Insight into the maintenance of odontogenic potential in mouse dental mesenchymal cells based on transcriptomic analysis

Background.Mouse dental mesenchymal cells (mDMCs) from tooth germs of cap or later stages are frequently used in the context of developmental biology or whole-tooth regeneration due to their odontogenic potential.In vitro-expanded mDMCs serve as an alternative cell source considering the difficulty in obtaining primary mDMCs; however, cultured mDMCs fail to support tooth development as a result of functional failures of specific genes or pathways. The goal of this study was to identify the genes that maintain the odontogenic potential of mDMCs in culture.Methods.We examined the odontogenic potential of freshly isolated versus cultured mDMCs from the lower first molars of embryonic day 14.5 mice. The transcriptome of mDMCs was detected using RNA sequencing and the data were validated by qRT-PCR. Differential expression analysis and pathway analysis were conducted to identify the genes that contribute to the loss of odontogenic potential.Results.Cultured mDMCs failed to develop into well-structured tooth when they were recombined with dental epithelium. Compared with freshly isolated mDMCs, we found that 1,004 genes were upregulated and 948 were downregulated in cultured mDMCs. The differentially expressed genes were clustered in the biological processes and signaling pathways associated with tooth development. Followingin vitroculture, genes encoding a wide array of components of MAPK, TGF-β/BMP, and Wnt pathways were significantly downregulated. Moreover, the activities ofBdnf,Vegfα,Bmp2, andBmp7were significantly inhibited in cultured mDMCs. Supplementation of VEGFα, BMP2, and BMP7 restored the expression of a subset of downregulated genes and induced mDMCs to form dentin-like structuresin vivo.Conclusions.Vegfα,Bmp2, andBmp7play a role in the maintenance of odontogenic potential in mDMCs.

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Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

10.1101/2019.12.16.878546 ◽

2019 ◽

Author(s):

Cassandra K. Hayne ◽

Casey A. Schmidt ◽

A. Gregory Matera ◽

Robin E. Stanley

Keyword(s):

Animal Cells ◽

Trna Splicing ◽

Polynucleotide Kinase ◽

Genes Encoding ◽

Intron Removal ◽

And Function ◽

Insight Into ◽

Negative Modulator

ABSTRACTThe splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34, and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

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Reconstitution of the human tRNA splicing endonuclease complex: insight into the regulation of pre-tRNA cleavage

Nucleic Acids Research ◽

10.1093/nar/gkaa438 ◽

2020 ◽

Vol 48 (14) ◽

pp. 7609-7622 ◽

Cited By ~ 3

Author(s):

Cassandra K Hayne ◽

Casey A Schmidt ◽

Maira I Haque ◽

A Gregory Matera ◽

Robin E Stanley

Keyword(s):

Animal Cells ◽

Trna Splicing ◽

Polynucleotide Kinase ◽

Genes Encoding ◽

Intron Removal ◽

And Function ◽

Insight Into ◽

Negative Modulator

Abstract The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1’s role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.

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YtxR, a Conserved LysR-Like Regulator That Induces Expression of Genes Encoding a Putative ADP-Ribosyltransferase Toxin Homologue in Yersinia enterocolitica

Journal of Bacteriology ◽

10.1128/jb.01159-06 ◽

2006 ◽

Vol 188 (23) ◽

pp. 8033-8043 ◽

Cited By ~ 14

Author(s):

Grace L. Axler-DiPerte ◽

Virginia L. Miller ◽

Andrew J. Darwin

Keyword(s):

