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 ...

National Institutes of Health (NIH)

Massachusetts General Hospital

Harvard Medical School

Shriners Hospital for Children

Peripheral nerve injury leads to sensory ganglion hyperexcitation, which increases neurotransmitter release and neuropathic pain. Botulinum toxin type A (BoNT/A) regulates pain transmission by reducing neurotransmitter release, thereby attenuating neuropathic pain. Despite multiple studies on the use of BoNT/A for managing neuropathic pain in the orofacial region, its exact mechanism of transport remains unclear. In this study, we investigated the effects of BoNT/A in managing neuropathic pain in two different animal models and its transport mechanism in the trigeminal nerve. Intraperitoneal administration of cisplatin induced bilateral neuropathic pain in the orofacial region, reducing the head withdrawal threshold to mechanical stimulation. Unilateral infraorbital nerve constriction (IONC) also reduced the ipsilateral head withdrawal threshold to mechanical stimulation. Unilateral peripheral administration of BoNT/A to the rat whisker pad attenuated cisplatin-induced pain behavior bilaterally. Furthermore, contralateral peripheral administration of BoNT/A attenuated neuropathy-induced behavior caused by IONC. We also noted the presence of BoNT/A in the blood using the mouse bioassay. In addition, the Alexa Fluor-488-labeled C-terminal half of the heavy chain of BoNT/A (BoNT/A-Hc) was localized in the neurons of the bilateral trigeminal ganglia following its unilateral administration. These findings suggest that axonal and hematogenous transport are involved in the therapeutic effects of peripherally administered BoNT/A in the orofacial region.

Peripherally Administered Botulinum Toxin Type A Localizes Bilaterally in Trigeminal Ganglia of Animal Model.

MicroRNA (miRNA) is a short (19-24 nucleotide) endogenous non-protein RNA that exists in the body and controls the translation process from genes to proteins. It has become useful as a diagnostic tool and a potential treatment target in cancer research. To explore the function of miRNA in contact dermatitis, female participants with a positive metal allergy diagnosis ( = 3) were enrolled along with additional female participants with no medical history of metal allergy ( = 3). A patch test was performed on each participant. Peripheral blood was collected from all the participants before the patch test and at days 3 and 7 after starting the patch test. After total RNA was obtained from peripheral blood leukocytes and cDNA was generated, microarray analysis was performed to analyze the large-scale circulating miRNA profile. Real-time polymerase chain reaction (RT-PCR) was then used to clarify the overall target miRNA expression. Downregulation of hsa-let-7d-5p, hsa-miR-24-3p, hsa-miR-23b-3p, hsa-miR-26b-5p, and hsa-miR-150-5p was found on day 7. Certain miRNAs were confirmed using RT-PCR. These peripheral blood miRNAs could be diagnostic biomarkers for metal allergies.

Identification of microRNA Signatures in Peripheral Blood of Young Women as Potential Biomarkers for Metal Allergy.

The relationship between caloric and nutrient intake and overall health has been extensively studied. However, little research has focused on the impact of the hardness of staple foods on health. In this study, we investigated the effects of a soft diet on brain function and behavior in mice from an early age. Mice fed a soft diet for six months exhibited increased body weight and total cholesterol levels, along with impaired cognitive and motor function, heightened nocturnal activity, and increased aggression. Interestingly, when these mice were switched back to a solid diet for three months, their weight gain ceased, total cholesterol levels stabilized, cognitive function improved, and aggression decreased, while their nocturnal activity remained high. These findings suggest that long-term consumption of a soft diet during early development can influence various behaviors associated with anxiety and mood regulation, including weight gain, cognitive decline, impaired motor coordination, increased nocturnal activity, and heightened aggression. Therefore, the hardness of food can impact brain function, mental well-being, and motor skills during the developmental stage. Early consumption of hard foods may be crucial for promoting and maintaining healthy brain function.

Long-Term Soft-Food Rearing in Young Mice Alters Brain Function and Mood-Related Behavior.

