Wang L, Huang J, Moore DC, Zuo C, Wu Q, Xie L, von der Mark K, Yuan X, Chen D, Warman ML, Ehrlich MG, Yang W.
PMID: 28983104 | DOI: 10.1038/s41598-017-12767-9
Transdifferentiation of hypertrophic chondrocytes into bone-forming osteoblasts has been reported, yet the underlying molecular mechanism remains incompletely understood. SHP2 is an ubiquitously expressed cytoplasmic protein tyrosine phosphatase. SHP2 loss-of-function mutations in chondroid cells are linked to metachondromatosis in humans and mice, suggesting a crucial role for SHP2 in the skeleton. However, the specific role of SHP2 in skeletal cells has not been elucidated. To approach this question, we ablated SHP2 in collagen 2α1(Col2α1)-Cre- and collagen 10α1(Col10α1)-Cre-expressing cells, predominantly proliferating and hypertrophic chondrocytes, using "Cre-loxP"-mediated gene excision. Mice lacking SHP2 in Col2α1-Cre-expressing cells die at mid-gestation. Postnatal SHP2 ablation in the same cell population caused dwarfism, chondrodysplasia and exostoses. In contrast, mice in which SHP2 was ablated in the Col10α1-Cre-expressing cells appeared normal but were osteopenic. Further mechanistic studies revealed that SHP2 exerted its influence partly by regulating the abundance of SOX9 in chondrocytes. Elevated and sustained SOX9 in SHP2-deficient hypertrophic chondrocytes impaired their differentiation to osteoblasts and impaired endochondral ossification. Our study uncovered an important role of SHP2 in bone development and cartilage homeostasis by influencing the osteogenic differentiation of hypertrophic chondrocytes and provided insight into the pathogenesis and potential treatment of skeletal diseases, such as osteopenia and osteoporosis.
Journal of developmental biology
Vonk, AC;Hasel-Kolossa, SC;Lopez, GA;Hudnall, ML;Gamble, DJ;Lozito, TP;
PMID: 35225965 | DOI: 10.3390/jdb10010012
(1) Background: Lizard tail regeneration provides a unique model of blastema-based tissue regeneration for large-scale appendage replacement in amniotes. Green anole lizard (Anolis carolinensis) blastemas contain fibroblastic connective tissue cells (FCTCs), which respond to hedgehog signaling to create cartilage in vivo. However, an in vitro model of the blastema has not previously been achieved in culture. (2) Methods: By testing two adapted tissue dissociation protocols and two optimized media formulations, lizard tail FCTCs were pelleted in vitro and grown in a micromass blastema organoid culture. Pellets were analyzed by histology and in situ hybridization for FCTC and cartilage markers alongside staged original and regenerating lizard tails. (3) Results: Using an optimized serum-free media and a trypsin- and collagenase II-based dissociation protocol, micromass blastema organoids were formed. Organoid cultures expressed FCTC marker CDH11 and produced cartilage in response to hedgehog signaling in vitro, mimicking in vivo blastema and tail regeneration. (4) Conclusions: Lizard tail blastema regeneration can be modeled in vitro using micromass organoid culture, recapitulating in vivo FCTC marker expression patterns and chondrogenic potential.
Brenna Ø, Furnes MW, Munkvold B, Kidd M, Sandvik AK, Gustafsson BI.