Yersinia Enterocolitica ◽

Transcription Initiation ◽

Photorhabdus Luminescens ◽

Expression Of Genes ◽

Dnase I Footprinting ◽

Genes Encoding ◽

Adp Ribosyltransferase ◽

Insight Into

ABSTRACT Yersinia enterocolitica causes human gastroenteritis, and many isolates have been classified as either “American” or “non-American” strains based on their geographic prevalence and virulence properties. In this study we describe identification of a transcriptional regulator that controls expression of the Y. enterocolitica ytxAB genes. The ytxAB genes have the potential to encode an ADP-ribosylating toxin with similarity to pertussis toxin. However, a ytxAB null mutation did not affect virulence in mice. Nevertheless, the ytxAB genes are conserved in many Y. enterocolitica strains. Interestingly, American and non-American strains have different ytxAB alleles encoding proteins that are only 50 to 60% identical. To obtain further insight into the ytxAB locus, we investigated whether it is regulated as part of a known or novel regulon. Transposon mutagenesis identified a LysR-like regulator, which we designated YtxR. Expression of ytxR from a nonnative promoter increased Φ(ytxA-lacZ) operon fusion expression up to 35-fold. YtxR also activated expression of its own promoter. DNase I footprinting showed that a His6-YtxR fusion protein directly interacted with the ytxA and ytxR control regions at similar distances upstream of their probable transcription initiation sites, identified by primer extension. Deletion analysis demonstrated that removal of the regions protected by His6-YtxR in vitro eliminated YtxR-dependent induction in vivo. The ytxAB locus is not present in most Yersinia species. In contrast, ytxR is conserved in multiple Yersinia species, as well as in the closely related organisms Photorhabdus luminescens and Photorhabdus asymbiotica. These observations suggest that YtxR may play a conserved role involving regulation of other genes besides ytxAB.

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Epithelial-mesenchymal interactions are required for msx 1 and msx 2 gene expression in the developing murine molar tooth

Development ◽

10.1242/dev.117.2.461 ◽

1993 ◽

Vol 117 (2) ◽

pp. 461-470 ◽

Cited By ~ 11

Author(s):

A.K. Jowett ◽

S. Vainio ◽

M.W. Ferguson ◽

P.T. Sharpe ◽

I. Thesleff

Keyword(s):

Gene Expression ◽

Tooth Development ◽

Homeobox Gene ◽

Oral Epithelium ◽

Tooth Formation ◽

Dental Papilla ◽

Tissue Interactions ◽

Dental Epithelium

Duplication of the msh-like homeobox gene of Drosophila may be related to the evolution of the vertebrate head. The murine homologues of this gene, msx 1 and msx 2 are expressed in the developing craniofacial complex including the branchial arches, especially in regions of epithelial-mesenchymal organogenesis including the developing tooth. By performing in vitro recombination experiments using homochronic dental and non-dental epithelial and mesenchymal tissues from E10 to E18 mouse embryos, we have found that the maintenance of homeobox gene expression in the tooth is dependent upon tissue interactions. In homotypic recombinants, dental-type tissue interactions occur, leading to expression of both genes in a manner similar to that seen during in vivo development. msx 1 is expressed exclusively in mesenchyme, both in the dental papilla and follicle. msx 2 is expressed in the dental epithelium and only in the mesenchyme of the dental papilla. In heterotypic recombinants, the dental epithelium is able to induce msx 1 expression in non-dental mesenchyme, this potential being lost at the bell stage. In these recombinants msx 2 was induced by presumptive dental epithelium prior to the bud stage but not thereafter. The expression of msx 1 and msx 2 in dental mesenchyme requires the presence of epithelium until the early bell stage. However, whereas non-dental, oral epithelium is capable of maintaining expression of msx 1 in dental mesenchyme throughout tooth development, induction of msx 2 was temporally restricted suggesting regulation by a specific epithelial-mesenchymal interaction related to the inductive events of tooth formation. msx 1 and msx 2, as putative transcription factors, may play a role in regulating the expression of other genes during tooth formation. We conclude that expression of msx 1 in jaw mesenchyme requires a non-specific epithelial signal, whereas msx 2 expression in either epithelium or mesenchyme requires reciprocal interactions between specialized dental cell populations.

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BMP4 rescues a non-cell-autonomous function of Msx1 in tooth development

Development ◽

10.1242/dev.127.21.4711 ◽

2000 ◽

Vol 127 (21) ◽

pp. 4711-4718 ◽

Cited By ~ 3

Author(s):

M. Bei ◽

K. Kratochwil ◽

R.L. Maas

Keyword(s):

