Cytoarchitecture and intercellular interactions in the trigeminal ganglion: Associations with neuropathic pain in the orofacial region.

BACKGROUND: Disorders of the trigeminal nerve, a sensory nerve of the orofacial region, often lead to complications in dental practice, including neuropathic pain, allodynia, and ectopic pain. Management of these complications requires an understanding of the cytoarchitecture of the trigeminal ganglion, where the cell bodies of the trigeminal nerve are located, and the mechanisms of cell-cell interactions. HIGHLIGHTS: In the trigeminal ganglion, the ganglion, satellite, Schwann, and immune cells coexist and interact. Cell-cell interactions are complex and occur through direct contact via gap junctions or through mediators such as adenosine triphosphate, nitric oxide, peptides, and cytokines. Interactions between the nervous and immune systems within the trigeminal ganglion may have neuroprotective effects during nerve injury or may exacerbate inflammation and produce chronic pain. Under pathological conditions of the trigeminal nerve, cell-cell interactions can cause allodynia and ectopic pain. Although cell-cell interactions that occur via mediators can act at some distance, they are more effective when the cells are close together. Therefore, information on the three-dimensional topography of the trigeminal ganglion cells is essential for understanding the pathophysiology of ectopic pain. CONCLUSIONS: A three-dimensional map of the somatotopic localization of trigeminal ganglion neurons revealed that ganglion cells innervating distant orofacial regions are often apposed to each other, interacting with and potentially contributing to ectopic pain. Elucidation of the complex network of mediators and their receptors responsible for intercellular communication within the trigeminal ganglion is essential for understanding ectopic pain.

Myosin II mediates Shh signals to shape dental epithelia via control of cell adhesion and movement.

The development of ectodermal organs begins with the formation of a stratified epithelial placode that progressively invaginates into the underlying mesenchyme as the organ takes its shape. Signaling by secreted molecules is critical for epithelial morphogenesis, but how that information leads to cell rearrangement and tissue shape changes remains an open question. Using the mouse dentition as a model, we first establish that non-muscle myosin II is essential for dental epithelial invagination and show that it functions by promoting cell-cell adhesion and persistent convergent cell movements in the suprabasal layer. Shh signaling controls these processes by inducing myosin II activation via AKT. Pharmacological induction of AKT and myosin II can also rescue defects caused by the inhibition of Shh. Together, our results support a model in which the Shh signal is transmitted through myosin II to power effective cellular rearrangement for proper dental epithelial invagination.

Developmental process of the modern house shrew's molars: implications for the evolution of the tribosphenic molar in Mesozoic mammals.

Phylogenetically, the tribosphenic molars-prototypes of multi-cusped cheek teeth in marsupial and placental mammals-are derived from the single-cusped conical teeth of reptiles through the addition of cusps. Ontogenetically, mammalian molars are formed through the interface between the dental epithelium and mesenchyme (future enamel-dentin junction), becoming geometrically complex by adding epithelial signaling centers, called enamel knots, which determine future cusp positions. To reevaluate cusp homologies in Mesozoic mammals from an ontogenetic perspective, this study tracked molar development in a living placental mammal species, the house shrew (Suncus murinus), whose molars are morphologically the least derived from tribosphenic prototypes. The development of shrew molars proceeded as if it replayed the evolutionary process of tribosphenic molars. The first formed enamel knots gave rise to the evolutionarily oldest cusps-upper paracone and lower protoconid. The order of formation of other enamel knots and their location in development seemed to trace the order of cusp appearance in evolution. The parallel relationship between ontogeny and phylogeny of mammalian molars, if any, suggests that a change in the timing between developmental events rather than a change in the morphogenetic mechanism itself, should have been a major causal factor for the evolutionary transformation of tooth morphology.

Potential Protective Effect of Dengue NS1 Human Monoclonal Antibodies against Dengue and Zika Virus Infections.