PMID: 27044258 | DOI: -
Guanylin (GUCA2A/Guca2a/GN) and uroguanylin (GUCA2B/Guca2b/UGN) are expressed in the gastrointestinal tract and have been implicated in ion and fluid homeostasis, satiety, abdominal pain, growth and intestinal barrier integrity. Their cellular sources are debated and include goblet cells, entero-/colonocytes, enteroendocrine (EE) cells and tuft cells. We therefore investigated the cellular sources of GN and UGN mRNAs in human and rat duodenal and colonic epithelium with in situ hybridization (ISH) to determine co-expression with Chromogranin A (CHGA/Chga/CgA; enterochromaffin [EC] cells), defensin alpha 6 (DEFA6/Defa6; Paneth cells), mucin 2 (MUC2/Muc2; goblet cells) and selected tuft cell markers. GUCA2A/Guca2a expression was localized to goblet cells and colonocytes in human and rat colon. In human duodenum, GUCA2A was expressed in Paneth cells and was scarce in villous epithelial cells. In rat duodenum, Guca2a was only localized to goblet cells. Guca2b was focally expressed in rat colon. In human and rat duodenum and in human colon, GUCA2B/Guca2b was expressed in dispersed solitary epithelial cells, some with a tuft cell-like appearance. Neither GUCA2A nor GUCA2B were co-expressed with CHGA in human duodenal cells. Consequently, EC cells are probably not the major source of human GN or UGN but other EE cells as a source of GN or UGN are not entirely excluded. No convincing overlap with tuft cell markers was found. For the first time, we demonstrate the cellular expression of GUCA2B in human duodenum. The specific cellular distribution of both GN and UGN differs between duodenum and colon and between human and rat intestines.
Chen, HJ;Barske, L;Talbot, JC;Dinwoodie, OM;Roberts, RR;Farmer, DT;Jimenez, C;Merrill, AE;Tucker, AS;Crump, JG;
PMID: 36905926 | DOI: 10.1016/j.devcel.2023.02.011
Organ development involves the sustained production of diverse cell types with spatiotemporal precision. In the vertebrate jaw, neural-crest-derived progenitors produce not only skeletal tissues but also later-forming tendons and salivary glands. Here we identify the pluripotency factor Nr5a2 as essential for cell-fate decisions in the jaw. In zebrafish and mice, we observe transient expression of Nr5a2 in a subset of mandibular postmigratory neural-crest-derived cells. In zebrafish nr5a2 mutants, nr5a2-expressing cells that would normally form tendons generate excess jaw cartilage. In mice, neural-crest-specific Nr5a2 loss results in analogous skeletal and tendon defects in the jaw and middle ear, as well as salivary gland loss. Single-cell profiling shows that Nr5a2, distinct from its roles in pluripotency, promotes jaw-specific chromatin accessibility and gene expression that is essential for tendon and gland fates. Thus, repurposing of Nr5a2 promotes connective tissue fates to generate the full repertoire of derivatives required for jaw and middle ear function.
Nan, P;Dong, X;Bai, X;Lu, H;Liu, F;Sun, Y;Zhao, X;
PMID: 34958892 | DOI: 10.1016/j.canlet.2021.12.025
The pancreatic ductal adenocarcinoma (PDAC) microenvironment contains dense desmoplastic stroma dominated by cancer-associated fibroblasts (CAFs) and is crucial to cancer development and progression. Several studies have revealed that thrombospondin 2 (THBS2) is a valuable serological-marker in PDAC. However, the detailed mechanism of the cancer-stroma interactome remains unclear. Here we showed that elevated THBS2 expression in PDAC was predominantly restricted to stroma and correlated with tumor progression and poor prognosis by quantitative proteomics and immunohistochemistry analyses. RNA in situ hybridization confirmed that CAFs but not neoplastic cells expressed THBS2 in precancerous lesions and its levels gradually increased with disease progression in genetically engineered mouse models. Mechanistically, cancer cell-secreted TGF-β1 activated CAFs to induce THBS2 expression via the p-Smad2/3 pathway. Consequently, CAF-derived THBS2 bound to the membrane receptors integrin αvβ3/CD36 and activated the MAPK pathway in PDAC cells to promote tumor growth and adhesion in vitro and in vivo. Inhibition of integrin αvβ3, CD36, MEK and JNK rescued THBS2-induced malignant phenotypes. In conclusion, the TGF-β1-THBS2-integrin αvβ3/CD36-MAPK cascade forms a complex feedback circuit to mediate reciprocal interactions of pancreatic cancer cells-CAFs. THBS2 may act as a novel therapeutic-target to block the cancer-stroma communication.
