Vascular architecture regulates mesenchymal stromal cell heterogeneity via P53-PDGF signaling in the mouse incisor.

Mesenchymal stem cells (MSCs) reside in niches to maintain tissue homeostasis and contribute to repair and regeneration. Although the physiological functions of blood and lymphatic vasculature are well studied, their regulation of MSCs as niche components remains largely unknown. Using adult mouse incisors as a model, we uncover the role of Trp53 in regulating vascular composition through THBS2 to maintain mesenchymal tissue homeostasis. Loss of Trp53 in GLI1+ progeny increases arteries and decreases other vessel types. Platelet-derived growth factors from arteries deposit in the MSC region and interact with PDGFRA and PDGFRB. Significantly, PDGFRA+ and PDGFRB+ cells differentially contribute to defined cell lineages in the adult mouse incisor. Collectively, our results highlight Trp53's importance in regulating the vascular niche for MSCs. They also shed light on how different arterial cells provide unique cues to regulate MSC subpopulations and maintain their heterogeneity. Furthermore, they provide mechanistic insight into MSC-vasculature crosstalk.

ARID1B maintains mesenchymal stem cell quiescence via inhibition of BCL11B-mediated non-canonical Activin signaling.

ARID1B haploinsufficiency in humans causes Coffin-Siris syndrome, associated with developmental delay, facial dysmorphism, and intellectual disability. The role of ARID1B has been widely studied in neuronal development, but whether it also regulates stem cells remains unknown. Here, we employ scRNA-seq and scATAC-seq to dissect the regulatory functions and mechanisms of ARID1B within mesenchymal stem cells (MSCs) using the mouse incisor model. We reveal that loss of Arid1b in the GLI1+ MSC lineage disturbs MSCs' quiescence and leads to their proliferation due to the ectopic activation of non-canonical Activin signaling via p-ERK. Furthermore, loss of Arid1b upregulates Bcl11b, which encodes a BAF complex subunit that modulates non-canonical Activin signaling by directly regulating the expression of activin A subunit, Inhba. Reduction of Bcl11b or non-canonical Activin signaling restores the MSC population in Arid1b mutant mice. Notably, we have identified that ARID1B suppresses Bcl11b expression via specific binding to its third intron, unveiling the direct inter-regulatory interactions among BAF subunits in MSCs. Our results demonstrate the vital role of ARID1B as an epigenetic modifier in maintaining MSC homeostasis and reveal its intricate mechanistic regulatory network in vivo, providing novel insights into the linkage between chromatin remodeling and stem cell fate determination.

Sensory nerve regulates progenitor cells via FGF-SHH axis in tooth root morphogenesis.

Nerves play important roles in organ development and tissue homeostasis. Stem/progenitor cells differentiate into different cell lineages responsible for building the craniofacial organs. The mechanism by which nerves regulate stem/progenitor cell behavior in organ morphogenesis has not yet been comprehensively explored. Here, we use tooth root development in mouse as a model to investigate how sensory nerves regulate organogenesis. We show that sensory nerve fibers are enriched in the dental papilla at the initiation of tooth root development. Through single cell RNA-sequencing analysis of the trigeminal ganglion and developing molar, we reveal several signaling pathways that connect the sensory nerve with the developing molar, of which FGF signaling appears to be one of the important regulators. Fgfr2 is expressed in the progenitor cells during tooth root development. Loss of FGF signaling leads to shortened roots with compromised proliferation and differentiation of progenitor cells. Furthermore, Hh signaling is impaired in Gli1-CreER;Fgfr2fl/fl mice. Modulation of Hh signaling rescues the tooth root defects in these mice. Collectively, our findings elucidate the nerve-progenitor crosstalk and reveal the molecular mechanism of the FGF-SHH signaling cascade during tooth root morphogenesis.

Lineage-specific mutation of Lmx1b provides new insights into distinct regulation of suture development in different areas of the calvaria.