Due to the lack of an effective therapeutic treatment to flavivirus, dengue virus (DENV) nonstructural protein 1 (NS1) has been considered to develop a vaccine owing to its lack of a role in antibody-dependent enhancement (ADE). However, both NS1 and its antibody have shown cross-reactivity to host molecules and have stimulated anti-DENV NS1 antibody-mediated endothelial damage and platelet dysfunction. To overcome the pathogenic events and reactogenicity, human monoclonal antibodies (HuMAbs) against DENV NS1 were generated from DENV-infected patients. Herein, the four DENV NS1-specific HuMAbs revealed the therapeutic effects in viral neutralization, reduction of viral replication, and enhancement of cell cytolysis of DENV and zika virus (ZIKV) via complement pathway. Furthermore, we demonstrate that DENV and ZIKV NS1 trigger endothelial dysfunction, leading to vascular permeability in vitro. Nevertheless, the pathogenic effects from NS1 were impeded by 2 HuMAbs (D25-4D4C3 and D25-2B11E7) and also protected the massive cytokines stimulation (interleukin [IL-]-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-13, IL-17, eotaxin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, Inducible protein-10, monocyte chemoattractant protein-1, macrophage inflammatory protein [MIP]-1 α, MIP-1ß, tumor necrosis factor-α, platelet-derived growth factor, and RANTES). Collectively, our findings suggest that the novel protective NS1 monoclonal antibodies generated from humans has multiple therapeutic benefits against DENV and ZIKV infections.

The potential of COVID-19 patients' sera to cause antibody-dependent enhancement of infection and IL-6 production.

Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many vaccine trials have been initiated. An important goal of vaccination is the development of neutralizing antibody (Ab) against SARS-CoV-2. However, the possible induction of antibody-dependent enhancement (ADE) of infection, which is known for other coronaviruses and dengue virus infections, is a particular concern in vaccine development. Here, we demonstrated that human iPS cell-derived, immortalized, and ACE2- and TMPRSS2-expressing myeloid cell lines are useful as host cells for SARS-CoV-2 infection. The established cell lines were cloned and screened based on their function in terms of susceptibility to SARS-CoV-2-infection or IL-6 productivity. Using the resulting K-ML2 (AT) clone 35 for SARS-CoV-2-infection or its subclone 35-40 for IL-6 productivity, it was possible to evaluate the potential of sera from severe COVID-19 patients to cause ADE and to stimulate IL-6 production upon infection with SARS-CoV-2.

Tissue-resident M2 macrophages directly contact primary sensory neurons in the sensory ganglia after nerve injury.

BACKGROUND: Macrophages in the peripheral nervous system are key players in the repair of nerve tissue and the development of neuropathic pain due to peripheral nerve injury. However, there is a lack of information on the origin and morphological features of macrophages in sensory ganglia after peripheral nerve injury, unlike those in the brain and spinal cord. We analyzed the origin and morphological features of sensory ganglionic macrophages after nerve ligation or transection using wild-type mice and mice with bone-marrow cell transplants. METHODS: After protecting the head of C57BL/6J mice with lead caps, they were irradiated and transplanted with bone-marrow-derived cells from GFP transgenic mice. The infraorbital nerve of a branch of the trigeminal nerve of wild-type mice was ligated or the infraorbital nerve of GFP-positive bone-marrow-cell-transplanted mice was transected. After immunostaining the trigeminal ganglion, the structures of the ganglionic macrophages, neurons, and satellite glial cells were analyzed using two-dimensional or three-dimensional images. RESULTS: The number of damaged neurons in the trigeminal ganglion increased from day 1 after infraorbital nerve ligation. Ganglionic macrophages proliferated from days 3 to 5. Furthermore, the numbers of macrophages increased from days 3 to 15. Bone-marrow-derived macrophages increased on day 7 after the infraorbital nerve was transected in the trigeminal ganglion of GFP-positive bone-marrow-cell-transplanted mice but most of the ganglionic macrophages were composed of tissue-resident cells. On day 7 after infraorbital nerve ligation, ganglionic macrophages increased in volume, extended their processes between the neurons and satellite glial cells, and contacted these neurons. Most of the ganglionic macrophages showed an M2 phenotype when contact was observed, and little neuronal cell death occurred. CONCLUSION: Most of the macrophages that appear after a nerve injury are tissue-resident, and these make direct contact with damaged neurons that act in a tissue-protective manner in the M2 phenotype. These results imply that tissue-resident macrophages signal to neurons directly through physical contact.

The Periodontium Damage Induces Neuronal Cell Death in the Trigeminal Mesencephalic Nucleus and Neurodegeneration in the Trigeminal Motor Nucleus in C57BL/6J Mice.

