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This study formulates three-dimensional (3D) residual flow, treating both stationary and transient waves. The zonal and meridional momentum equations contain four terms: the geostrophic wind tendency, Coriolis force for the residual horizontal flow, product of the geostrophic wind and potential vorticity other than the constant planetary vorticity, and friction. The thermodynamic equation contains three terms: the potential temperature tendency, advection of the basic potential temperature by the residual vertical flow, and diabatic heating. The zonal mean of the 3D residual flow equals the time mean of the residual flow of the transformed Eulerian-mean equations. The new residual flow is the sum of that derived by Plumb for transient waves and the quadratic terms of the time-mean fields, which correspond approximately to the Stokes correction due to stationary waves. The 3D residual flow and momentum equations are symmetric in the zonal and meridional directions, in contrast with those formulated by Kinoshita et al., which treat the time-mean zonal-mean zonal wind as the basic wind. The newly derived formulas are applied to the climatology of the 3D structure of the deep branch of the Brewer–Dobson circulation. In the Northern Hemisphere in December–February, the residual flows are directed inward toward the polar vortex strongly over east Siberia, where the downward flow is maximized, and weakly over the Atlantic; meanwhile, they are directed outward from the vortex over North America and Europe. A longitudinal dependence of the poleward flow is also observed in the Southern Hemisphere in June–August.

PURPOSE Since cone-beam computed tomography was developed, a number of radiological studies on the bifid mandibular canals (BMCs) and trifid mandibular canals (TMCs) have been reported. However, many of the suggested subtypes of the BMC described in the literature seem to be normal branches of the inferior alveolar nerve. This might be due to a lack of revisiting classic anatomical studies in the field of radiology. Therefore, such studies are revisited here. METHODS A database search using PubMed and Google Scholar was conducted on BMC and TMC. Eighty-nine articles underwent full-text assessment. The reported three classifications of BMC and the six modified classifications were reviewed and compared to the intramandibular inferior alveolar nerve branches. RESULTS Some subtypes of BMC and TMC simply represent normal inferior alveolar nerve branches, i.e., retromolar branch, molar branch (alveolar branch/dental branch), large mental branch, or communicating branch. Others such as Naitoh's type III BMC and forward canal might be a true BMC. CONCLUSION We found that the bifid mandibular canal is an additional intramandibular canal running parallel to the mandibular canal with/without confluence with the main canal through comparison of classifications of BMC/TMC between the radiology and anatomy fields.

Population aging is a global phenomenon and with it, the number of bone fractures increases due to higher incidences of osteoporosis. Bone fractures in the elderly increase the risk of bedridden status and mortality. Therefore, the control of osteoporosis and bone fracture is important for healthy life expectancy, and the fundamental understanding of its pathogenesis and its application in treatment is of great social significance. To solve these clinical problems, it is necessary to integrate clinical medicine and basic research. Bone regeneration after a fracture is an essential function of the living body. The prevailing view is that a small number of resident skeletal stem cells are solely responsible for regenerative capacity. Although these cells have long been considered to be in the bone marrow, it has been shown that they are also present in the growth plate and periosteum. More recently, distinct types of cells in the bone marrow, including bone marrow stromal cells, osteoblast progenitor cells, and osteoblasts, have been shown to participate in bone regeneration. Interestingly, the cellular plasticity of differentiated cells, rather than active recruitment of resident stem cell populations, may largely account for regeneration of bone tissues; terminally differentiated cells de-differentiate into a stem cell-like state, and then re-differentiate into regenerating bone. In this review, we discuss the clinical risk and preventive therapy of bone fractures and the current concept of bone regeneration in basic mechanical insights, which may prove useful to both clinicians and researchers.

Sufficient knowledge of anatomy is critical for oral and maxillofacial surgeons to provide the best treatment to their patients. The authors have recently established the "Clinical Anatomy Research Association in Oral and Maxillofacial Surgery." There is no doubt as to the benefits of collaboration between oral and maxillofacial surgeons/radiologists and anatomists. In this article, we share what was accomplished at the first annual online conference and discuss our mission for the future.