The calvaria (top part of the skull) is made of pieces of bone as well as multiple soft tissue joints called sutures. The latter is crucial to the growth and morphogenesis of the skull, and thus a loss of calvarial sutures can lead to severe congenital defects in humans. During embryogenesis, the calvaria develops from the cranial mesenchyme covering the brain, which contains cells originating from the neural crest and the mesoderm. While the mechanism that patterns the cranial mesenchyme into bone and sutures is not well understood, function of Lmx1b, a gene encoding a LIM-domain homeodomain transcription factor, plays a key role in this process. In the current study, we investigated a difference in the function of Lmx1b in different parts of the calvaria using neural crest-specific and mesoderm-specific Lmx1b mutants. We found that Lmx1b was obligatory for development of the interfrontal suture and the anterior fontanel along the dorsal midline of the skull, but not for the posterior fontanel over the midbrain. Also, Lmx1b mutation in the neural crest-derived mesenchyme, but not the mesoderm-derived mesenchyme, had a non-cell autonomous effect on coronal suture development. Furthermore, overexpression of Lmx1b in the neural crest lineage had different effects on the position of the coronal suture on the apical part and the basal part. Other unexpected phenotypes of Lmx1b mutants led to an additional finding that the coronal suture and the sagittal suture are of dual embryonic origin. Together, our data reveal a remarkable level of regional specificity in regulation of calvarial development.

Canonical Wnt signaling regulates soft palate development by mediating ciliary homeostasis.

Craniofacial morphogenesis requires complex interactions involving different tissues, signaling pathways, secreted factors and organelles. The details of these interactions remain elusive. In this study, we have analyzed the molecular mechanisms and homeostatic cellular activities governing soft palate development to improve regenerative strategies for individuals with cleft palate. We have identified canonical Wnt signaling as a key signaling pathway primarily active in cranial neural crest (CNC)-derived mesenchymal cells surrounding soft palatal myogenic cells. Using Osr2-Cre;ß-cateninfl/fl mice, we show that Wnt signaling is indispensable for mesenchymal cell proliferation and subsequently for myogenesis through mediating ciliogenesis. Specifically, we have identified that Wnt signaling directly regulates expression of the ciliary gene Ttll3. Impaired ciliary disassembly leads to differentiation defects in mesenchymal cells and indirectly disrupts myogenesis through decreased expression of Dlk1, a mesenchymal cell-derived pro-myogenesis factor. Moreover, we show that siRNA-mediated reduction of Ttll3 expression partly rescues mesenchymal cell proliferation and myogenesis in the palatal explant cultures from Osr2-Cre;ß-cateninfl/fl embryos. This study highlights the role of Wnt signaling in palatogenesis through the control of ciliary homeostasis, which establishes a new mechanism for Wnt-regulated craniofacial morphogenesis.

Sensory nerve niche regulates mesenchymal stem cell homeostasis via FGF/mTOR/autophagy axis.

Mesenchymal stem cells (MSCs) reside in microenvironments, referred to as niches, which provide structural support and molecular signals. Sensory nerves are niche components in the homeostasis of tissues such as skin, bone marrow and hematopoietic system. However, how the sensory nerve affects the behavior of MSCs remains largely unknown. Here we show that the sensory nerve is vital for mesenchymal tissue homeostasis and maintenance of MSCs in the continuously growing adult mouse incisor. Loss of sensory innervation leads to mesenchymal disorder and a decrease in MSCs. Mechanistically, FGF1 from the sensory nerve directly acts on MSCs by binding to FGFR1 and activates the mTOR/autophagy axis to sustain MSCs. Modulation of mTOR/autophagy restores the MSCs and rescues the mesenchymal tissue disorder of Fgfr1 mutant mice. Collectively, our study provides insights into the role of sensory nerves in the regulation of MSC homeostasis and the mechanism governing it.

TGF-ß signaling and Creb5 cooperatively regulate Fgf18 to control pharyngeal muscle development.