Dental Pulp ◽

Tooth Development ◽

Survival Function ◽

Tooth Germ ◽

Tooth Formation ◽

Kidney Capsule ◽

Dependent Signal ◽

Dental Epithelium ◽

Tooth Germs

The development of many organs depends on sequential epithelial-mesenchymal interactions, and the developing tooth germ provides a powerful model for elucidating the nature of these inductive tissue interactions. In Msx1-deficient mice, tooth development arrests at the bud stage when Msx1 is required for the expression of Bmp4 and Fgf3 in the dental mesenchyme (Bei, M. and Maas, R. (1998) Development 125, 4325–4333). To define the tissue requirements for Msx1 function, we performed tissue recombinations between wild-type and Msx1 mutant dental epithelium and mesenchyme. We show that through the E14.5 cap stage of tooth development, Msx1 is required in the dental mesenchyme for tooth formation. After the cap stage, however, tooth development becomes Msx1 independent, although our experiments identify a further late function of Msx1 in odontoblast and dental pulp survival. These results suggest that prior to the cap stage, the dental epithelium receives an Msx1-dependent signal from the dental mesenchyme that is necessary for tooth formation. To further test this hypothesis, Msx1 mutant tooth germs were first cultured with either BMP4 or with various FGFs for two days in vitro and then grown under the kidney capsule of syngeneic mice to permit completion of organogenesis and terminal differentiation. Previously, using an in vitro culture system, we showed that BMP4 stimulated the growth of Msx1 mutant dental epithelium (Chen, Y., Bei, M. Woo, I., Satokata, I. and Maas, R. (1996). Development 122, 3035–3044). Using the more powerful kidney capsule grafting procedure, we now show that when added to explanted Msx1-deficient tooth germs prior to grafting, BMP4 rescues Msx1 mutant tooth germs all the way to definitive stages of enamel and dentin formation. Collectively, these results establish a transient functional requirement for Msx1 in the dental mesenchyme that is almost fully supplied by BMP4 alone, and not by FGFs. In addition, they formally prove the postulated downstream relationship of BMP4 with respect to Msx1, establish the non-cell-autonomous nature of Msx1 during odontogenesis, and disclose an additional late survival function for Msx1 in odontoblasts and dental pulp.

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The homeobox gene Hox 7.1 has specific regional and temporal expression patterns during early murine craniofacial embryogenesis, especially tooth development in vivo and in vitro

Development ◽

10.1242/dev.111.2.269 ◽

1991 ◽

Vol 111 (2) ◽

pp. 269-285 ◽

Cited By ~ 10

Author(s):

A. Mackenzie ◽

G.L. Leeming ◽

A.K. Jowett ◽

M.W. Ferguson ◽

P.T. Sharpe

Keyword(s):

Neural Crest ◽

Tooth Development ◽

Expression Patterns ◽

Homeobox Gene ◽

Branchial Arch ◽

Developing Brain ◽

Stage Of Development ◽

Tooth Germs

Hox 7.1 is a murine homeobox-containing gene expressed in a range of neural-crest-derived tissues and areas of putative epithelial-mesenchymal interactions during embryogenesis. We have examined the expression of Hox 7.1 during craniofacial development in the mouse embryo between days 8 and 16 of development. Whereas facial expression at day 10 of gestation is broadly localised in the neural-crest-derived mesenchyme of the medial nasal, lateral nasal, maxillary and mandibular processes, by day 12 expression is restricted to the mesenchyme immediately surrounding the developing tooth germs in the maxillary and mandibular processes. Hox 7.1 expression in the mesenchyme of the dental papilla and follicle is maximal at the cap stage of development and progressively declines in the bell stage prior to differentiation of odontoblasts and ameloblasts. Hox 7.1 expression in tooth germs is independent of overall embryonic stage of development but is dependent on stage of development of the individual tooth. Similar patterns of transient Hox 7.1 expression can also be detected in tooth germs in vitro in organ cultures of day 11 first branchial arch explants cultured for up to 7 days. Hox 7.1 is also expressed early in development (days 10/11) in the epithelium of the developing anterior pituitary (Rathke's pouch), the connective tissue capsule and meninges of the developing brain, and specific regions of neuroepithelium in the developing brain.

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Induction and Differentiation of Dental Epithelium in Vivo and in Vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100053425 ◽

1982 ◽

Vol 40 ◽

pp. 142-145

Author(s):

E. J. Kollar

Keyword(s):

Connective Tissue ◽

Oral Mucosa ◽

Basal Lamina ◽

Functional Derivatives ◽

Specific Source ◽

Dental Epithelium ◽

Connective Tissue Stroma

The differentiation and maintenance of many specialized epithelial structures are dependent on the underlying connective tissue stroma and on an intact basal lamina. These requirements are especially stringent in the development and maintenance of the skin and oral mucosa. The keratinization patterns of thin or thick cornified layers as well as the appearance of specialized functional derivatives such as hair and teeth can be correlated with the specific source of stroma which supports these differentiated expressions.