Miura Y, Ota S, Peterlin M, McDevitt G, Kanazawa S.
| DOI: 10.1002/jbm4.10132
Specific MHC class II genes result in a high susceptibility to rheumatoid arthritis (RA), with co‐stimulatory molecules working together with MHC class II during the progression of the disease. To elucidate the involvement of the B7.1 co‐stimulatory molecule in RA, we analyzed the phenotype of B7.1 transgenic (named D1BC) mice and the sequential differentiation of synovial fibroblasts (SFs) by studying the expression of chondrogenic and osteogenic lineage markers together with lineage tracing experiment using B7.1 transgene in vivo. The B7.1 transgene was driven by a collagen type II (CII) promoter and enhancer in the D1BC mouse. A low‐dose of bovine CII (bCII) was used to induce chronic articular inflammation with interstitial pneumonitis. Joint damage was analyzed by histopathological examination and computed tomography. B7.1 was expressed in articular cartilage and SFs of D1BC mice. Chronic inflammatory arthritis in bCII‐D1BC mouse shared common features with those found in patients with RA, such as pannus formation, bone destruction, osteoporosis, and joint ankylosis. A subpopulation of SFs (Runx2+, Sox9+, Col10a1+, Osx+ and CX‐) in the pannus was classified as osteochondrogenic lineage rather than mesenchymal stromal lineage. These cells underwent differentiation into osteogenic lineage via hypertrophic chondrocytes at the end of the chronic phase. The ectopic expression of B7.1 in chondrocytes and SFs leads to an increased susceptibility to chronic inflammatory arthritis and subsequent new bone formation, reminiscent of ankylosis. The regulation of cartilage remodeling in pannus tissue is an important consideration in the treatment of RA.
Microbial exposure during early human development primes fetal immune cells
Mishra, A;Lai, GC;Yao, LJ;Aung, TT;Shental, N;Rotter-Maskowitz, A;Shepherdson, E;Singh, GSN;Pai, R;Shanti, A;Wong, RMM;Lee, A;Khyriem, C;Dutertre, CA;Chakarov, S;Srinivasan, KG;Shadan, NB;Zhang, XM;Khalilnezhad, S;Cottier, F;Tan, ASM;Low, G;Chen, P;Fan, Y;Hor, PX;Lee, AKM;Choolani, M;Vermijlen, D;Sharma, A;Fuks, G;Straussman, R;Pavelka, N;Malleret, B;McGovern, N;Albani, S;Chan, JKY;Ginhoux, F;
PMID: 34077752 | DOI: 10.1016/j.cell.2021.04.039
The human fetal immune system begins to develop early during gestation; however, factors responsible for fetal immune-priming remain elusive. We explored potential exposure to microbial agents in utero and their contribution toward activation of memory T cells in fetal tissues. We profiled microbes across fetal organs using 16S rRNA gene sequencing and detected low but consistent microbial signal in fetal gut, skin, placenta, and lungs in the 2nd trimester of gestation. We identified several live bacterial strains including Staphylococcus and Lactobacillus in fetal tissues, which induced in vitro activation of memory T cells in fetal mesenteric lymph node, supporting the role of microbial exposure in fetal immune-priming. Finally, using SEM and RNA-ISH, we visualized discrete localization of bacteria-like structures and eubacterial-RNA within 14th weeks fetal gut lumen. These findings indicate selective presence of live microbes in fetal organs during the 2nd trimester of gestation and have broader implications toward the establishment of immune competency and priming before birth. Crown
Single-Cell RNA-seq Reveals Angiotensin-Converting Enzyme 2 and Transmembrane Serine Protease 2 Expression in TROP2+ Liver Progenitor Cells: Implications in Coronavirus Disease 2019-Associated Liver Dysfunction
Seow, JJW;Pai, R;Mishra, A;Shepherdson, E;Lim, TKH;Goh, BKP;Chan, JKY;Chow, PKH;Ginhoux, F;DasGupta, R;Sharma, A;
PMID: 33968947 | DOI: 10.3389/fmed.2021.603374