The communication between myogenic cells and their surrounding connective tissues is indispensable for muscle morphogenesis. During late embryonic development in mice, myogenic progenitors migrate to discrete sites to form individual muscles. The detailed mechanism of this process remains unclear. Using mouse levator veli palatini (LVP) development as a model, we systematically investigated how a distinct connective tissue subpopulation, perimysial fibroblasts, communicates with myogenic cells to regulate mouse pharyngeal myogenesis. Using single-cell RNAseq data analysis, we identified that TGF-ß signaling is a key regulator for the perimysial fibroblasts. Loss of TGF-ß signaling in the neural crest-derived palatal mesenchyme leads to defects in perimysial fibroblasts and muscle malformation in the soft palate in Osr2Cre;Tgfbr1fl/fl mice. In particular, Creb5, a transcription factor expressed in the perimysial fibroblasts, cooperates with TGF-ß signaling to activate expression of Fgf18. Moreover, Fgf18 supports pharyngeal muscle development in vivo and exogenous Fgf18 can partially rescue myogenic cell numbers in Osr2Cre;Tgfbr1fl/fl samples, illustrating that TGF-ß-regulated Fgf18 signaling is required for LVP development. Collectively, our findings reveal the mechanism by which TGF-ß signaling achieves its functional specificity in defining the perimysial-to-myogenic signals for pharyngeal myogenesis.

Spatiotemporal single-cell regulatory atlas reveals neural crest lineage diversification and cellular function during tooth morphogenesis.

Cranial neural crest cells are an evolutionary innovation of vertebrates for craniofacial development and function, yet the mechanisms that govern the cell fate decisions of postmigratory cranial neural crest cells remain largely unknown. Using the mouse molar as a model, we perform single-cell transcriptome profiling to interrogate the cell fate diversification of postmigratory cranial neural crest cells. We reveal the landscape of transcriptional heterogeneity and define the specific cellular domains during the progression of cranial neural crest cell-derived dental lineage diversification, and find that each domain makes a specific contribution to distinct molar mesenchymal tissues. Furthermore, IGF signaling-mediated cell-cell interaction between the cellular domains highlights the pivotal role of autonomous regulation of the dental mesenchyme. Importantly, we reveal cell-type-specific gene regulatory networks in the dental mesenchyme and show that Foxp4 is indispensable for the differentiation of periodontal ligament. Our single-cell atlas provides comprehensive mechanistic insight into the cell fate diversification process of the cranial neural crest cell-derived odontogenic populations.

KDM6B interacts with TFDP1 to activate P53 signaling in regulating mouse palatogenesis.

Epigenetic regulation plays extensive roles in diseases and development. Disruption of epigenetic regulation not only increases the risk of cancer, but can also cause various developmental defects. However, the question of how epigenetic changes lead to tissue-specific responses during neural crest fate determination and differentiation remains understudied. Using palatogenesis as a model, we reveal the functional significance of Kdm6b, an H3K27me3 demethylase, in regulating mouse embryonic development. Our study shows that Kdm6b plays an essential role in cranial neural crest development, and loss of Kdm6b disturbs P53 pathway-mediated activity, leading to complete cleft palate along with cell proliferation and differentiation defects in mice. Furthermore, activity of H3K27me3 on the promoter of Trp53 is antagonistically controlled by Kdm6b, and Ezh2 in cranial neural crest cells. More importantly, without Kdm6b, the transcription factor TFDP1, which normally binds to the promoter of Trp53, cannot activate Trp53 expression in palatal mesenchymal cells. Furthermore, the function of Kdm6b in activating Trp53 in these cells cannot be compensated for by the closely related histone demethylase Kdm6a. Collectively, our results highlight the important role of the epigenetic regulator KDM6B and how it specifically interacts with TFDP1 to achieve its functional specificity in regulating Trp53 expression, and further provide mechanistic insights into the epigenetic regulatory network during organogenesis.

Arid1a-Plagl1-Hh signaling is indispensable for differentiation-associated cell cycle arrest of tooth root progenitors.