Lower third molar removal is the most commonly performed dental surgical procedure. Nevertheless, it is difficult to ensure that all the informed consent forms given to patients are based on the best evidence as many newer publications could change the conclusions of previous research. Therefore, the goal of this review article is to cover existing meta-analyses, randomized control trials, and related articles in order to collect data for improved and more current informed consent.

PURPOSE OF REVIEW: Skeletal stem cells (SSCs) are considered to play important roles in bone development and repair. These cells have been historically defined by their in vitro potential for self-renewal and differentiation into “trilineage” cells; however, little is known about their in vivo identity. Here, we discuss recent progress on SSCs and how they potentially contribute to bone development and repair. RECENT FINDINGS: Bone is composed of diverse tissues, which include cartilage and its perichondrium, cortical bone and its periosteum, and bone marrow and its trabecular bone and stromal compartment. We are now at the initial stage of understanding the precise identity of SSCs in each bone tissue. The emerging concept is that functionally dedicated SSCs are encased by their own unique cellular and extracellular matrix microenvironment, and locally support its own compartment. SUMMARY: Diverse groups of SSCs are likely to work in concert to achieve development and repair of the highly functional skeletal organ.

Background & Aims Liver lobules are typically subdivided into 3 metabolic zones: zones 1, 2, and 3. However, the contribution of zonal differences in hepatocytes to liver regeneration, as well as to carcinogenic susceptibility, remains unclear. Methods We developed a new method for sustained genetic labelling of zone 3 hepatocytes and performed fate tracing to monitor these cells in multiple mouse liver tumour models. Results We first examined changes in the zonal distribution of the Wnt target gene Axin2 over time using Axin2-CreERT2;Rosa26-Lox-Stop-Lox-tdTomato mice (Axin2;tdTomato). We found that following tamoxifen administration at 3 weeks of age, approximately one-third of total hepatocytes that correspond to zone 3 were labelled in Axin2;tdTomato mice; the tdTomato+ cell distribution closely matched that of the zone 3 marker CYP2E1. Cell fate analysis revealed that zone 3 hepatocytes maintained their own lineage but rarely proliferated beyond their liver zonation during homoeostasis; this indicated that our protocol enabled persistent genetic labelling of zone 3 hepatocytes. Using this system, we found that zone 3 hepatocytes generally had high neoplastic potential, which was promoted by constitutive activation of Wnt/β-catenin signalling in the pericentral area. However, the frequency of zone 3 hepatocyte-derived tumours varied depending on the regeneration pattern of the liver parenchyma in response to liver injury. Notably, Axin2-expressing hepatocytes undergoing chronic liver injury significantly contributed to liver regeneration and possessed high neoplastic potential. Additionally, we revealed that the metabolic phenotypes of liver tumours were acquired during tumorigenesis, irrespective of their spatial origin. Conclusions Hepatocytes receiving Wnt/β-catenin signalling from their microenvironment have high neoplastic potential, and Wnt/β-catenin signalling is a potential drug target for the prevention of hepatocellular carcinoma. Lay summary Lineage tracing revealed that zone 3 hepatocytes residing in the pericentral niche have high neoplastic potential. Under chronic liver injury, hepatocytes receiving Wnt/β-catenin signalling broadly exist across all hepatic zones and significantly contribute to liver tumorigenesis as well as liver regeneration. Wnt/β-catenin signalling is a potential drug target for the prevention of hepatocellular carcinoma., Graphical abstract, Highlights • We developed a new method for sustained genetic labelling of Zone 3 hepatocytes. • Lineage tracing revealed that Zone 3 hepatocytes generally have high neoplastic potential. • The frequency of Zone 3 hepatocyte-derived tumours varied depending on the regeneration pattern of liver parenchyma. • Under chronic liver injury, hepatocytes receiving Wnt/β-catenin signalling significantly contributed to tumorigenesis. • Wnt/β-catenin signalling is a potential drug target for the prevention of HCC.