The recent coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2. COVID-19 was first reported in China (December 2019) and is now prevalent across the globe. Entry of severe acute respiratory syndrome coronavirus 2 into mammalian cells requires the binding of viral Spike (S) proteins to the angiotensin-converting enzyme 2 receptor. Once entered, the S protein is primed by a specialized serine protease, transmembrane serine protease 2 in the host cell. Importantly, besides the respiratory symptoms that are consistent with other common respiratory virus infections when patients become viremic, a significant number of COVID-19 patients also develop liver comorbidities. We explored whether a specific target cell-type in the mammalian liver could be implicated in disease pathophysiology other than the general deleterious response to cytokine storms. Here, we used single-cell RNA-seq to survey the human liver and identified potentially implicated liver cell-type for viral ingress. We analyzed ~300,000 single cells across five different (i.e., human fetal, healthy, cirrhotic, tumor, and adjacent normal) liver tissue types. This study reports on the co-expression of angiotensin-converting enzyme 2 and transmembrane serine protease 2 in a TROP2+ liver progenitor population. Importantly, we detected enrichment of this cell population in the cirrhotic liver when compared with tumor tissue. These results indicated that in COVID-19-associated liver dysfunction and cell death, a viral infection of TROP2+ progenitors in the liver might significantly impair liver regeneration in patients with liver cirrhosis.
Yoshida T, Takizawa N, Matsuda T, Yamada H, Kitada M, Tanaka S
PMID: 31949236 | DOI: 10.1038/s41598-019-57351-5
Adrenal cortex autotransplantation with ACTH stimulation may be an alternative therapy for patients with bilateral adrenalectomy to avoid adrenal crisis, but its underlying mechanism has not been elucidated. Previously, we detected Dhh upregulation in rat adrenocortical autografts after transplantation. Here, we investigated potential regulators such as Gata4, Gata6, Sry and Sox9 which affect Dhh transcription in adrenocortical autografts with or without ACTH stimulation. In ACTH-stimulated autografts, Gata4 and Gata6 were downregulated compared to control autografts. This response was linked to rDhh repression. A reporter assay using the upstream region of rDhh and a GATA binding motif revealed that rDhh promoters were significantly upregulated by co-transfection with Gata4 or Gata6 or both. Sry and Sox9 expression in autografts with or without ACTH stimulation were verified by PCR and RNAscope analyses. The ovarian differentiation factors Foxl2 and Rspo1 were also upregulated in the autografts. Gata4 and Gata6 were found to be significant factors in the regulation of rDhh expression and could be associated with adrenocortical autograft maintenance. Gonadal primordia with bipotential testicular and ovarian functions may also be present in these autografts.
"Boldog E, Bakken TE, Hodge RD, Novotny M, Aevermann BD, Baka J, Bordé S, Close JL, Diez-Fuertes F, Ding SL, Faragó N, Kocsis AK, Kovács B, Maltzer Z, McCorrison JM, Miller JA, Molnár G, Oláh G, Ozsvár A, Rózsa M, Shehata SI, Smith KA, Sunkin SM, Tran D
PMID: 30150662 | DOI: 10.1038/s41593-018-0205-2
We describe convergent evidence from transcriptomics, morphology, and physiology for a specialized GABAergic neuron subtype in human cortex. Using unbiased single-nucleus RNA sequencing, we identify ten GABAergic interneuron subtypes with combinatorial gene signatures in human cortical layer 1 and characterize a group of human interneurons with anatomical features never described in rodents, having large 'rosehip'-like axonal boutons and compact arborization. These rosehip cells show an immunohistochemical profile (GAD1+CCK+, CNR1-SST-CALB2-PVALB-) matching a single transcriptomically defined cell type whose specific molecular marker signature is not seen in mouse cortex. Rosehip cells in layer 1 make homotypic gap junctions, predominantly target apical dendritic shafts of layer 3 pyramidal neurons, and inhibit backpropagating pyramidal action potentials in microdomains of the dendritic tuft. These cells are therefore positioned for potent local control of distal dendritic computation in cortical pyramidal neurons.