Chromatin remodelers often show broad expression patterns in multiple cell types yet can elicit cell-specific effects in development and diseases. Arid1a binds DNA and regulates gene expression during tissue development and homeostasis. However, it is unclear how Arid1a achieves its functional specificity in regulating progenitor cells. Using the tooth root as a model, we show that loss of Arid1a impairs the differentiation-associated cell cycle arrest of tooth root progenitors through Hedgehog (Hh) signaling regulation, leading to shortened roots. Our data suggest that Plagl1, as a co-factor, endows Arid1a with its cell-type/spatial functional specificity. Furthermore, we show that loss of Arid1a leads to increased expression of Arid1b, which is also indispensable for odontoblast differentiation but is not involved in regulation of Hh signaling. This study expands our knowledge of the intricate interactions among chromatin remodelers, transcription factors, and signaling molecules during progenitor cell fate determination and lineage commitment.

Arid1a regulates cell cycle exit of transit-amplifying cells by inhibiting the Aurka-Cdk1 axis in mouse incisor.

Stem cells self-renew or give rise to transit-amplifying cells (TACs) that differentiate into specific functional cell types. The fate determination of stem cells to TACs and their transition to fully differentiated progeny is precisely regulated to maintain tissue homeostasis. Arid1a, a core component of the switch/sucrose nonfermentable complex, performs epigenetic regulation of stage- and tissue-specific genes that is indispensable for stem cell homeostasis and differentiation. However, the functional mechanism of Arid1a in the fate commitment of mesenchymal stem cells (MSCs) and their progeny is not clear. Using the continuously growing adult mouse incisor model, we show that Arid1a maintains tissue homeostasis through limiting proliferation, promoting cell cycle exit and differentiation of TACs by inhibiting the Aurka-Cdk1 axis. Loss of Arid1a overactivates the Aurka-Cdk1 axis, leading to expansion of the mitotic TAC population but compromising their differentiation ability. Furthermore, the defective homeostasis after loss of Arid1a ultimately leads to reduction of the MSC population. These findings reveal the functional significance of Arid1a in regulating the fate of TACs and their interaction with MSCs to maintain tissue homeostasis.

Lhx6 regulates canonical Wnt signaling to control the fate of mesenchymal progenitor cells during mouse molar root patterning.

Mammalian tooth crown formation has long served as a model for investigating how patterning and morphogenesis are orchestrated during development. However, the mechanism underlying root patterning and morphogenesis remains poorly understood. In this study, we find that Lhx6 labels a subpopulation of root progenitor cells in the apical dental mesenchyme, which is closely associated with furcation development. Loss of Lhx6 leads to furcation and root number defects, indicating that Lhx6 is a key root patterning regulator. Among the multiple cellular events regulated by Lhx6 is the odontoblast fate commitment of progenitor cells, which it controls in a cell-autonomous manner. Specifically, Lhx6 loss leads to elevated expression of the Wnt antagonist Sfrp2 and down-regulation of Wnt signaling in the furcation region, while overactivation of Wnt signaling in Lhx6+ progenitor cells partially restore the furcation defects in Lhx6-/- mice. Collectively, our findings have important implications for understanding organ morphogenesis and future strategies for tooth root regeneration.

Reciprocal interaction between mesenchymal stem cells and transit amplifying cells regulates tissue homeostasis.

Interaction between adult stem cells and their progeny is critical for tissue homeostasis and regeneration. In multiple organs, mesenchymal stem cells (MSCs) give rise to transit amplifying cells (TACs), which then differentiate into different cell types. However, whether and how MSCs interact with TACs remains unknown. Using the adult mouse incisor as a model, we present in vivo evidence that TACs and MSCs have distinct genetic programs and engage in reciprocal signaling cross talk to maintain tissue homeostasis. Specifically, an IGF-WNT signaling cascade is involved in the feedforward from MSCs to TACs. TACs are regulated by tissue-autonomous canonical WNT signaling and can feedback to MSCs and regulate MSC maintenance via Wnt5a/Ror2-mediated non-canonical WNT signaling. Collectively, these findings highlight the importance of coordinated bidirectional signaling interaction between MSCs and TACs in instructing mesenchymal tissue homeostasis, and the mechanisms identified here have important implications for MSC-TAC interaction in other organs.

