spatial-transcriptomics-early-tooth-morphogenesis-formalin-fixed

Spatial Transcriptomics of Early Tooth Morphogenesis in Formalin-fixed Paraffin-embedded Mouse Embryonic Tissue

The developing tooth comprises diverse and highly specialized cell populations that work together to maintain proper form and function. Elucidating the ...

multiplex-fluorescent-mrna-situ-hybridization-developing-craniofacial

Multiplex Fluorescent mRNA <em>In Situ</em> Hybridization of Developing Craniofacial Tissues in FFPE Mouse Samples

Craniofacial development depends on the coordinated activity of diverse, specialized cell populations that interact within intricate tissue architectures ...

Massachusetts General Hospital

Harvard Medical School

Shriners Hospital for Children

The small ubiquitin-related modifier SUMO reversibly modifies many proteins, including promoter-specific transcription factors. Genetic studies in both humans and mice indicate that the Msx1 transcription factor is associated with specific disorders, including cleft palate. We show that Msx1 conjugation to SUMO-1 in vivo is enhanced by an E3 SUMO ligase, PIAS1, suggesting that sumoylation of Msx1 is a new mechanism for modulating the molecular function of Msx1 during organogenesis.

Modification of Msx1 by SUMO-1.

Late tooth morphogenesis is characterized by a series of events that determine crown morphogenesis and the histodifferentiation of epithelial cells into enamel-secreting ameloblasts and of mesenchymal cells into dentin-secreting odontoblasts. Functional ameloblasts are tall, columnar, polarized cells that synthesize and secrete a number of enamel-specific proteins. After depositing the full thickness of enamel matrix, ameloblasts shrink in size and regulate enamel maturation. Amelogenesis imperfecta (AI) is a heterogeneous group of inherited defects in enamel formation. Clinically, AI presents as a spectrum of enamel malformations that are categorized as hypoplastic, hypocalcified, or hypomaturation types, based upon the thickness and hardness of the enamel. The different types of AI are inherited, either as X-linked, autosomal-dominant, or autosomal-recessive traits. Recently, several gene mutations have been identified to cause the subtypes of AI. How these genes, however, coordinate their function to control amelogenesis is not understood. In this review, we discuss the role of genes that play definitive role on the determination of ameloblast cell fate and life cycle based on studies in transgenic animals.

Molecular genetics of ameloblast cell lineage.

Organogenesis depends upon a well-ordered series of inductive events involving coordination of molecular pathways that regulate the generation and patterning of specific cell types. Key questions in organogenesis involve the identification of the molecular mechanisms by which proteins interact to organize distinct pattern formation and cell fate determination. Tooth development is an excellent context for investigating this complex problem because of the wealth of information emerging from studies of model organisms and human mutations. Since there are no obvious sources of stem cells in adult human teeth, any attempt to create teeth de novo will probably require the reprogramming of other cell types. Thus, the fundamental understanding of the control mechanisms responsible for normal tooth patterning in the embryo will help us understand cell fate specificity and may provide valuable information towards tooth organ regeneration.

Molecular genetics of tooth development.

Proliferative scarring is a human disease with neither available effective treatment nor relevant animal model. One of the hypotheses for scar formation involves deregulation of fibroblast signaling and delayed apoptosis. Here, we introduce a new chemical-free method for fibroblast density control in culture by intermittently delivered pulsed electric fields (IDPEF), which cause irreversible damage to cell membranes. Using 5-100 pulses with electric field strength of 150 V/mm, pulse duration 70 µs, and frequency of 1 Hz, we investigated the effects of PEF application on growth, death, and regeneration of normal human dermal fibroblasts in culture. We found that the fraction of fibroblasts that survive depends on the number of pulses applied and follows a Weibull distribution. We have successfully developed an IDPEF protocol that controls fibroblasts density in culture. Specifically, through application of IDPEF every 72 h for 12 days, we maintain a normal human dermal fibroblast density in the 3.1 ± 0.2 × 10(5) -1.4 ± 0.2 × 10(5)  cell/mL range. Our results suggest that IDPEFs may prove useful as a non-chemical method for fibroblast density control in human wound healing.

Regeneration and control of human fibroblast cell density by intermittently delivered pulsed electric fields.