A number of studies have previously shown variations of inferior alveolar, however, only a few reports focused on nearby the foramen ovale. In a formalin fixed cadaver, we identified three minor branches (anterior, middle, and posterior branches) arising from the main trunk of the mandibular nerve adjacent to the foramen ovale, passing lateral to the maxillary artery (MA), and joining the inferior alveolar nerve. The diameter of the branches was 0.68 mm, 1.43 mm, and 0.40 mm, respectively. The branches traveled inside the lateral pterygoid muscle (LPM) or between the LPM and tensor/levator veli palatini. Moreover, all of the branches were superficial to MA. Knowledge of such a variation might be helpful to dentists during, for example, anesthetic blockade and various oral surgeries.

A photocatalytic strategy for regioselective synthesis of phenanthridine derivatives from N-(2-arylbenzylidenamino)pyridinium salts has been developed. Utilization of an Ir photocatalyst, [Ir{dF(CF3)ppy}2(dtbbpy)]PF6 (dF(CF3)ppy = 3,5-difluoro-2-(5-(trifluoromethyl)-2-pyridyl)phenyl, dtbbpy = 4,4′-di-tert-butyl-2,2′-bipyridine), is a key to successful reactions. The excited Ir catalyst does not serve as a 1e-redox reagent but as an energy donor toward the pyridinium salts. The present system can be also applied to one-pot synthesis of Trisphaeridine, an anti-cancer drug.

OBJECTIVES: Primary failure of eruption (PFE) is a genetic disorder exhibiting the cessation of tooth eruption. Loss-of-function mutations in parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor (PTH/PTHrP receptor, PPR) were reported as the underlying cause of this disorder in humans. We showed in a PFE mouse model that PTHrP-PPR signaling is responsible for normal dental follicle cell differentiation and tooth eruption. However, the mechanism underlying the eruption defect in PFE remains undefined. In this descriptive study, we aim to chronologically observe tooth eruption and root formation of mouse PFE molars through 3D microCT analyses. SETTING AND SAMPLE POPULATION: Two individuals with PFE were recruited at Showa University. A mouse PFE model was generated by deleting PPR specifically in PTHrP-expressing dental follicle and divided into three groups, PPR(fl/fl);R26R(tdTomato/+)(Control), PTHrP-creER;PPR(fl/+);R26R(tdTomato/+)(cHet), and PTHrP-creER;PRR(fl/fl);R26R(tdTomato/+)(cKO). MATERIALS AND METHODS: Images from human PFE subjects were acquired by CBCT. All groups of mouse samples were studied at postnatal days 14, 25, 91, and 182 after a tamoxifen pulse at P3, and superimposition of 3D microCT images among three groups was rendered. RESULTS: Mouse and human PFE molars exhibited a similar presentation in the 3D CT analyses. The quantitative analysis in mice demonstrated a statistically significant decrease in the eruption height of cKO first and second molars compared to other groups after postnatal day 25. Additionally, cKO molars demonstrated significantly shortened roots with dilacerations associated with the reduced interradicular bone height. CONCLUSIONS: Mouse PFE molars erupt at a much slower rate compared to normal molars, associated with shortened and dilacerated roots and defective interradicular bones.