Proprioception from masticatory apparatus and periodontal ligaments comes through the trigeminal mesencephalic nucleus (Vmes). We evaluated the effects of tooth loss on neurodegeneration of the Vmes and trigeminal motor nucleus (Vmo). Bilateral maxillary molars of 2-month-old C57BL/6J mice were extracted under anesthesia. Neural projections of the Vmes to the periodontium were confirmed by injecting Fluoro-Gold (FG) retrogradely into the extraction sockets, and for the anterograde labeling adeno-associated virus encoding green fluorescent protein (AAV-GFP) was applied. For immunohistochemistry, Piezo2, ATF3, Caspase 3, ChAT and TDP-43 antibodies were used. At 1 month after tooth extraction, the number of Piezo2-immunoreactive (IR) Vmes neurons were decreased significantly. ATF3-IR neurons were detected on day 5 after tooth extraction. Dead cleaved caspase-3-IR neurons were found among Vmes neurons on days 7 and 12. In the Vmo, neuronal cytoplasmic inclusions (NCIs) formation type of TDP-43 increased at 1 and 2 months after extraction. These indicate the existence of neural projections from the Vmes to the periodontium in mice and that tooth loss induces the death of Vmes neurons followed by TDP-43 pathology in the Vmo. Therefore, tooth loss induces Vmes neuronal cell death, causing Vmo neurodegeneration and presumably affecting masticatory function.

A potent neutralizing mouse monoclonal antibody specific to dengue virus type 1 Mochizuki strain recognized a novel epitope around the N-67 glycan on the envelope protein: A possible explanation of dengue virus evolution regarding the acquisition of N-67 glycan.

The analysis of neutralizing epitope of dengue virus (DENV) is important for the development of an effective dengue vaccine. A potent neutralizing mouse monoclonal antibody named 7F4 was previously reported and, here, we further analyzed the detailed epitope of this antibody. 7F4 recognized a novel conformational epitope close to the N-67 glycan on the envelope protein. This antibody was specific to the DENV that lacks N-67 glycan, including the Mochizuki strain. Interestingly, the Mochizuki strain acquired N-67 glycan by 7F4 selective pressure. Considering that most of the currently circulating DENVs possess N-67 glycan, DENVs may have evolved to escape from antibodies targeting 7F4 epitope, suggesting the potency of this neutralizing epitope. In addition, this study demonstrated the existence of the epitopes close to 7F4 epitope and their crucial role in neutralization. In conclusion, the epitopes close to the N-67 glycan are attractive targets for the dengue vaccine antigen. Further analysis of this epitope is warranted.

A Novel Sub-Lineage of Chikungunya Virus East/Central/South African Genotype Indian Ocean Lineage Caused Sequential Outbreaks in Bangladesh and Thailand.

In recent decades, chikungunya virus (CHIKV) has become geographically widespread. In 2004, the CHIKV East/Central/South African (ECSA) genotype moved from Africa to Indian ocean islands and India followed by a large epidemic in Southeast Asia. In 2013, the CHIKV Asian genotype drove an outbreak in the Americas. Since 2016, CHIKV has re-emerged in the Indian subcontinent and Southeast Asia. In the present study, CHIKVs were obtained from Bangladesh in 2017 and Thailand in 2019, and their nearly full genomes were sequenced. Phylogenetic analysis revealed that the recent CHIKVs were of Indian Ocean Lineage (IOL) of genotype ECSA, similar to the previous outbreak. However, these CHIKVs were all clustered into a new distinct sub-lineage apart from the past IOL CHIKVs, and they lacked an alanine-to-valine substitution at position 226 of the E1 envelope glycoprotein, which enhances CHIKV replication in Aedes albopictus. Instead, all the re-emerged CHIKVs possessed mutations of lysine-to-glutamic acid at position 211 of E1 and valine-to-alanine at position 264 of E2. Molecular clock analysis suggested that the new sub-lineage CHIKV was introduced to Bangladesh around late 2015 and Thailand in early 2017. These results suggest that re-emerged CHIKVs have acquired different adaptations than the previous CHIKVs.

Clinical Evaluation of Endoscopic Bronchial Occlusion with an Endobronchial Watanabe Spigot for the Management of Intractable Pneumothorax, Pyothorax with Bronchial Fistula, and Postoperative Air Leakage.

