Probes for INS

The vestibular lamina (VL) forms the oral vestibule, creating a gap between the teeth, lips and cheeks. In a number of ciliopathies, formation of the vestibule is defective, leading to the creation of multiple frenula. In contrast to the neighbouring dental lamina, which forms the teeth, little is known about the genes that pattern the VL. Here, we establish a molecular signature for the usually non-odontogenic VL in mice and highlight several genes and signalling pathways that may play a role in its development. For one of these, the Sonic hedgehog (Shh) pathway, we show that co-receptors Gas1, Cdon and Boc are highly expressed in the VL and act to enhance the Shh signal from the forming incisor region. In Gas1 mutant mice, expression of Gli1 was disrupted and the VL epithelium failed to extend due to a loss of proliferation. This defect was exacerbated in Boc/Gas1 double mutants and could be phenocopied using cyclopamine in culture. Signals from the forming teeth, therefore, control development of the VL, coordinating the development of the dentition and the oral cavity.

This year in review on osteoarthritis biology summarizes a series of research articles published between the 2020 and 2021 Osteoarthritis Research Society International (OARSI) World Congress. Research hightlights were selected and discussed based on the new discoveries of OA's cellular molecular mechanism, anatomical signatures, potential therapeutic targets, and regenerative therapy. The recently developed potential therapeutic targets are summarized, and the research focuses on TGFβ and WNT signaling in joint tissue homeostasis, joint aging and the dynamic of synolytics in OA joint, and the roles of TRP2, LDHA, OSCAR in cartilage homeostasis and OA joints are highlighted. Subsquencially, new anatomical structures and OA features are introduced, such as synovitis-induced venous portal circulation, horiozontal fissures between cartilage and subchondral bone, the cellular derivation of osteophytes formation, OA subtypes, and subchondral remodeling and pain biology. Then, research on the possibility of tissue regeneration in OA joints are discussed; skeletal stem cells in OA cartilage regeneration, and preclinical results of regenerative therapy for meniscus tear and osteochondral tissue morphoghesis are included. At last, the clinical evidence of the importance of delivery site of bone marrow stem cells for OA treatment is discussed. These findings represent advances in our understanding of OA pathophysiology.

Prdm1 mutant mice are one of the rare mutant strains that do not develop whisker hair follicles while still displaying a pelage. Here, we show that Prdm1 is expressed at the earliest stage of whisker development in clusters of mesenchymal cells before placode formation. Its conditional knockout in the murine soma leads to the loss of expression of Bmp2, Shh, Bmp4, Krt17, Edar, and Gli1, though leaving the β-catenin-driven first dermal signal intact. Furthermore, we show that Prdm1 expressing cells not only act as a signaling center but also as a multipotent progenitor population contributing to the several lineages of the adult whisker. We confirm by genetic ablation experiments that the absence of macro vibrissae reverberates on the organization of nerve wiring in the mystacial pads and leads to the reorganization of the barrel cortex. We demonstrate that Lef1 acts upstream of Prdm1 and identify a primate-specific deletion of a Lef1 enhancer named Leaf. This loss may have been significant in the evolutionary process, leading to the progressive defunctionalization and disappearance of vibrissae in primates.

In adults, hepatocytes are mainly replenished from the existing progenitor pools of hepatocytes and cholangiocytes during chronic liver injury. However, it is unclear whether other cell types in addition to classical hepatocytes and cholangiocytes contribute to hepatocyte regeneration after chronic liver injuries. Here, we identified a new biphenotypic cell population that contributes to hepatocyte regeneration during chronic liver injuries. We found that a cell population expressed Gli1 and EpCAM (EpCAM+Gli1+), which was further characterized with both epithelial and mesenchymal identities by single-cell RNA sequencing. Genetic lineage tracing using dual recombinases revealed that Gli1+ nonhepatocyte cell population could generate hepatocytes after chronic liver injury. EpCAM+Gli1+ cells exhibited a greater capacity for organoid formation with functional hepatocytes in vitro and liver regeneration upon transplantation in vivo. Collectively, these findings demonstrate that EpCAM+Gli1+ cells can serve as a new source of liver progenitor cells and contribute to liver repair and regeneration.

The field of osteoarthritis (OA) biology is rapidly evolving and brilliant progress has been made this year as well. Landmark studies of OA biology published in 2021 and early 2022 were selected through PubMed search by personal opinion. These papers were classified by their molecular mechanisms, and it was largely divided into the intracellular signaling mechanisms and the inter-compartment interaction in chondrocyte homeostasis and OA progression. The intracellular signaling mechanisms involving OA progression included (1) Piezo1/transient receptor potential channels of the vanilloid subtype (TRPV) 4-mediated calcium signaling, (2) mechanical load-F-box and WD repeat domain containing 7 (FBXW7) in chondrocyte senescence, (3) mechanical loading-primary cilia-hedgehog signaling, (4) low grade inflammation by toll-like receptor (TLR)-CD14-lipopolysaccharide-binding protein (LBP) complex and inhibitor of NF-κB kinase (IKK) β-nuclear factor kappa B (NF-κB) signaling, (5) selenium pathway and reactive oxygen species (ROS) production, (6) G protein-coupled receptor (GPCR) and cyclic adenosine monophosphate (cAMP) signaling, (7) peroxisome proliferator-activated receptor α (PPARα)-acyl-CoA thioesterase 12 (ACOT12)-mediated de novo lipogenesis and (8) hypoxia-disruptor of telomeric silencing 1-like (DOT1L)-H3-lysine 79 (H3K79) methylation pathway. The studies on inter-compartment or intercellular interaction in OA progression included the following subjects; (1) the anabolic role of lubricin, glycoprotein from superficial zone cells, (2) osteoclast-chondrocyte interaction via exosomal miRNA and sphingosine 1-phosphate (S1P), (3) senescent fibroblast-like synoviocyte and chondrocyte interaction, (4) synovial macrophage and chondrocyte interaction through Flightless I, (5) αV integrin-mediated transforming growth factor beta (TGFβ) activation by mechanical loading, and (6) osteocytic TGFβ in subchondral bone thickening. Despite the disastrous Covid-19 pandemic, many outstanding studies have expanded the boundary of OA biology. They provide both critical insight into the pathophysiology as well as clues for the treatment of OA.