Multiple genetic and environmental etiologies contribute to the pathogenesis of cleft palate, which constitutes the most common among the inherited disorders of the craniofacial complex. Insights into the molecular mechanisms regulating osteogenic differentiation and patterning in the palate during embryogenesis are limited and needed for the development of innovative diagnostics and cures. This study utilized the  mouse model with a consistent phenotype of cleft secondary palate to investigate the role of  in the process of palatal osteogenesis. While prior research had identified upregulation of Wnt pathway modulators  and  in  palate mesenchyme, limitations of spatial resolution and technology restricted a more robust analysis. Here, data from single-nucleus transcriptomics and chromatin accessibility assays validated by  highly multiplex targeted single-cell spatial profiling technology suggest a distinct relationship between  and osteogenic populations. Loss of  results in spatially restricted osteogenic domains bounded by , which normally interfaces with  in the mesenchyme. These results suggest that Pax9-dependent Wnt signaling modulators influence osteogenic programming during palate formation, potentially contributing to the observed cleft palate phenotype.

Spatial Multiomics Reveal the Role of Wnt Modulator, Dkk2, in Palatogenesis.

Tooth loss and decreased masticatory function reportedly affect cognitive function; tooth loss allegedly induces astrogliosis and aging of astrocytes in the hippocampus and hypothalamus, which is a response specific to the central nervous system owing to homeostasis in different brain regions. Capsaicin, a component of red peppers, has positive effects on brain disorders in mice. Decreased expression of transient receptor potential vanilloid 1, a receptor of capsaicin, is associated with the development of dementia. In this study, we investigated the effect of capsaicin administration in aged mice (C57BL/6N mice) with reduced masticatory function owing to the extraction of maxillary molars to investigate preventive/therapeutic methods for cognitive decline attributed to age-related masticatory function loss. The results demonstrated that mice with impaired masticatory function showed decreased motor and cognitive function at the behavioral level. At the genetic level, neuroinflammation, microglial activity, and astrogliosis, such as increased glial fibrillary acidic protein levels, were observed in the mouse brain. The mice with extracted molars fed on a diet containing capsaicin for 3 months demonstrated improved behavioral levels and astrogliosis, which suggest that capsaicin is useful in maintaining brain function in cases of poor oral function and prosthetic difficulties.

Long-Term Capsaicin Administration Ameliorates the Dysfunction and Astrogliosis of the Brain in Aged Mice with Missing Maxillary Molars.

The differentiation of osteoblasts and the subsequent formation of bone marks an important terminal phase in palate formation that leads to the separation of the oral and nasal cavities. While the developmental events that precede palatal osteogenesis are well explored, major gaps remain in our understanding of the molecular mechanisms that lead to the bony union of fusing palatal shelves. Herein, the timeline of osteogenic transcriptional programming is unveiled in the embryonic palate by way of integrated bulk, single-cell, and spatially resolved RNA-seq analyses. We define spatially restricted expression patterns of key marker genes, both regulatory and structural, that are differentially expressed during palatal fusion, including the identification of several novel genes (  ) spatially restricted in expression to the palate, providing a relevant framework for future studies that identify new candidate genes for cleft palate anomalies in humans as well as the timing of mammalian embryonic palatal osteogenesis.

Integrated spatiotemporal transcriptomic resolution of embryonic palate osteogenesis.

The terminal differentiation of osteoblasts and subsequent formation of bone marks an important phase in palate development that leads to the separation of the oral and nasal cavities. While the morphogenetic events preceding palatal osteogenesis are well explored, major gaps remain in our understanding of the molecular mechanisms driving the formation of this bony union of the fusing palate. Through bulk, single-nucleus, and spatially resolved RNA-sequencing analyses of the developing secondary palate, we identify a shift in transcriptional programming between embryonic days 14.5 and 15.5 pinpointing the onset of osteogenesis. We define spatially restricted expression patterns of key osteogenic marker genes that are differentially expressed between these developmental timepoints. Finally, we identify genes in the palate highly expressed by palate nasal epithelial cells, also enriched within palatal osteogenic mesenchymal cells. This investigation provides a relevant framework to advance palate-specific diagnostic and therapeutic biomarker discovery.