Objective The present study aimed to evaluate the clinical effectiveness of endoscopic bronchial occlusion (EBO) with endobronchial Watanabe spigots (EWSs) for the management of prolonged pulmonary air leaks, such as intractable pneumothorax, pyothorax with bronchial fistula, and postoperative air leakage. Methods This was a retrospective study. Between April 2005 and March 2018, we recruited 21 patients with intractable pneumothorax (10 cases), pyothorax with bronchial fistula (7 cases), and postsurgical pulmonary fistula (4 cases) in whom appropriate drainage for 2 weeks had been unsuccessful and who were unsuitable for surgery. An EWS was inserted using a flexible bronchoscope via an endotracheal or a tracheostomy tube. Results The mean number of sessions with EWS procedures was 1.94, and the mean number of inserted EWS per patient was 6.5. In addition to EWS procedures, pleural washing and pleural adhesion therapy were performed in all cases with pyothorax, whereas pleural adhesion therapy was performed in three patients with pneumothorax. The successful treatment rate was 85.7%. Reduction of air leakage was observed in 19/21 patients. The mean duration of reduction of air leaks was 4.1 days (median, 1; range, 0-24 days) following EWS procedures. The mean duration from tube insertion to chest tube removal was 43.4 days (median, 29; range, 16-105 days). Complications included spigot migration and infection (aspergillosis); no complications caused significant mortality. Conclusion Performing EBO using an EWS appears to be a reasonable option for the management of intractable pneumothorax, pyothorax with pulmonary fistula, and postoperative air leakage.

Severe tracheal stenosis after first administration of pembrolizumab rescued by Dumon Y-stent in a lung cancer patient.

A 70-year-old woman diagnosed with advanced, non-resectable programmed cell death ligand 1-positive-non-small-cell lung carcinoma was treated with pembrolizumab as first-line therapy. Soon after therapy initiation, she presented with severe dyspnea, and chest computed tomography revealed a soft tissue mass in the lower trachea of the right main bronchus. During bronchoscopy, she became severely hypoxic, and we performed endoscopic tumor ablation and Dumon Y-stent placement. We considered this severe deterioration caused by pseudoprogression, and suggest that it is necessary to perform bronchoscopy and to prepare for the bronchial intervention when treating patients with immune checkpoint inhibitors.

Neuropeptides and ATP signaling in the trigeminal ganglion.

Peripheral nociceptive stimuli from orofacial structures are largely transmitted by the trigeminal nerve. According to the peripheral noxious stimuli, neurons in the trigeminal ganglion (TG) produce neuropeptides such as substance P, and calcitonin-gene-related peptide, etc. Beside the production of neuropeptides, there exists unique non-synaptic interaction system between maxillary and mandibular neurons in the TG. Neurons in the TG are surrounded by satellite glial cells (SGCs), which initially receive the signal from TG neurons. These activated SGCs secrete a transmitter to activate adjacent SGCs or TG neurons, thereby amplifying the signal, for example, from mandibular neurons to maxillary neurons in the TG. Similar to the dorsal root ganglion, in the TG, microglia/macrophage-like cells (MLCs) are activated by uptake of a transmitter from TG neurons or SGCs. This communication between neurons, SGCs, and MLCs results in responses such as ectopic pain, hyperesthesia, or allodynia. The focus of this review is the cooperative interaction of the maxillary and mandibular nerves in the TG by neuropeptides, and adenosine 3'-phosphate (ATP) signaling from neurons to SGCs and MLCs. Stimulated neurons either secrete ATP by means of vesicular nucleotide transporters, or secrete neuropeptides from the neuronal cell body to mediate signal transmission.

Hepatocellular carcinoma with intracranial metastasis in a Japanese macaque (Macaca fuscata).

BACKGROUND: A 23-year-old male Japanese macaque (Macaca fuscata) showed left ptosis, which progressed to exophthalmos. METHODS: The macaque underwent a clinical examination, CT and MRI, and was euthanized. Necropsy and histopathological examination were performed after euthanasia. RESULTS: The CT revealed and MRI confirmed an intracranial mass at the skull base with orbital extension. At necropsy, there were a large hepatic mass and an intracranial mass compressing the left temporal lobe of the brain. Histopathological and immunohistological examinations revealed that the masses were hepatocellular carcinoma (HCC) and a metastatic lesion. In both the primary and metastatic lesions, neoplastic hepatocytes were arranged mainly in a trabecular pattern. Immunohistochemically, the tumor cells were positive for cytokeratin (AE1/AE3 and CAM5.2) and hepatocyte paraffin 1 and negative for cytokeratin 7 and 20 and vimentin. CONCLUSION: To our knowledge, this is the first case report of HCC with intracranial metastasis in a macaque.