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Plant-Derived Extracellular Vesicles: Current Findings,Challenges, and Future Applications

Membranes ◽

10.3390/membranes11060411 ◽

2021 ◽

Vol 11 (6) ◽

pp. 411

Author(s):

Nader Kameli ◽

Anya Dragojlovic-Kerkache ◽

Paul Savelkoul ◽

Frank R. Stassen

Keyword(s):

Human Health ◽

Extracellular Vesicles ◽

Preliminary Results ◽

In Vivo Experiments ◽

Health And Disease ◽

Conducting Research ◽

Protocol Standardization ◽

Insight Into

In recent years, plant-derived extracellular vesicles (PDEVs) have gained the interest of many experts in fields such as microbiology and immunology, and research in this field has exponentially increased. These nano-sized particles have provided researchers with a number of interesting findings, making their application in human health and disease very promising. Both in vitro and in vivo experiments have shown that PDEVs can exhibit a multitude of effects, suggesting that these vesicles may have many potential future applications, including therapeutics and nano-delivery of compounds. While the preliminary results are promising, there are still some challenges to face, such as a lack of protocol standardization, as well as knowledge gaps that need to be filled. This review aims to discuss various aspects of PDEV knowledge, including their preliminary findings, challenges, and future uses, giving insight into the complexity of conducting research in this field.

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MESENCHYMAL TNFR1 EXPRESSION PRESERVES THE COLONIC EPITHELIAL STEM CELL NICHE

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa347.018 ◽

2021 ◽

Vol 27 (Supplement_1) ◽

pp. S7-S8

Author(s):

Safina Gadeock ◽

Cambrian Liu ◽

Brent Polk

Keyword(s):

Stem Cell ◽

Stem Cell Niche ◽

Mesenchymal Cells ◽

Epithelial Stem Cells ◽

Epithelial Stem Cell ◽

Long Term Treatment ◽

Lgr5 Expression ◽

Cell Niche

Abstract Tumor necrosis factor (TNF) is a highly expressed cytokine in inflammatory bowel disease (IBD). Although TNF can induce colonic epithelial dysfunction and apoptosis, recent studies suggest that TNF signalling promotes epithelial wound repair and stem cell function. Here we investigated the role of TNF receptor 1 (TNFR1) in mediating TNF’s effects on colonic epithelial stem cells, integral to mucosal healing in colitis. We demonstrate that Tnfr1-/- mice exhibit loss in Lgr5 expression (-52%, p<0.02; N=6) compared to wildtype (WT) controls. However, the opposite result was found in vitro, wherein murine Tnfr1-/- colonoids demonstrated a significant increase in Lgr5 expression (66%, p<0.007; N=6) compared to WT colonoids. Similarly, human colonoids treated with an anti-TNFR1 antibody also demonstrated an increase in Lgr5 expression, relative to IgG controls. To resolve the contradiction in the in vivo versus in vitro environment, we hypothesized that mesenchymal TNFR1 expression regulates the epithelial stem cell niche. To determine the relationships between these cell types, we co-cultured WT or Tnfr1-/- colonoids with WT or Tnfr1-/- colonic myofibroblasts (CMFs). We found that epithelial Lgr5 expression was significantly higher (by 52%, p<0.05; N=3) when co-cultured with WT compared to TNFR1-/- myofibroblasts. The loss of TNFR1 expression in vivo increases the number of αSMA+ mesenchymal cells by nearly 56% (N=6) but considerably reduces the pericryptal PDGFRα+ cells, suggesting modifications in mesenchymal populations that contribute to the epithelial stem cell niche. Functionally, primary Tnfr1-/--CMFs displayed PI3k (p<0.001; N=3) and MAPK (p<0.01; N=3)-dependent increases in migration, proliferation, and differentiation, but RNA profiling demonstrated by diminished levels of stem cell niche factors, Rspo3 (-80%, p<0.0001; N=6) and Wnt2b (-63%, p<0.008; N=6) compared to WT-CMFs. Supplementation with 50ng recombinant Rspo3 for 5 d to Lgr5-GFP organoids co-cultured with TNFR1-/--CMFs restored Lgr5 expression to wildtype levels. Therefore, TNFR1-mediated TNF signalling in mesenchymal cells promotes their ability to support an epithelial stem cell niche. These results should motivate future studies of the stem cell niche in the context of long-term treatment with anti-TNF therapies.

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In vivo transcriptome analysis provides insights into host-dependent expression of virulence factors by Yersinia entomophaga MH96, during infection of Galleria mellonella

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkaa024 ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Amber R Paulson ◽

Maureen O’Callaghan ◽

Xue-Xian Zhang ◽

Paul B Rainey ◽

Mark R H Hurst

Keyword(s):

Galleria Mellonella ◽

Functional Enrichment ◽

Natural Environments ◽

Insecticidal Toxin ◽

Heat Stable ◽

Expression Of Genes ◽

Genes Encoding ◽

Cell Densities

Abstract The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.

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