AIM This study aimed to evaluate changes in prenatal testing among women with twin pregnancies before and after the introduction of noninvasive prenatal testing (NIPT). To date, no consensus on prenatal testing for twin pregnancies has been reached in Japan. METHODS Women pregnant with twins who requested prenatal testing at Kyushu Medical Center from 2005 to 2018 were included in this study. Genetic counseling was provided to all participants. Their chosen methods of testing were collected and classified as invasive diagnosis (ID), noninvasive screening (NIS), and no test requested (NR). Parity, chorionicity, and methods of conception were assessed as attributes. The study period was divided into three terms according to testing availability in our center. RESULTS After NIPT was introduced in our center, the use of ID methods decreased and eventually disappeared while NIS came to the forefront. NR was also the preferred choice of women with twin pregnancies before the introduction of NIPT and decreased but did not disappear after introducing NIPT. Women with twin pregnancies who underwent assisted reproduction initially showed hesitation to undergo testing but showed a strong preference for NIS after the introduction of NIPT. Differences in choice according to parity, chorionicity, and methods of conception were found before the introduction of NIPT but disappeared after introducing NIPT. CONCLUSION Increasing information about NIPT has apparently influenced the attitudes of women with twin pregnancies to prenatal testing in Japan. In particular, those who conceive through assisted reproductive technologies exhibited a strong preference for NIPT.

Chondrocytes in the resting zone of the postnatal growth plate are characterized by slow cell cycle progression, and encompass a population of parathyroid hormone-related protein (PTHrP)-expressing skeletal stem cells that contribute to the formation of columnar chondrocytes. However, how these chondrocytes are maintained in the resting zone remains undefined. We undertook a genetic pulse-chase approach to isolate slow cycling, label-retaining chondrocytes (LRCs) from the growth plate using a chondrocyte-specific doxycycline-controllable Tet-Off system regulating expression of histone 2B-linked GFP. Comparative RNA-seq analysis identified significant enrichment of inhibitors and activators for Wnt/β-catenin signaling in LRCs and non-LRCs, respectively. Activation of Wnt/β-catenin signaling in PTHrP+resting chondrocytes usingPthrp-creERandApc-floxed allele impaired their ability to form columnar chondrocytes. Therefore, slow-cycling chondrocytes are maintained in a canonical Wnt-inhibitory environment within the resting zone, unraveling a novel mechanism regulating maintenance and differentiation of PTHrP+skeletal stem cells of the postnatal growth plate.

Bone marrow houses a multifunctional stromal cell population expressing C-X-C motif chemokine ligand 12 (CXCL12), termed CXCL12-abundant reticular (CAR) cells, that regulates osteogenesis and adipogenesis. The quiescent pre-adipocyte-like subset of CXCL12+ stromal cells ("Adipo-CAR" cells) is localized to sinusoidal surfaces and particularly enriched for hematopoiesis-supporting cytokines. However, detailed characteristics of these CXCL12+ pre-adipocyte-like stromal cells and how they contribute to marrow adipogenesis remain largely unknown. Here we highlight CXCL12-dependent physical coupling with hematopoietic cells as a potential mechanism regulating the adipogenic potential of CXCL12+ stromal cells. Single-cell computational analyses of RNA velocity and cell signaling reveal that Adipo-CAR cells exuberantly communicate with hematopoietic cells through CXCL12-CXCR4 ligand-receptor interactions but do not interconvert with Osteo-CAR cells. Consistent with this computational prediction, a substantial fraction of Cxcl12-creER+ pre-adipocyte-like cells intertwines with hematopoietic cells in vivo and in single-cell preparation in a protease-sensitive manner. Deletion of CXCL12 in these cells using Col2a1-cre leads to a reduction of stromal-hematopoietic coupling and extensive marrow adipogenesis in adult bone marrow, which appears to involve direct conversion of CXCL12+ cells to lipid-laden marrow adipocytes without altering mesenchymal progenitor cell fates. Therefore, these findings suggest that CXCL12+ pre-adipocyte-like marrow stromal cells prevent their premature differentiation by maintaining physical coupling with hematopoietic cells in a CXCL12-dependent manner, highlighting a possible cell-non-autonomous mechanism that regulates marrow adipogenesis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Simple Summary Metabolic reprogramming is gaining attentions as a hallmark of cancers. However, lipid metabolism has been difficult to analyze due to technical problems. In recent years, lipidomics techniques such as mass spectrometry have advanced and allowed us to analyze detailed lipid profiles of cancers. Moreover, it has become clear that changes in lipid metabolism also play an important role in the interaction between the cancers and the surrounding microenvironment. This review summarizes the latest research progress of reprogrammed lipid metabolism and also lipidomics technologies applied in cancer research. Abstract Lipids in our body, which are mainly composed of fatty acids, triacylglycerides, sphingolipids, phospholipids, and cholesterol, play important roles at the cellular level. In addition to being energy sources and structural components of biological membranes, several types of lipids serve as signaling molecules or secondary messengers. Metabolic reprogramming has been recognized as a hallmark of cancer, but changes in lipid metabolism in cancer have received less attention compared to glucose or glutamine metabolism. However, recent innovations in mass spectrometry- and chromatography-based lipidomics technologies have increased our understanding of the role of lipids in cancer. Changes in lipid metabolism, so-called “lipid metabolic reprogramming”, can affect cellular functions including the cell cycle, proliferation, growth, and differentiation, leading to carcinogenesis. Moreover, interactions between cancer cells and adjacent immune cells through altered lipid metabolism are known to support tumor growth and progression. Characterization of cancer-specific lipid metabolism can be used to identify novel metabolic targets for cancer treatment, and indeed, several clinical trials are currently underway. Thus, we discuss the latest findings on the roles of lipid metabolism in cancer biology and introduce current advances in lipidomics technologies, focusing on their applications in cancer research.