Neutralizing and enhancing antibody responses to five genotypes of dengue virus type 1 (DENV-1) in DENV-1 patients.

Dengue virus (DENV) has four distinct serotypes, DENV-1-4, with four to six genotypes in each serotype. The World Health Organization recommends tetravalent formulations including one genotype of each serotype as safe and effective dengue vaccines. Here, we investigated the impact of genotype on the neutralizing antibody responses to DENV-1 in humans. Convalescent sera collected from patients with primary infection of DENV-1 were examined for neutralizing antibody against single-round infectious particles of the five DENV-1 genotypes (GI-GV). In both GI- and GIV-infected patients, their neutralizing antibody titres against the five genotypes were similar, differing ≤4-fold from the homogenotypic responses. The enhancing activities against the five genotypes were also similar in these sera. Thus, the genotype strains of DENV-1 showed no significant antigenic differences in these patients, suggesting that GI- or GIV-derived vaccine antigens should induce equivalent levels of neutralizing antibodies against all DENV-1 genotypes.

A mouse monoclonal antibody against dengue virus type 1 Mochizuki strain targeting envelope protein domain II and displaying strongly neutralizing but not enhancing activity.

Dengue fever and its more severe form, dengue hemorrhagic fever, are major global concerns. Infection-enhancing antibodies are major factors hypothetically contributing to increased disease severity. In this study, we generated 26 monoclonal antibodies (MAbs) against the dengue virus type 1 Mochizuki strain. We selected this strain because a relatively large number of unique and rare amino acids were found on its envelope protein. Although most MAbs showing neutralizing activities exhibited enhancing activities at subneutralizing doses, one MAb (D1-IV-7F4 [7F4]) displayed neutralizing activities without showing enhancing activities at lower concentrations. In contrast, another MAb (D1-V-3H12 [3H12]) exhibited only enhancing activities, which were suppressed by pretreatment of cells with anti-FcγRIIa. Although antibody engineering revealed that antibody subclass significantly affected 7F4 (IgG3) and 3H12 (IgG1) activities, neutralizing/enhancing activities were also dependent on the epitope targeted by the antibody. 7F4 recognized an epitope on the envelope protein containing E118 (domain II) and had a neutralizing activity 10- to 1,000-fold stronger than the neutralizing activity of previously reported human or humanized neutralizing MAbs targeting domain I and/or domain II. An epitope-blocking enzyme-linked immunosorbent assay (ELISA) indicated that a dengue virus-immune population possessed antibodies sharing an epitope with 7F4. Our results demonstrating induction of these antibody species (7F4 and 3H12) in Mochizuki-immunized mice may have implications for dengue vaccine strategies designed to minimize induction of enhancing antibodies in vaccinated humans.

Phylogenetic analysis of dengue virus types 1 and 3 isolated in Jakarta, Indonesia in 1988.

Dengue viruses are mosquito-borne viruses that cause dengue fever and dengue hemorrhagic fever, both of which are globally important diseases. These viruses have evolved in a transmission cycle between human hosts and mosquito vectors in various tropical and subtropical environments. We previously isolated three strains of dengue type 1 virus (DENV1) and 14 strains of dengue type 3 virus (DENV3) during an outbreak of dengue fever and dengue hemorrhagic fever in Jakarta, Indonesia in 1988. Here, we compared the nucleotide sequences of the entire envelope protein-coding region among these strains. The isolates were 97.6-100% identical for DENV1 and 98.8-100% identical for DENV3. All DENV1 isolates were included in two different clades of genotype IV and all DENV3 isolates were included in a single clade of genotype I. For DENV1, three Yap Island strains isolated in 2004 were the only strains closely related to the present isolates; the recently circulated Indonesian strains were in different clades. Molecular clock analyses estimated that ancestors of the genotype IV strains of DENV1 have been indigenous in Indonesia since 1948. We predict that they diverged frequently around 1967 and that their offspring distributed to Southeast Asia, the Western Pacific, and Africa. For DENV3, the clade containing all the present isolates also contained strains isolated from other Indonesian regions and other countries including Malaysia, Singapore, China, and East Timor from 1985-2010. Molecular clock analyses estimated that the common ancestor of the genotype I strains of DENV3 emerged in Indonesia around 1967 and diverged frequently until 1980, and that their offspring distributed mainly in Southeast Asia. The first dengue outbreak in 1968 and subsequent outbreaks in Indonesia might have influenced the divergence and distribution of the DENV1 genotype IV strains and the DENV3 genotype I strains in many countries.