Multimodal spatiotemporal transcriptomic resolution of embryonic palate osteogenesis.

Mouse and human genetic studies indicate key roles of the  ligand in odontogenesis. Previous studies have identified effectors and regulators of the Wnt signaling pathway actively expressed during key stages of tooth morphogenesis. However, limitations in multiplexing and spatial resolution hindered a more comprehensive analysis of these signaling molecules. Here, profiling of transcriptomes using fluorescent multiplex  hybridization and single-cell RNA-sequencing (scRNA-seq) provide robust insight into the synchronized expression patterns of , , and  simultaneously during tooth development. First, we identified  transcripts restricted to the epithelium at the stage of tooth bud morphogenesis, contrasting that of  and  localization to the dental mesenchyme. By embryonic day 15.5 (E15.5), a marked shift of  expression from dental epithelium to mesenchyme was noted, while  and  expression remained enriched in the mesenchyme. By postnatal day 0 (P0), co-localization patterns of , , and  were observed in both terminally differentiating and secreting odontoblasts of molars and incisors. Interestingly,  exhibited robust expression in fully differentiated ameloblasts at the developing cusp tip of both molars and incisors, an observation not previously noted in prior studies. At P7 and 14, after the mineralization of dentin and enamel,  expression was limited to odontoblasts. Meanwhile, Wnt modulators showed reduced or absent signals in molars. In contrast, strong signals persisted in ameloblasts (for ) and odontoblasts (for , , and ) towards the proximal end of incisors, near the cervical loop. Our scRNA-seq analysis used CellChat to further contextualize Wnt pathway-mediated communication between cells by examining ligand-receptor interactions among different clusters. The co-localization pattern of , , and  in both terminally differentiating and secreting odontoblasts of molars and incisors potentially signifies the crucial ligand-modulator interaction along the gradient of cytodifferentiation starting from each cusp tip towards the apical region. These data provide cell type-specific insight into the role of Wnt ligands and mediators during epithelial-mesenchymal interactions in odontogenesis.

Profiles of Wnt pathway gene expression during tooth morphogenesis.

Orthodontic maxillary expansion relies on intrinsic mid-palatal suture mechanobiology to induce guided osteogenesis, yet establishment of the mid-palatal suture within the continuous secondary palate and causes of maxillary insufficiency remain poorly understood. In contrast, advances in cranial suture research hold promise to improve surgical repair of prematurely fused cranial sutures in craniosynostosis to potentially restore the obliterated signaling environment and ensure continual success of the intervention. We hypothesized that mid-palatal suture establishment is governed by shared principles with calvarial sutures and involves functional linkage between expanding primary ossification centres with the midline mesenchyme. We characterized establishment of the mid-palatal suture from late embryonic to early postnatal timepoints. Suture establishment was visualized using histological techniques and multimodal transcriptomics. We identified that mid-palatal suture formation depends on a spatiotemporally controlled signalling milieu in which tendon-associated genes play a significant role. We mapped relationships between extracellular matrix-encoding gene expression, tenocyte markers, and novel suture patency candidate genes. We identified similar expression patterns in FaceBase-deposited scRNA-seq datasets from cranial sutures. These findings demonstrate shared biological principles for suture establishment, providing further avenues for future development and understanding of maxillofacial interventions.

Tendon-associated gene expression precedes osteogenesis in mid-palatal suture establishment.