Runx2-Twist1 interaction coordinates cranial neural crest guidance of soft palate myogenesis.

Cranial neural crest (CNC) cells give rise to bone, cartilage, tendons, and ligaments of the vertebrate craniofacial musculoskeletal complex, as well as regulate mesoderm-derived craniofacial muscle development through cell-cell interactions. Using the mouse soft palate as a model, we performed an unbiased single-cell RNA-seq analysis to investigate the heterogeneity and lineage commitment of CNC derivatives during craniofacial muscle development. We show that Runx2, a known osteogenic regulator, is expressed in the CNC-derived perimysial and progenitor populations. Loss of Runx2 in CNC-derivatives results in reduced expression of perimysial markers (Aldh1a2 and Hic1) as well as soft palate muscle defects in Osr2-Cre;Runx2fl/fl mice. We further reveal that Runx2 maintains perimysial marker expression through suppressing Twist1, and that myogenesis is restored in Osr2-Cre;Runx2fl/fl;Twist1fl/+ mice. Collectively, our findings highlight the roles of Runx2, Twist1, and their interaction in regulating the fate of CNC-derived cells as they guide craniofacial muscle development through cell-cell interactions.

Cranial Suture Regeneration Mitigates Skull and Neurocognitive Defects in Craniosynostosis.

Craniosynostosis results from premature fusion of the cranial suture(s), which contain mesenchymal stem cells (MSCs) that are crucial for calvarial expansion in coordination with brain growth. Infants with craniosynostosis have skull dysmorphology, increased intracranial pressure, and complications such as neurocognitive impairment that compromise quality of life. Animal models recapitulating these phenotypes are lacking, hampering development of urgently needed innovative therapies. Here, we show that Twist1+/- mice with craniosynostosis have increased intracranial pressure and neurocognitive behavioral abnormalities, recapitulating features of human Saethre-Chotzen syndrome. Using a biodegradable material combined with MSCs, we successfully regenerated a functional cranial suture that corrects skull deformity, normalizes intracranial pressure, and rescues neurocognitive behavior deficits. The regenerated suture creates a niche into which endogenous MSCs migrated, sustaining calvarial bone homeostasis and repair. MSC-based cranial suture regeneration offers a paradigm shift in treatment to reverse skull and neurocognitive abnormalities in this devastating disease.

Ror2-mediated non-canonical Wnt signaling regulates Cdc42 and cell proliferation during tooth root development.

The control of size and shape is an important part of regulatory process during organogenesis. Tooth formation is a highly complex process that fine-tunes the size and shape of the tooth, which are crucial for its physiological functions. Each tooth consists of a crown and one or more roots. Despite comprehensive knowledge of the mechanism that regulates early tooth crown development, we have limited understanding of the mechanism regulating root patterning and size during development. Here, we show that Ror2-mediated non-canonical Wnt signaling in the dental mesenchyme plays a crucial role in cell proliferation, and thereby regulates root development size in mouse molars. Furthermore, Cdc42 acts as a potential downstream mediator of Ror2 signaling in root formation. Importantly, activation of Cdc42 can restore cell proliferation and partially rescue the root development size defects in Ror2 mutant mice. Collectively, our findings provide novel insights into the function of Ror2-mediated non-canonical Wnt signaling in regulating tooth morphogenesis, and suggest potential avenues for dental tissue engineering.

Spatiotemporal cellular movement and fate decisions during first pharyngeal arch morphogenesis.

Cranial neural crest (CNC) cells contribute to different cell types during embryonic development. It is unknown whether postmigratory CNC cells undergo dynamic cellular movement and how the process of cell fate decision occurs within the first pharyngeal arch (FPA). Our investigations demonstrate notable heterogeneity within the CNC cells, refine the patterning domains, and identify progenitor cells within the FPA. These progenitor cells undergo fate bifurcation that separates them into common progenitors and mesenchymal cells, which are characterized by Cdk1 and Spry2/Notch2 expression, respectively. The common progenitors undergo further bifurcations to restrict them into osteogenic/odontogenic and chondrogenic/fibroblast lineages. Disruption of a patterning domain leads to specific mandible and tooth defects, validating the binary cell fate restriction process. Different from the compartment model of mandibular morphogenesis, our data redefine heterogeneous cellular domains within the FPA, reveal dynamic cellular movement in time, and describe a sequential series of binary cell fate decision-making process.