Vero cells infected with vaccinia viruses expressing Japanese encephalitis virus envelope protein induce polykaryocyte formation under neutral conditions.

Flavivirus membranes fuse with cellular membranes by low pH-induced mechanisms. Although Japanese encephalitis virus (JEV) has similar mechanisms, fusion induced under neutral conditions has been observed. We report herein polykaryocyte formation using Vero cells infected with recombinant vaccinia viruses, vP829 expressing JEV premembrane (prM) and envelope (E), or vP555 expressing prM, E, and the nonstructural proteins NS1 and NS2A. Polykaryocytes were detected under pH 7.0 as early as 9 h after infection. Higher fusion indices were shown with vP555 than vP829. A monoclonal antibody to E suppressed vP829/vP555-induced polykaryocyte formation. Polykaryocytes were also formed under alkaline conditions (pH 8.0).

Interlaboratory validation of the modified murine local lymph node assay based on adenosine triphosphate measurement.

INTRODUCTION: The murine local lymph node assay (LLNA) is a well-established alternative to the guinea pig maximization test (GPMT) or Buehler test (BT) for the assessment of the skin sensitizing ability of drugs and chemicals. Daicel Chemical Industries Ltd. has developed a modified LLNA based on the adenosine triphosphate (ATP) content (LLNA-DA). We conducted 2 interlaboratory validation studies to evaluate the reliability and relevance of LLNA-DA. METHODS: The experiment involved 17 laboratories, wherein 14 chemicals were examined under blinded conditions. In the first study, 3 chemicals were examined in 10 laboratories and the remaining 9 were examined in 3 laboratories. In the second study, 1 chemical was examined in 7 laboratories and the remaining 4 chemicals were examined in 4 laboratories. The data were expressed as the ATP content for each chemical-treated group, and the stimulation index (SI) for each chemical-treated group was determined as the increase in the ATP content relative to the concurrent vehicle control group. An SI of 3 was set as the cut-off value for exhibiting skin sensitization activity. RESULTS: The results of the first study obtained in the experiments conducted for the 3 chemicals that were examined in all the 10 laboratories and for 5 of the remaining 9 chemicals were sufficiently consistent with small variations in their SI values. The sensitivity, specificity, and accuracy of LLNA-DA against those of GPMT/BT were 7/8 (87.5%), 3/3 (100%), and 10/11 (90.9%), respectively. In the second study, all the 5 chemicals studied demonstrated acceptably small interlaboratory variations. DISCUSSION: In the first study, a large variation was observed for 2 chemicals; in the second study, this variation was small. It was attributed to the application of dimethylsulfoxide as the solvent for the metallic salts. In conclusion, these 2 studies provide good evidence for the reliability of the LLNA-DA.

Development of heterodont dentition in house shrew (Suncus murinus).

Mammalian heterodont dentition comprises incisors, canines, premolars, and molars. Although there has been intensive research, the patterning of these specific tooth types has not yet been elucidated. In order for the gene expression data to be linked with tooth type determination, it is first necessary to determine precisely the incisor-, canine-, premolar-, and molar-forming regions in the jaw primordia. To accomplish this, we studied dentition development in the house shrew (Suncus murinus), which has retained all the tooth types, using three-dimensional reconstructions from serial histological sections and the Sonic hedgehog (Shh) expression patterns. Before the appearance of morphological signs of odontogenesis, Shh expression localized to the presumptive tooth-forming regions, in which the mesial and distal expression domains corresponded to the incisor- and premolar-forming regions, respectively. The upper incisor region was found to extend across the boundary between the frontonasal and the maxillary processes. The canine-forming regions later appeared in the intermediate portions of the maxillary and the mandibular processes. The molar-forming regions later appeared distal to the initially demarcated tooth-forming regions by secondary extension of the distal ends. The demarcation visualized by the Shh expression pattern in the jaw primordia of the house shrew probably represents the basic developmental pattern of mammalian heterodont dentition.