Although both the 2014 and 2020 World Health Organization (WHO) criteria require unequivocal glandular and squamous differentiation for a diagnosis of cervical adenosquamous carcinoma (ASC), in practice, ASC diagnoses are often made in tumors that lack unequivocal squamous and/or glandular differentiation. Considering the ambiguous etiologic, morphologic, and clinical features and outcomes associated with ASCs, we sought to redefine these tumors. We reviewed slides from 59 initially diagnosed ASCs (including glassy cell carcinoma and related lesions) to confirm an ASC diagnosis only in the presence of unequivocal malignant glandular and squamous differentiation. Select cases underwent immunohistochemical profiling as well as human papillomavirus (HPV) testing by in situ hybridization. Of the 59 cases originally classified as ASCs, 34 retained their ASC diagnosis, 9 were reclassified as pure invasive stratified mucin-producing carcinomas, 10 as invasive stratified mucin-producing carcinomas with other components (such as HPV-associated mucinous, usual-type, or ASCs), and 4 as HPV-associated usual or mucinous adenocarcinomas with benign-appearing squamous metaplasia. Two glassy adenocarcinomas were reclassified as poorly differentiated HPV-associated carcinomas based on morphology and immunophenotype. There were no significant immunophenotypic differences between ASCs and pure invasive stratified mucin-producing carcinomas with regard to HPV and other markers including p16 expression. Although limited by a small sample size, survival outcomes seemed to be similar between all groups. ASCs should be diagnosed only in the presence of unequivocal malignant glandular and squamous differentiation. The 2 putative glassy cell carcinomas studied did not meet our criteria for ASC and categorizing them as such should be reconsidered.

Oropharyngeal squamous cell carcinoma is frequently associated with high-risk HPV infection, which confers a good prognosis. Immunohistochemistry for p16 is used as a surrogate for HPV status, but discrepant results are occasionally seen. Here, we report a case with a unique pattern of partial loss of p16.A 63 year old male presented with a base of tongue nonkeratinizing squamous cell carcinoma and a large metastatic neck mass. The primary lesion and multiple regions of the metastatic mass were assessed with p16 immunohistochemistry, RNA in situ hybridization for high-risk HPV, and HPV16 genome sequencing.The primary lesion was p16 negative, and the metastatic neck mass had large, confluent regions that were either strongly p16 positive or entirely p16 negative. All of these regions were positive for high-risk HPV with identical HPV16 genomes.This unusual case illustrates a potential diagnostic pitfall, and it raises important questions regarding molecular mechanisms and prognostic implications of p16 staining in oropharyngeal squamous cell carcinoma.

Human papillomavirus (HPV)-independent primary endometrial squamous cell carcinoma (PESCC) is a rare but aggressive subtype of endometrial carcinoma for which little is known about the genomic characteristics. Traditional criteria have restricted the diagnosis of PESCC to cases without any cervical involvement. However, given that modern ancillary techniques can detect HPV and characteristic genetic alterations that should identify the more common mimics in the differential diagnosis, including endometrial endometrioid carcinoma with extensive squamous differentiation and HPV-associated primary cervical squamous cell carcinoma, those criteria may benefit from revision. To further characterize PESCC, we identified 5 cases of pure squamous cell carcinoma dominantly involving the endometrium that had the potential to be PESCC: 1 case involving only the endometrium and 4 cases with some involvement of the cervix. Clinicopathologic features were assessed and immunohistochemical analysis (p16, estrogen receptor, progesterone receptor, and p53), HPV RNA in situ hybridization (high-risk and low-risk cocktails and targeted probes for 16 and 18), and molecular studies were performed. All tumors showed aberrant/mutation-type p53 expression, were negative for estrogen receptor, progesterone receptor, and p16, and had no detectable HPV. Per whole-exome sequencing, 4 of the 5 tumors demonstrated comutations in TP53 and CDKN2A (p16). Four patients died of disease within 20 months (range, 1 to 20 mo; mean, 9 mo), and 1 patient had no evidence of disease at 38 months. PESCC represents a unique, clinically aggressive subtype of endometrial cancer with TP53 and CDKN2A comutations. This characteristic profile, which is similar to HPV-independent squamous cell carcinoma of the vulva, is distinct from endometrioid carcinoma with extensive squamous differentiation and HPV-associated primary cervical squamous cell carcinoma and can be used to distinguish PESCC from those mimics even when cervical involvement is present. Diagnostic criteria for PESCC should be relaxed to allow for cervical involvement when other pathologic features are consistent with, and ancillary techniques are supportive of classification as such.