Peribiliary glands (PBGs), clusters of epithelial cells residing in the submucosal compartment of extrahepatic bile ducts, have been suggested as biliary epithelial stem/progenitor cell niche; however, evidence to support this claim is limited because of a lack of PBG-specific markers. We therefore sought to identify PBG-specific markers to investigate the potential role of PBGs as stem/progenitor cell niches, as well as an origin of cancer.We examined the expression pattern of the Wnt target gene Axin2 in extrahepatic bile ducts. We then applied lineage tracing to investigate whether Axin2-expressing cells from PBGs contribute to biliary regeneration and carcinogenesis using Axin2-CreWnt signaling activation, marked by Axin2, was limited to PBGs located in the periampullary region. Lineage tracing showed that Axin2-expressing periampullary PBG cells are capable of self-renewal and supplying new biliary epithelial cells (BECs) to the luminal surface. Additionally, the expression pattern of Axin2 and the mature ductal cell marker CK19 were mutually exclusive in periampullary region, and fate tracing of CK19A specific cell population receiving Wnt-activating signal in periampullary PBGs functions as biliary epithelial stem/progenitor cells and also the cellular origin of ampullary carcinoma.

Introduction The mandibular canal, as it was formerly named in Terminologia Anatomica (TA), has also been called the inferior alveolar (nerve) canal in many scientific publications. This study was conducted to investigate how these terms have been understood in different regions and different areas of expertise and to discuss the appropriate future application of the term "mandibular canal." Methods A literature search was conducted using PubMed, and articles using different terms for this structure were classified into two groups, inferior alveolar canal/inferior alveolar nerve canal (IAC/IANC) and the mandibular canal (MC). The 50 most recent articles in each group were included. Publication year, journal title, country of the first author, and affiliation of all authors were recorded in both groups for all 100 articles. Results There was a significant difference between the IAC/IANC and MC groups in the numbers of anatomy journals, other journals, and anatomy affiliations. Turkey published most frequently with a total of 15 articles, followed by Iran with 10 articles, and China/India/United States with seven each. When the six countries of the first author that had three or more publications in each group were compared, only Turkey appeared in both groups; otherwise, different countries were in the two groups. Conclusions Based on the results of this analysis, and considering that the tentative new term "inferior alveolar foramen" is used in the latest TA, we suggest that the mandibular canal should be renamed the "inferior alveolar canal."

