly expressed in the dental epithelium. Conditional deletion of Dicer-1 in the epithelium (Dcr(K14)(-)(/)(-)) resulted in rather mild but significant aberrations in tooth shape and enamel formation. The cusp patterns of the Dcr(K14)(-)(/)(-) molar crowns resembled the patterns of both ancestral muroid rodents and mouse mutants with modulated signal pathways. In the Dcr(K14)(-)(/)(-) incisors, longitudinal grooves formed on the labial surface and these were shown to result from ectopic budding of the progenitor epithelium in the cervical loop. In addition, ameloblast differentiation was impaired and resulted in deficient enamel formation in molars and incisors. To help the identification of candidate target genes of the selected tooth enriched miRNAs, we constructed a new ectodermal organ oriented database, miRTooth. The predicted targets of the selected miRNAs included several components of the main morphogenetic signal pathways regulating tooth development. Based on our findings we suggest that miRNAs modulate tooth morphogenesis largely by fine tuning conserved signaling networks and that miRNAs may have played important roles during tooth evolution.

Sox2 marks epithelial competence to generate teeth in mammals and reptiles

Tooth renewal is initiated from epithelium associated with existing teeth. The development of new teeth requires dental epithelial cells that have competence for tooth formation, but specific marker genes for these cells have not been identified. Here, we analyzed expression patterns of the transcription factor Sox2 in two different modes of successional tooth formation: tooth replacement and serial addition of primary teeth. We observed specific Sox2 expression in the dental lamina that gives rise to successional teeth in mammals with one round of tooth replacement as well as in reptiles with continuous tooth replacement. Sox2 was also expressed in the dental lamina during serial addition of mammalian molars, and genetic lineage tracing indicated that Sox2+ cells of the first molar give rise to the epithelial cell lineages of the second and third molars. Moreover, conditional deletion of Sox2 resulted in hyperplastic epithelium in the forming posterior molars. Our results indicate that the Sox2+ dental epithelium has competence for successional tooth formation and that Sox2 regulates the progenitor state of dental epithelial cells. The findings imply that the function of Sox2 has been conserved during evolution and that tooth replacement and serial addition of primary teeth represent variations of the same developmental process. The expression patterns of Sox2 support the hypothesis that dormant capacity for continuous tooth renewal exists in mammals.

BMP2 and BMP7 play antagonistic roles in feather induction.

Feathers, like hairs, first appear as primordia consisting of an epidermal placode associated with a dermal condensation that is necessary for the continuation of their differentiation. Previously, the BMPs have been proposed to inhibit skin appendage formation. We show that the function of specific BMPs during feather development is more complex. BMP2 and BMP7, which are expressed in both the epidermis and the dermis, are involved in an antagonistic fashion in regulating the formation of dermal condensations, and thus are both necessary for subsequent feather morphogenesis. BMP7 is expressed earlier and functions as a chemoattractant that recruits cells into the condensation, whereas BMP2 is expressed later, and leads to an arrest of cell migration, likely via its modulation of the EIIIA fibronectin domain and α4 integrin expression. Based on the observed cell proliferation, chemotaxis and the timing of BMP2 and BMP7 expression, we propose a mathematical model, a reaction-diffusion system, which not only simulates feather patterning, but which also can account for the negative effects of excess BMP2 or BMP7 on feather formation.

Identification and Validation of Human Papillomavirus Encoded microRNAs

We report here identification and validation of the first papillomavirus encoded microRNAs expressed in human cervical lesions and cell lines. We established small RNA libraries from ten human papillomavirus associated cervical lesions including cancer and two human papillomavirus harboring cell lines. These libraries were sequenced using SOLiD 4 technology. We used the sequencing data to predict putative viral microRNAs and discovered nine putative papillomavirus encoded microRNAs. Validation was performed for five candidates, four of which were successfully validated by qPCR from cervical tissue samples and cell lines: two were encoded by HPV 16, one by HPV 38 and one by HPV 68. The expression of HPV 16 microRNAs was further confirmed by in situ hybridization, and colocalization with p16INK4A was established. Prediction of cellular target genes of HPV 16 encoded microRNAs suggests that they may play a role in cell cycle, immune functions, cell adhesion and migration, development, and cancer. Two putative viral target sites for the two validated HPV 16 miRNAs were mapped to the E5 gene, one in the E1 gene, two in the L1 gene and one in the LCR region. This is the first report to show that papillomaviruses encode their own microRNA species. Importantly, microRNAs were found in libraries established from human cervical disease and carcinoma cell lines, and their expression was confirmed in additional tissue samples. To our knowledge, this is also the first paper to use in situ hybridization to show the expression of a viral microRNA in human tissue.

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.

The Dynamic Interest in Topics within the Biomedical Scientific Community

The increase in the size of the scientific community created an explosion in scientific production. We have analyzed the dynamics of biomedical scientific output during 1957–2007 by applying a bibliometric analysis of the PubMed database using different keywords representing specific biomedical topics. With the assumption that increased scientific interest will result in increased scientific output, we compared the output of specific topics to that of all scientific output. This analysis resulted in three broad categories of topics; those that follow the general trend of all scientific output, those that show highly variable output, and attractive topics which are new and grow explosively. The analysis of the citation impact of the scientific output resulted in a typical longtail distribution: the majority of journals and articles are of very low impact. This distribution has remained unchanged since 1957, although the interests of scientists must have shifted in this period. We therefore analyzed the distribution of articles in top journals and lower impact journals over time for the attractive topics. Novelty is rewarded by publication in top journals. Over time more articles are published in low impact journals progressively creating the longtail distribution, signifying acceptance of the topic by the community. There can be a gap of years between novelty and acceptance. Within topics temporary novelty is created with new subtopics. In conclusion, the longtail distribution is the foundation of the scientific output of the scientific community and can be used to examine different aspects of science practice.