An improved understanding of the molecular pathways that drive tooth morphogenesis and enamel secretion is needed to generate teeth from organ cultures for therapeutic implantation or to determine the pathogenesis of primary disorders of dentition (Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., and Wrana, J. L. (1997) J. Biol. Chem. 272, 27678-27685). Here we present a novel ectodermal dysplasia phenotype associated with conditional deletion of p38α MAPK in ectodermal appendages using K14-cre mice (p38α(K14) mice). These mice display impaired patterning of dental cusps and a profound defect in the production and biomechanical strength of dental enamel because of defects in ameloblast differentiation and activity. In the absence of p38α, expression of amelogenin and β4-integrin in ameloblasts and p21 in the enamel knot was significantly reduced. Mice lacking the MAP2K MKK6, but not mice lacking MAP2K MKK3, also show the enamel defects, implying that MKK6 functions as an upstream kinase of p38α in ectodermal appendages. Lastly, stimulation with BMP2/7 in both explant culture and an ameloblast cell line confirm that p38α functions downstream of BMPs in this context. Thus, BMP-induced activation of the p38α MAPK pathway is critical for the morphogenesis of tooth cusps and the secretion of dental enamel.

p38α MAPK is required for tooth morphogenesis and enamel secretion.

Previous studies indicate that ion channels are mediators of bioelectricity promoting wound closure/regeneration in nonmammalian, lower vertebrate systems. The role of ion channels however in regeneration of wounds in mammalian systems that do not regenerate as adults is not yet defined. Using a mammalian model system that allows us to determine differentially expressed genes when skin regenerates and when skin does not regenerate after wound induction, we identified two potassium channels, kcnh2 and kcnj8, to be (1) differentially expressed between the two states and (2) highly expressed after wound induction at the nonregenerative state. We also found that kcnh2 small molecule inhibitor enhanced wound healing while kcnj8 small molecule inhibitor did not. In contrast, kcnj8 activator accelerated wound healing and even augmented the effect of kcnh2 inhibition. These results provide evidence for the first time that potassium channels may mediate skin wound healing and regeneration interactively.

Kcnh2 and Kcnj8 interactively regulate skin wound healing and regeneration.

Skin scar formation is a complex process that results in the formation of dense extracellular matrix (ECM) without normal skin appendages such as hair and glands. The absence of a scarless healing model in adult mammals prevents the development of successful therapies. We show that irreversible electroporation of skin drives its regeneration with all accessory organs in normal adult rats. Pulsed electric fields at 500 V, with 70 μs pulse duration and 1000 pulses delivered at 3 Hz, applied through two electrodes separated by 2 mm lead to massive cell death. However, the ECM architecture of the skin was preserved. Six months after the ablation, the epidermis, sebaceous glands, panniculus carnosus, hair follicles, microvasculature and arrector pili muscle were altogether re-formed in the entire ablated area. These results suggest a key role of the ECM architecture in the differentiation, migration and signalling of cells during scarless wound healing. Copyright © 2016 John Wiley & Sons, Ltd.

Skin regeneration with all accessory organs following ablation with irreversible electroporation.

Dental stem cells (DSCs) have emerged as a promising tool for basic research and clinical practice. A variety of adult stem cell (ASC) populations can be isolated from different areas within the dental tissue, which, due to their cellular and molecular characteristics, could give rise to different outcomes when used in potential applications. In this study, we performed a high-throughput molecular comparison of two primary human adult dental stem cell (hADSC) sub-populations: Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) and Periodontal Ligament Stem Cells (PDLSCs). A detailed proteomic mapping of SHEDs and PDLSCs, via employment of nano-LC tandem-mass spectrometry (MS/MS) revealed 2032 identified proteins in SHEDs and 3235 in PDLSCs. In total, 1516 proteins were expressed in both populations, while 517 were unique for SHEDs and 1721 were exclusively expressed in PDLSCs. Further analysis of the recorded proteins suggested that SHEDs predominantly expressed molecules that are involved in organizing the cytoskeletal network, cellular migration and adhesion, whereas PDLSCs are highly energy-producing cells, vastly expressing proteins that are implicated in various aspects of cell metabolism and proliferation. Applying the Rho-GDI signaling pathway as a paradigm, we propose potential biomarkers for SHEDs and for PDLSCs, reflecting their unique features, properties and engaged molecular pathways.

A High-Resolution Proteomic Landscaping of Primary Human Dental Stem Cells: Identification of SHED- and PDLSC-Specific Biomarkers.

Editorial: On Tissue Engineering and Regenerative Medicine of Skin and Its Appendages.

Ion channels are a large family of transmembrane proteins, accessible by soluble membrane-impermeable molecules, and thus are targets for development of therapeutic drugs. Ion channels are the second most common target for existing drugs, after G protein-coupled receptors, and are expected to make a big impact on precision medicine in many different diseases including wound repair and regeneration. Research has shown that endogenous bioelectric signaling mediated by ion channels is critical in non-mammalian limb regeneration. However, the role of ion channels in regeneration of limbs in mammalian systems is not yet defined.

Potassium channels as potential drug targets for limb wound repair and regeneration.