Ruscitto A, Morel MM, Shawber CJ, Reeve G, Lecholop MK, Bonthius D, Yao H, Embree MC
PMID: 32030828 | DOI: 10.1096/fj.201902287R
Temporomandibular joint osteoarthritis (TMJ OA) leads to permanent cartilage destruction, jaw dysfunction, and compromises the quality of life. However, the pathological mechanisms governing TMJ OA are poorly understood. Unlike appendicular articular cartilage, the TMJ has two distinct functions as the synovial joint of the craniofacial complex and also as the site for endochondral jaw bone growth. The established dogma of endochondral bone ossification is that hypertrophic chondrocytes undergo apoptosis, while invading vasculature with osteoprogenitors replace cartilage with bone. However, contemporary murine genetic studies support the direct differentiation of chondrocytes into osteoblasts and osteocytes in the TMJ. Here we sought to characterize putative vasculature and cartilage to bone transdifferentiation using healthy and diseased TMJ tissues from miniature pigs and humans. During endochondral ossification, the presence of fully formed vasculature expressing CD31+ endothelial cells and ?-SMA+ vascular smooth muscle cells were detected within all cellular zones in growing miniature pigs. Arterial, endothelial, venous, angiogenic, and mural cell markers were significantly upregulated in miniature pig TMJ tissues relative to donor matched knee meniscus fibrocartilage tissue. Upon surgically creating TMJ OA in miniature pigs, we discovered increased vasculature and putative chondrocyte to osteoblast transformation dually marked by COL2 and BSP or RUNX2 within the vascular bundles. Pathological human TMJ tissues also exhibited increased vasculature, while isolated diseased human TMJ cells exhibited marked increased in vasculature markers relative to control 293T cells. Our study provides evidence to suggest that the TMJ in higher order species are in fact vascularized. There have been no reports of cartilage to bone transdifferentiation or vasculature in human-relevant TMJ OA large animal models or in human TMJ tissues and cells. Therefore, these findings may potentially alter the clinical management of TMJ OA by defining new drugs that target angiogenesis or block the cartilage to bone transformation
Kennedy, EA;Aggarwal, S;Dhar, A;Karst, SM;Wilen, CB;Baldridge, MT;
PMID: 37188813 | DOI: 10.1038/s41564-023-01383-1
Norovirus (NoV) is the leading global cause of viral gastroenteritis. Young children bear the highest burden of disease and play a key role in viral transmission throughout the population. However, which host factors contribute to age-associated variability in NoV severity and shedding are not well-defined. The murine NoV (MNoV) strain CR6 causes persistent infection in adult mice and targets intestinal tuft cells. Here we find that natural transmission of CR6 from infected dams occurred only in juvenile mice. Direct oral CR6 inoculation of wild-type neonatal mice led to accumulation of viral RNA in the ileum and prolonged shedding in the stool that was replication-independent. This viral exposure induced both innate and adaptive immune responses including interferon-stimulated gene expression and MNoV-specific antibody responses. Interestingly, viral uptake depended on passive ileal absorption of luminal virus, a process blocked by cortisone acetate administration, which prevented ileal viral RNA accumulation. Neonates lacking interferon signalling in haematopoietic cells were susceptible to productive infection, viral dissemination and lethality, which depended on the canonical MNoV receptor CD300LF. Together, our findings reveal developmentally associated aspects of persistent MNoV infection, including distinct tissue and cellular tropism, mechanisms of interferon regulation and severity of infection in the absence of interferon signalling. These emphasize the importance of defining viral pathogenesis phenotypes across the developmental spectrum and highlight passive viral uptake as an important contributor to enteric infections in early life.