Aging is associated with a decline in oral immune function, marked by reduced levels of antimicrobial peptides such as defensins. Capsaicin, a bioactive component found in chili peppers, has been theorized to modulate immune responses through specific receptor pathways. This study examined the effects of aging on oral defensin levels and the potential mitigating role of capsaicin, mediated by the immune response in oral tissues. We conducted a comparative analysis between young and aged mice, with or without capsaicin supplementation, for 3 months. The effect of capsaicin was also studied in vitro in senescence-induced human oral keratinocytes. We found that aging did not reduce defensin levels uniformly but did so in some instances. Capsaicin treatment increased defensin levels in these cases, potentially through transient receptor potential cation channel subfamily V member 1 (TRPV1)-mediated pathways in the oral cavity. Capsaicin supplementation may counteract age-related declines in oral defensin levels, enabling the maintenance of oral immune function during aging.

Capsaicin modulates TRPV1, induces β-defensin expression, and regulates NF-κB in oral senescent cells and a murine model.

Despite advances in understanding the morphological disruptions that lead to defects in palate formation, the precise perturbations within the signaling microenvironment of palatal clefts remain poorly understood. To explore in greater depth the genomic basis of palatal clefts, we designed and implemented the first single cell spatial RNA-sequencing study in a cleft palate model, utilizing the   murine model at multiple developmental timepoints, which exhibits a consistent cleft palate defect. Visium HD, an emerging platform for true single-cell resolution spatially resolved transcriptomics, was employed using custom bins of 2×2 μm spatial gene expression data. Validation of spatial gene expression was then validated using custom designed Xenium In Situ mRNA spatial profiling and RNAscope Multiplex assays. Functional enrichment analysis revealed a palate cell-specific perturbation in Wnt signaling effector function in tandem with disrupted expression of extracellular matrix genes in developing mesenchyme. As a key step toward laying the framework for identifying key molecular targets these data can be used for translational studies aimed at developing effective therapies for human palatal clefts.

Single Cell Spatial Transcriptomics of the Murine Embryonic Palate Links Pax9 to Patterning and Organization of Extracellular Matrix Components.

Understanding how intricate cellular networks and signaling pathways communicate during the formation of craniofacial tissues like the palate and tooth has been the subject of intense investigation for several decades. Both organ systems undergo patterning morphogenesis and the subsequent terminal differentiation of matrix-producing cells that form biomineralized matrices like bone, enamel, dentin, and cementum. Until recently, gene expression profiles could only be assessed for a select number of cells without the context of the entire milieu of genes expressed by neighboring cells and tissues. Today, the cutting-edge field of spatial transcriptomics offers a remarkable suite of innovative technologies of multiplex gene analyses and imaging that can assess the expression of a vast library of genes that are present in situ during normal and abnormal conditions. In this review, we summarize some key technologies which have in recent years enabled an unprecedented breadth and depth of transcriptomic analyses in craniofacial development. We focus in detail on select methods that our research group has applied to better understand the cellular and molecular events that drive palate and tooth development. Our overall goal is to unravel the complexities of these unique biological systems to provide meaningful biological insights into the cellular and molecular events that drive normal development. As a work-in-progress, we strive for a deeper understanding of the temporal and spatial gene expression profiles within cells and tissues during normal and abnormal palate and tooth development. Such knowledge provides the framework for further studies that can characterize the function of new or novel genes that have the potential of serving as therapeutic targets for correcting disorders like cleft palate and tooth agenesis.

Leveraging Spatial Transcriptomics to Decode Craniofacial Development.

Despite advances in understanding the morphological disruptions that lead to defects in palate formation, the precise perturbations within the signaling microenvironment of palatal clefts remain poorly understood. To explore in greater depth the genomic basis of palatal clefts, we designed and implemented the first single cell spatial RNA-sequencing study in a cleft palate model, utilizing the Pax9 murine model at multiple developmental timepoints, which exhibits a consistent cleft palate defect. Visium HD, an emerging platform for true single-cell resolution spatially resolved transcriptomics, was employed using custom bins of 2 × 2 μm spatial gene expression data. Validation of spatial gene expression was then validated using custom designed Xenium In Situ mRNA spatial profiling and RNAscope Multiplex assays. Functional enrichment analysis revealed a palate cell-specific perturbation in Wnt signaling effector function in tandem with disrupted expression of extracellular matrix genes in developing mesenchyme. As a key step toward laying the framework for identifying key molecular targets these data can be used for translational studies aimed at developing effective therapies for human palatal clefts.