Ameloblasts are epithelially derived cells responsible for enamel formation through a process known as amelogenesis. Amongst the several transcription factors that are expressed during amelogenesis, both  and  transcription factors play important role.  and  mouse mutants, exhibit similar amelogenesis defects, namely enamel hypoplasia, while humans with amelogenesis imperfecta (AI) carry mutations in the human homologues of MSX2 or SP6 genes. These across species similarities in function indicate that these two transcription factors may reside in the same developmental pathway. In this paper, we test whether they work in a coordinated manner to exert their effect during amelogenesis.

An - Pathway Operates During Late Stages of Tooth Development to Control Amelogenesis.

In these studies, we explored for the first time the molecular relationship between the paired-domain-containing transcription factor, , and the ectodysplasin () signaling pathway during mouse incisor formation. Mice that were deficient in both  and  were generated, and the status of dentition analyzed in all progeny using gross evaluation and histomorphometric means. When compared to wildtype controls,  mice lack mandibular incisors. Interestingly,  and  signaling are down-regulated while  and  appear unaffected. These findings suggest that -dependent signaling involves the  pathway and that this genetic relationship is important for mandibular incisor development. Studies of records of humans affected by mutations in  lead to the congenital absence of posterior dentition but interestingly involve agenesis of mandibular central incisors. The latter phenotype is exhibited by individuals with  or  mutations. Thus, it is likely that , in addition to playing a role in the formation of more complex dentition, is also involved with  signaling in the initiation of odontogenesis within the incisal domain.

's Interaction With the Ectodysplasin Signaling Pathway During the Patterning of Dentition.

The precise formation of mineralized dental tissues such as enamel and/or dentin require tight transcriptional control of the secretion of matrix proteins. Here, we have investigated the transcriptional regulation of the second most prominent enamel matrix protein, enamelin, and its regulation through the major odontogenic transcription factor, MSX2.

Enam expression is regulated by Msx2.

The  gene belongs to the NK2 class of homeobox genes that play important role in vertebrate development and share overlapping expression patterns.  are involved in several pathways that lead to cell differentiation, migration, and maturation of the cells, indicating their essential role in the formation and homeostasis of the organism. Here we report that the  mouse mutants have an orofacial phenotype affecting among other structures and the developing teeth. Specifically, we confirm that while incisors and upper molars are normal, the mandibular molars of the mutants have abnormal crown shape. Second, we provide for the first time evidence that the absence of  affects the differentiation process of the two most essential dental cell populations, namely the ameloblasts and the odontoblasts. Macroscopic, histological, 3D micro-CT of the lower molars of  mouse mutants exhibit enamel and dentin phenotypes with the expression of the  and  genes to be selectively reduced in  deficient secretory ameloblasts and dentinoblasts, respectively.

ArticleTotal : 2	Year
Publication title Cited by 1	2026
Publication title	2026

Article	Year
Modification of Msx1 by SUMO-1. Biochemical and biophysical research communications\| PubMed ID: 16678795	2006
Molecular genetics of ameloblast cell lineage. Journal of experimental zoology. Part B, Molecular and developmental evolution\| PubMed ID: 19090561	2009
Molecular genetics of tooth development. Current opinion in genetics & development\| PubMed ID: 19875280	2009
Regeneration and control of human fibroblast cell density by intermittently delivered pulsed electric fields. Biotechnology and bioengineering\| PubMed ID: 23297079	2013
p38α MAPK is required for tooth morphogenesis and enamel secretion. The Journal of biological chemistry\| PubMed ID: 25406311	2015
Kcnh2 and Kcnj8 interactively regulate skin wound healing and regeneration. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society\| PubMed ID: 26220146	2015
Skin regeneration with all accessory organs following ablation with irreversible electroporation. Journal of tissue engineering and regenerative medicine\| PubMed ID: 27976527	2018
A High-Resolution Proteomic Landscaping of Primary Human Dental Stem Cells: Identification of SHED- and PDLSC-Specific Biomarkers. International journal of molecular sciences\| PubMed ID: 29304003	2018
Editorial: On Tissue Engineering and Regenerative Medicine of Skin and Its Appendages. Frontiers in physiology\| PubMed ID: 31244678	2019
Potassium channels as potential drug targets for limb wound repair and regeneration. Precision clinical medicine\| PubMed ID: 32257531	2020
An - Pathway Operates During Late Stages of Tooth Development to Control Amelogenesis. Frontiers in physiology\| PubMed ID: 33192593	2020
's Interaction With the Ectodysplasin Signaling Pathway During the Patterning of Dentition. Frontiers in physiology\| PubMed ID: 33329029	2020
Enam expression is regulated by Msx2. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 37191055	2023
Homeobox Gene Plays an Essential Role in Later Stages of Orofacial Differentiation. Annals of pediatrics & child health\| PubMed ID: 40416342	2025

Marianna Bei

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