FaceBase 3: analytical tools and FAIR resources for craniofacial and dental research.

The FaceBase Consortium was established by the National Institute of Dental and Craniofacial Research in 2009 as a 'big data' resource for the craniofacial research community. Over the past decade, researchers have deposited hundreds of annotated and curated datasets on both normal and disordered craniofacial development in FaceBase, all freely available to the research community on the FaceBase Hub website. The Hub has developed numerous visualization and analysis tools designed to promote integration of multidisciplinary data while remaining dedicated to the FAIR principles of data management (findability, accessibility, interoperability and reusability) and providing a faceted search infrastructure for locating desired data efficiently. Summaries of the datasets generated by the FaceBase projects from 2014 to 2019 are provided here. FaceBase 3 now welcomes contributions of data on craniofacial and dental development in humans, model organisms and cell lines. Collectively, the FaceBase Consortium, along with other NIH-supported data resources, provide a continuously growing, dynamic and current resource for the scientific community while improving data reproducibility and fulfilling data sharing requirements.

Runx2+ Niche Cells Maintain Incisor Mesenchymal Tissue Homeostasis through IGF Signaling.

Stem cell niches provide a microenvironment to support the self-renewal and multi-lineage differentiation of stem cells. Cell-cell interactions within the niche are essential for maintaining tissue homeostasis. However, the niche cells supporting mesenchymal stem cells (MSCs) are largely unknown. Using single-cell RNA sequencing, we show heterogeneity among Gli1+ MSCs and identify a subpopulation of Runx2+/Gli1+ cells in the adult mouse incisor. These Runx2+/Gli1+ cells are strategically located between MSCs and transit-amplifying cells (TACs). They are not stem cells but help to maintain the MSC niche via IGF signaling to regulate TAC proliferation, differentiation, and incisor growth rate. ATAC-seq and chromatin immunoprecipitation reveal that Runx2 directly binds to Igfbp3 in niche cells. This Runx2-mediated IGF signaling is crucial for regulating the MSC niche and maintaining tissue homeostasis to support continuous growth of the adult mouse incisor, providing a model for analysis of the molecular regulation of the MSC niche.

Runx2 Regulates Mouse Tooth Root Development Via Activation of WNT Inhibitor NOTUM.

Progenitor cells are crucial in controlling organ morphogenesis. Tooth development is a well-established model for investigating the molecular and cellular mechanisms that regulate organogenesis. Despite advances in our understanding of how tooth crown formation is regulated, we have limited understanding of tooth root development. Runt-related transcription factor 2 (RUNX2) is a well-known transcription factor in osteogenic differentiation and early tooth development. However, the function of RUNX2 during tooth root formation remains unknown. We revealed in this study that RUNX2 is expressed in a subpopulation of GLI1+ root progenitor cells, and that loss of Runx2 in these GLI1+ progenitor cells and their progeny results in root developmental defects. Our results provide in vivo evidence that Runx2 plays a crucial role in tooth root development and in regulating the differentiation of root progenitor cells. Furthermore, we identified that Gli1, Pcp4, NOTUM, and Sfrp2 are downstream targets of Runx2 by integrating bulk and single-cell RNA sequencing analyses. Specifically, ablation of Runx2 results in downregulation of WNT inhibitor NOTUM and upregulation of canonical WNT signaling in the odontoblastic site, which disturbs normal odontoblastic differentiation. Significantly, exogenous NOTUM partially rescues the impaired root development in Runx2 mutant molars. Collectively, our studies elucidate how Runx2 achieves functional specificity in regulating the development of diverse organs and yields new insights into the network that regulates tooth root development. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).