Single cell spatial transcriptomics links Wnt signaling disruption to extracellular matrix development in a cleft palate model.

Interleukin-10 (IL-10) is a cytokine that can exert an analgesic effect on trigeminal neuropathic pain (TNP). However, its precise mechanism remains unclear. In this study, we investigated the antinociceptive effects of recombinant IL-10 (rIL-10) in a rat model of infraorbital nerve constriction. Using male Sprague-Dawley rats, we administered rIL-10 or phosphate-buffered saline to the intra-trigeminal ganglion and observed a peak analgesic effect at 4 h post-injection in the rIL-10 group. Real-time PCR demonstrated significant upregulation of the Proopiomelanocortin (Pomc) gene in rIL-10-treated rats, with immunofluorescence staining confirming increased expression of bioactive peptide β-endorphin (β-END) in the same group. In situ hybridization assay further localized Pomc expression to satellite glial cells and neurons in the trigeminal ganglion, with β-END exhibiting a similar distribution. To elucidate the signaling mechanism, we co-administered a STAT3 inhibitor, Stattic, which abolished the analgesic effect of rIL-10, suppressed Pomc upregulation, and reduced β-END expression. These findings indicate that the STAT3 pathway is a critical mediator of rIL-10-induced analgesia, with Pomc and β-END as potential molecular effectors.

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

Article	Year
Peripherally Administered Botulinum Toxin Type A Localizes Bilaterally in Trigeminal Ganglia of Animal Model. Toxins\| PubMed ID: 34678997	2021
Identification of microRNA Signatures in Peripheral Blood of Young Women as Potential Biomarkers for Metal Allergy. Biomedicines\| PubMed ID: 36830814	2023
Long-Term Soft-Food Rearing in Young Mice Alters Brain Function and Mood-Related Behavior. Nutrients\| PubMed ID: 37242280	2023
Spatial Multiomics Reveal the Role of Wnt Modulator, Dkk2, in Palatogenesis. bioRxiv : the preprint server for biology\| PubMed ID: 37292772	2024
Long-Term Capsaicin Administration Ameliorates the Dysfunction and Astrogliosis of the Brain in Aged Mice with Missing Maxillary Molars. Nutrients\| PubMed ID: 37299434	2023
Integrated spatiotemporal transcriptomic resolution of embryonic palate osteogenesis. bioRxiv : the preprint server for biology\| PubMed ID: 37333290	2023
Multimodal spatiotemporal transcriptomic resolution of embryonic palate osteogenesis. Nature communications\| PubMed ID: 37709732	2023
Profiles of Wnt pathway gene expression during tooth morphogenesis. Frontiers in physiology\| PubMed ID: 38274045	2023
Tendon-associated gene expression precedes osteogenesis in mid-palatal suture establishment. bioRxiv : the preprint server for biology\| PubMed ID: 38798531	2024
Capsaicin modulates TRPV1, induces β-defensin expression, and regulates NF-κB in oral senescent cells and a murine model. Genes to cells : devoted to molecular & cellular mechanisms\| PubMed ID: 39266282	2024
Single Cell Spatial Transcriptomics of the Murine Embryonic Palate Links Pax9 to Patterning and Organization of Extracellular Matrix Components. Research square\| PubMed ID: 40034445	2025
Leveraging Spatial Transcriptomics to Decode Craniofacial Development. Genes\| PubMed ID: 40428379	2025
Single cell spatial transcriptomics links Wnt signaling disruption to extracellular matrix development in a cleft palate model. Scientific reports\| PubMed ID: 40804270	2025
Antinociceptive role of IL-10/STAT3 signaling in trigeminal neuropathic pain male rat model through Pomc and β-endorphin. Neuroscience letters\| PubMed ID: 41421421	2026

Resmi Raju

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