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

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.

Spatial Transcriptomic Analysis of Pituitary Corticotroph Tumors.

Context: Spatial transcriptomic (ST) analysis of tumors provides a novel approach to studying gene expression along with the localization of tumor cells in their environment to uncover spatial interactions. Design: We present ST analysis of corticotroph pituitary neuroendocrine tumors (PitNETs) from formalin-fixed, paraffin-embedded tissues. ST data were compared to immunohistochemistry results. Gene expression profiles were reviewed for cluster annotations, and differentially expressed genes were used for pathway analysis. Results: Seven tumors were used for ST analysis. In situ annotation of tumor tissue was inferred from the gene expression profiles and was in concordance with the annotation made by a pathologist. Furthermore, relative gene expression in the tumor corresponded to common protein staining used in the evaluation of PitNETs, such as reticulin and Ki-67 index. Finally, we identified intratumor heterogeneity; clusters within the same tumor may present with different transcriptomic profiles, unveiling potential intratumor cell variability. Conclusion: Together, our results provide the first attempt to clarify the spatial cell profile in PitNETs.

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

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 Pax9-/- mouse model with a consistent phenotype of cleft secondary palate to investigate the role of Pax9 in the process of palatal osteogenesis. While prior research had identified upregulation of Wnt pathway modulators Dkk1 and Dkk2 in Pax9-/- 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 in situ highly multiplex targeted single-cell spatial profiling technology suggest a distinct relationship between Pax9+ and osteogenic populations. Loss of Pax9 results in spatially restricted osteogenic domains bounded by Dkk2, which normally interfaces with Pax9 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.

Multimodal 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.

Spatial Transcriptomic Analysis of Pituitary Corticotroph Tumors Unveils Intratumor Heterogeneity.

Spatial transcriptomic (ST) analysis of tumors provides a novel approach on studying gene expression along with the localization of tumor cells in their environment to uncover spatial interactions. Herein, we present ST analysis of corticotroph pituitary neuroendocrine tumors (PitNETs) from formalin-fixed, paraffin-embedded (FFPE) tissues. We report that the in situ annotation of tumor tissue can be inferred from the gene expression profiles and is in concordance with the annotation made by a pathologist. Furthermore, relative gene expression in the tumor corresponds to common protein staining used in the evaluation of PitNETs, such as reticulin and Ki-67 index. Finally, we identify intratumor heterogeneity; clusters within the same tumor may present with different secretory capacity and transcriptomic profiles, unveiling potential intratumor cell variability with possible therapeutic interest. Together, our results provide the first attempt to clarify the spatial cell profile in PitNETs.

Integrated spatiotemporal transcriptomic resolution of embryonic palate osteogenesis.

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 ( Deup1, Dynlrb2, Lrrc23 ) 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.

Physiology and Clinical Manifestations of Pathologic Cranial Suture Widening.

Cranial sutures are complex structures integrating mechanical forces with osteogenesis which are often affected in craniofacial syndromes. While premature fusion is frequently described, rare pathological widening of cranial sutures is a comparatively understudied phenomenon. This narrative review aims to bring to light the biologically variable underlying causes of widened sutures and persistent fontanelles leading to a common outcome. The authors herein present four syndromes, selected from a literature review, and their identified biological mechanisms in the context of altered suture physiology, exploring the roles of progenitor cell differentiation, extracellular matrix production, mineralization, and bone resorption. This article illustrates the gaps in understanding of complex craniofacial disorders, and the potential for further unification of genetics, cellular biology, and clinical pillars of health science research to improve treatment outcomes for patients.

Profiles of Wnt pathway gene expression during tooth morphogenesis.

Mouse and human genetic studies indicate key roles of the Wnt10a 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 in situ hybridization and single-cell RNA-sequencing (scRNA-seq) provide robust insight into the synchronized expression patterns of Wnt10a, Dkk1, and Sost simultaneously during tooth development. First, we identified Wnt10a transcripts restricted to the epithelium at the stage of tooth bud morphogenesis, contrasting that of Sost and Dkk1 localization to the dental mesenchyme. By embryonic day 15.5 (E15.5), a marked shift of Wnt10a expression from dental epithelium to mesenchyme was noted, while Sost and Dkk1 expression remained enriched in the mesenchyme. By postnatal day 0 (P0), co-localization patterns of Wnt10a, Dkk1, and Sost were observed in both terminally differentiating and secreting odontoblasts of molars and incisors. Interestingly, Wnt10a 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, Wnt10a expression was limited to odontoblasts. Meanwhile, Wnt modulators showed reduced or absent signals in molars. In contrast, strong signals persisted in ameloblasts (for Wnt10a) and odontoblasts (for Wnt10a, Sost, and Dkk1) 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 Wnt10a, Dkk1, and Sost 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.