Developmental and epileptic encephalopathy (DEE) represents a group of neurodevelopmental disorders characterized by infantile-onset intractable seizures and unfavorable prognosis of psychomotor development. To date, hundreds of genes have been linked to the onset of DEE. GNAO1 is a DEE-associated gene encoding the alpha-O1 subunit of guanine nucleotide-binding protein (GαO ). Despite the increasing number of reported children with GNAO1 encephalopathy, the molecular mechanisms underlying their neurodevelopmental phenotypes remain elusive. We herein present that co-immunoprecipitation and mass spectrometry analyses identified another DEE-associated protein, SPTAN1, as an interacting partner of GαO . Silencing of endogenous Gnao1 attenuated the neurite outgrowth and calcium-dependent signaling. Inactivation of GNAO1 in human-induced pluripotent stem cells gave rise to anomalous brain organoids that only weakly expressed SPTAN1 and Ankyrin-G. Furthermore, GNAO1-deficient organoids failed to conduct synchronized firing to adjacent neurons. These data indicate that GαO and other DEE-associated proteins organize the cytoskeletal remodeling and functional polarity of neurons in the developing brain.

The growth plate is an essential component of endochondral bone development. Not surprisingly, the growth plate and its surrounding structure, the perichondrium, contain a wealth of skeletal stem cells (SSCs) and progenitor cells that robustly contribute to bone development. Recent in vivo lineage-tracing studies using mouse genetic models provide substantial insight into the diversity and versatility of these skeletal stem and progenitor cell populations, particularly shedding light on the importance of the transition from cartilage to bone. Chondrocytes and perichondrial cells are inseparable twins that develop from condensing undifferentiated mesenchymal cells during the fetal stage; although morphologically and functionally distinct, these cells ultimately serve for the same goal, that is, to make bone bigger and stronger. Even in the postnatal stage, a small subset of growth plate chondrocytes can transform into osteoblasts and marrow stromal cells; this is in part fueled by a unique type of SSCs maintained in the resting zone of the growth plate, which continue to self-renew for the long term. Here, we discuss diverse skeletal stem and progenitor cell populations in the growth plate and the perichondrium and their transition from cartilage to bone.

Focusing on the interannual variabilities in the zonal mean fields and Rossby wave forcing in austral winter, an interhemispheric coupling in the stratosphere is examined using reanalysis data: the Modern-Era Retrospective Analysis for Research and Applications, version 2 (MERRA-2). In the present study, the Eliassen–Palm (EP) flux divergence averaged over the latitude and height regions of 50–30∘ S and 0.3–1 hPa, respectively, are used as a proxy of the Rossby wave forcing, where the absolute value of the EP flux divergence is maximized in the winter in the Southern Hemisphere (SH). The interannual variabilities in the zonal mean temperature and zonal wind are significantly correlated with the SH Rossby wave forcing in the stratosphere in both the SH and Northern Hemisphere (NH). The interannual variability in the strength of the poleward residual mean flow in the SH stratosphere is also correlated with the strength of the wave forcing. This correlation is significant even around the Equator at an altitude of 40 km and at NH low latitudes of 20–40 km. The temperature anomaly is consistent with this residual mean flow anomaly. The relation between the cross-equatorial flow and the zonal mean absolute angular momentum gradient (M‾y) is examined in the meridional cross section. The M‾y around the Equator at the altitude of 40 km is small when the wave forcing is strong, which provides a pathway for the cross-equatorial residual mean flow. These results indicate that an interhemispheric coupling is present in the stratosphere through the meridional circulation modulated by the Rossby wave forcing.