What is the biological basis of pattern formation of skin lesions

Abstract:  Pattern recognition is at the heart of clinical dermatology and dermatopathology. Yet, while every practitioner of the art of dermatological diagnosis recognizes the supreme value of diagnostic cues provided by defined patterns of ‘efflorescences’, few contemplate on the biological basis of pattern formation in and of skin lesions. Vice versa, developmental and theoretical biologists, who would be best prepared to study skin lesion patterns, are lamentably slow to discover this field as a uniquely instructive testing ground for probing theoretical concepts on pattern generation in the human system. As a result, we have at best scraped the surface of understanding the biological basis of pattern formation of skin lesions, and widely open questions dominate over definitive answer. As a symmetry‐breaking force, pattern formation represents one of the most fundamental principles that nature enlists for system organization. Thus, the peculiar and often characteristic arrangements that skin lesions display provide a unique opportunity to reflect upon – and to experimentally dissect – the powerful organizing principles at the crossroads of developmental, skin and theoretical biology, genetics, and clinical dermatology that underlie these – increasingly less enigmatic – phenomena. The current ‘Controversies’ feature offers a range of different perspectives on how pattern formation of skin lesions can be approached. With this, we hope to encourage more systematic interdisciplinary research efforts geared at unraveling the many unsolved, yet utterly fascinating mysteries of dermatological pattern formation. In short: never a dull pattern!

Tooth evolution and dental defects: from genetic regulation network to micro-RNA fine-tuning.

Teeth, like all epithelial appendages, form via a sequential and reciprocal series of inductive signals between the epithelium and the underlying mesenchyme. The genes involved in this signaling network regulating ectodermal organ development have been highly conserved during evolution and are gaining more understanding in great detail. The specific functions of numerous genes during embryogenesis are known, and the involvement of their mutations in the pathogenesis of congenital defects is being extensively studied. Recently, the micro-RNA (miRNA) pathway has been associated with various aspects of embryogenesis including ectodermal organ formation and odontogenesis. In this review, I presented the genetic network involved during tooth formation and evolution, and several mutations that give rise to dental defects. The possible impact of fine-tuning and network regulation by miRNAs on development, evolution of teeth, and defects are, therefore, discussed.

Dermal condensation formation in the chick embryo: Requirement for integrin engagement and subsequent stabilization by a possible Notch/integrin interaction

During embryonic development, feathers appear first as primordia consisting of an epidermal placode associated with a dermal condensation. When 7‐day chick embryo dorsal skin fragments showing three rows of feather primordia are cultured, they undergo a complete reorganization, which involves the down‐regulation of morphogenetic genes and dispersal of dermal fibroblasts, leading to the disappearance of primordia. This loss of organisation is followed by de novo differentiation events. We have used this model to study potential factors involved in the formation of dermal condensations. Activation of Integrins by extracellular Manganese or intracellular Calcium prevents the initial disappearance of the dermal condensations. New primordia formation occurs even after inhibition of the Notch pathway albeit with some fusion between primordia. In conclusion, dermal fibroblast migration requires β1‐Integrin whereas the stability of dermal condensations could depend on Notch/Integrin interaction. Developmental Dynamics 236:755–768, 2007.

Mesenchymal Wnt/β‐Catenin Signaling Controls Epithelial Stem Cell Homeostasis in Teeth by Inhibiting the Antiapoptotic Effect of Fgf10

Continuous growth of rodent incisors relies on epithelial stem cells (SCs) located in the SC niche called labial cervical loop (LaCL). Here, we found a population of apoptotic cells residing in a specific location of the LaCL in mouse incisor. Activated Caspase 3 and Caspase 9, expressed in this location colocalized in part with Lgr5 in putative SCs. The addition of Caspase inhibitors to incisors ex vivo resulted in concentration dependent thickening of LaCL. To examine the role of Wnt signaling in regulation of apoptosis, we exposed the LaCL of postnatal day 2 (P2) mouse incisor ex vivo to BIO, a known activator of Wnt/β‐catenin signaling. This resulted in marked thinning of LaCL as well as enhanced apoptosis. We found that Wnt/β‐catenin signaling was intensely induced by BIO in the mesenchyme surrounding the LaCL, but, unexpectedly, no β‐catenin activity was detected in the LaCL epithelium either before or after BIO treatment. We discovered that the expression of Fgf10, an essential growth factor for incisor epithelial SCs, was dramatically downregulated in the mesenchyme around BIO‐treated LaCL, and that exogenous Fgf10 could rescue the thinning of the LaCL caused by BIO. We conclude that the homeostasis of the epithelial SC population in the mouse incisor depends on a proper rate of apoptosis and that this apoptosis is controlled by signals from the mesenchyme surrounding the LaCL. Fgf10 is a key mesenchymal signal limiting apoptosis of incisor epithelial SCs and its expression is negatively regulated by Wnt/β‐catenin. Stem Cells 2015;33:1670–1681