Formation of functional skeletal tissues requires highly organized steps of mesenchymal progenitor cell differentiation. The dental follicle (DF) surrounding the developing tooth harbors mesenchymal progenitor cells for various differentiated cells constituting the tooth root–bone interface and coordinates tooth eruption in a manner dependent on signaling by parathyroid hormone-related peptide (PTHrP) and the PTH/PTHrP receptor (PPR). However, the identity of mesenchymal progenitor cells in the DF and how they are regulated by PTHrP-PPR signaling remain unknown. Here, we show that the PTHrP-PPR autocrine signal maintains physiological cell fates of DF mesenchymal progenitor cells to establish the functional periodontal attachment apparatus and orchestrates tooth eruption. A single-cell RNA-seq analysis revealed cellular heterogeneity of PTHrP + cells, wherein PTHrP + DF subpopulations abundantly express PPR. Cell lineage analysis using tamoxifen-inducible PTHrP-creER mice revealed that PTHrP + DF cells differentiate into cementoblasts on the acellular cementum, periodontal ligament cells, and alveolar cryptal bone osteoblasts during tooth root formation. PPR deficiency induced a cell fate shift of PTHrP + DF mesenchymal progenitor cells to nonphysiological cementoblast-like cells precociously forming the cellular cementum on the root surface associated with up-regulation of Mef2c and matrix proteins, resulting in loss of the proper periodontal attachment apparatus and primary failure of tooth eruption, closely resembling human genetic conditions caused by PPR mutations. These findings reveal a unique mechanism whereby proper cell fates of mesenchymal progenitor cells are tightly maintained by an autocrine system mediated by PTHrP-PPR signaling to achieve functional formation of skeletal tissues.

Skeletal stem cells regulate bone growth and homeostasis by generating diverse cell types, including chondrocytes, osteoblasts and marrow stromal cells. The emerging concept postulates that there exists a distinct type of skeletal stem cell that is closely associated with the growth plate1-4, which is a type of cartilaginous tissue that has critical roles in bone elongation5. The resting zone maintains the growth plate by expressing parathyroid hormone-related protein (PTHrP), which interacts with Indian hedgehog (Ihh) that is released from the hypertrophic zone6-10, and provides a source of other chondrocytes11. However, the identity of skeletal stem cells and how they are maintained in the growth plate are unknown. Here we show, in a mouse model, that skeletal stem cells are formed among PTHrP-positive chondrocytes within the resting zone of the postnatal growth plate. PTHrP-positive chondrocytes expressed a panel of markers for skeletal stem and progenitor cells, and uniquely possessed the properties of skeletal stem cells in cultured conditions. Cell-lineage analysis revealed that PTHrP-positive chondrocytes in the resting zone continued to form columnar chondrocytes in the long term; these chondrocytes underwent hypertrophy, and became osteoblasts and marrow stromal cells beneath the growth plate. Transit-amplifying chondrocytes in the proliferating zone-which was concertedly maintained by a forward signal from undifferentiated cells (PTHrP) and a reverse signal from hypertrophic cells (Ihh)-provided instructive cues to maintain the cell fates of PTHrP-positive chondrocytes in the resting zone. Our findings unravel a type of somatic stem cell that is initially unipotent and acquires multipotency at the post-mitotic stage, underscoring the malleable nature of the skeletal cell lineage. This system provides a model in which functionally dedicated stem cells and their niches are specified postnatally, and maintained throughout tissue growth by a tight feedback regulation system.

Craniofacial development requires a set of patterning codes that define the identities of postmigratory mesenchymal cells in a region-specific manner, in which locally expressed morphogens, including fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs), provide instructive cues. Msx2, a bona fide target of BMP signaling, is a transcription factor regulating Runx2 and osterix (Osx), whose mutations are associated with cranial deformities in humans. Here we show that Msx2 defines osteo-chondro precursor cells in specific regions of the craniofacial mesenchyme at the postmigratory stage, particularly in the mandibular process and the posterior cranial vault. Analysis of Msx2-creER mice revealed that early mesenchymal cells in proximity to the BMP4-expressing mesenchyme were marked upon tamoxifen injection, and their descendants contributed to diverse types of mesenchymal cells in the later stage, such as chondrocytes and perichondrial cells of the transient cartilage, as well as osteoblasts and suture mesenchymal cells. By contrast, Osx-creER marked osteoblast precursors at the later stage, and their descendants continued to become osteoblasts well into the postnatal stage. Therefore, Msx2 marks spatially restricted populations of mesenchymal precursor cells with diverse differentiation potential, suggesting that extrinsic molecular cues can dictate the nature of postmigratory mesenchymal cells in craniofacial development.