ABSTRACT: Hox and ParaHox genes encode transcription factors with conserved similar expression patterns in divergent animals. The Pdx (Xlox) homeobox gene, for example, is expressed in a sharp spatial domain in the endodermal cell layer of the gut in chordates, echinoderms, annelids and molluscs. The significance of comparable gene expression patterns is unclear because it is not known if downstream transcriptional targets are also conserved. We thus conducted experiments to show that a classic transcriptional target of Pdx1 in vertebrates, the insulin gene, is also a direct target of Pdx in the Pacific oyster. We report that oyster has a diversity of insulin-related genes including one co-expressed with Pdx in the endodermal layer of oyster digestive tissue. Transcriptome analysis reveals functional similarity of this tissue to vertebrate pancreas. Using ATAC-seq we identify a Pdx homeodomain binding site upstream of the endodermally-expressed oyster insulin-related gene and using cell culture demonstrate that oyster Pdx acts as a transcriptional activator through this site. These data argue that a classic homeodomain-target gene interaction dates back to the base of Bilateria.
Project description:The homeodomain transcription factor, Pdx-1, has important roles in pancreatic development and M-NM-2-cell function and survival. In the present study, we demonstrate that adenovirus-mediated overexpression of Pdx-1 in rat or human islets also stimulates cell replication. Moreover, co-overexpression of Pdx-1 with another homeodomain transcription factor, Nkx6.1, has an additive effect on proliferation compared to either factor alone, implying discrete activating mechanisms. Consistent with this, Nkx6.1 stimulates mainly M-NM-2-cell proliferation, whereas Pdx-1 stimulates both M-NM-1- and M-NM-2-cell proliferation. Furthermore, cyclins D1/D2 are upregulated by Pdx-1 but not by Nkx6.1, and inhibition of cdk4 blocks Pdx-1- but not Nkx6.1-stimulated islet cell proliferation. Genes regulated by Pdx-1 and not Nkx6.1 were identified by microarray analysis. Two members of the transient receptor potential cation (TRPC) channel family, TRPC3 and TRPC6, are upregulated by Pdx-1 overexpression, and siRNA-mediated knockdown of TRPC3/6 or TRPC6 alone inhibits Pdx-1-induced but not Nkx6.1-induced islet cell proliferation. Pdx-1 also stimulates ERK1/2 phosphorylation, an effect partially blocked by knockdown of TRPC3/6, and blockade of ERK1/2 activation with a MEK1/2 inhibitor partially impairs Pdx-1-stimulated proliferation. These studies define a pathway by which overexpression of Pdx-1 activates islet cell proliferation that is distinct from and additive to a pathway activated by Nkx6.1. We identified genes that were upregulated or downregulated at 48 h with Pdx-1 overexpression as compared to untreated and M-NM-2gal controls. We set up a microarray using primary rat islets that were left untreated or transduced with adenoviruses overexpressing M-NM-2gal or Pdx-1 for 48 h.
Project description:We previously put forward a multi-layer systems toxicology framework for in vitro assessment of e-liquids that is meant to complement the battery of classical assays for genotoxicity testing. The framework started with the first layer to screen e-liquids for their potential toxicity, followed by the second layer to investigate the toxicity-related mechanism of a selected e-liquid(s), and finally the third layer to evaluate the toxicity-related mechanism of the corresponding aerosol(s). In this work, we leveraged this framework to assess the biological impact of an e-liquid MESH™ “Classic Tobacco” and its aerosol in comparison with the impact of 3R4F reference cigarettes. In the first layer, we evaluated the cytotoxicity profile of the MESH Classic Tobacco liquid (containing humectants, nicotine, and flavors) and its Base liquid (containing humectant and nicotine only) in comparison with total particulate matter (TPM) of 3R4F cigarette smoke (CS). In the second layer, we explored changes in specific markers using high content screening assays to identify potential toxicity-related mechanisms of the MESH Classic Tobacco and Base liquids beyond cell viability compared with the 3R4F TPM-induced effects. In the third layer, we compared the biological impact of exposure to the MESH Classic Tobacco aerosol with CS using human organotypic buccal and small airway epithelial cultures. The results showed that the cytotoxicity profile of the MESH Classic Tobacco liquid was similar to the Base liquid but lower than the toxicity of 3R4F TPM at comparable nicotine concentrations. When compared with CS exposure, MESH Classic Tobacco aerosol exposure did not cause tissue damage and elicited lower changes in the global mRNA, global microRNA, and protein markers. The global mRNA changes following Classic Tobacco aerosol exposure indicated perturbations in processes related to cell fate, cell stress, and inflammatory response that were less than 20% of the perturbations following CS exposure. In the context of tobacco-harm reduction strategy, the framework is suitable to assess the potential reduced impact of EC aerosol relative to CS.
Project description:We previously put forward a multi-layer systems toxicology framework for in vitro assessment of e-liquids that is meant to complement the battery of classical assays for genotoxicity testing. The framework started with the first layer to screen e-liquids for their potential toxicity, followed by the second layer to investigate the toxicity-related mechanism of a selected e-liquid(s), and finally the third layer to evaluate the toxicity-related mechanism of the corresponding aerosol(s). In this work, we leveraged this framework to assess the biological impact of an e-liquid MESH™ “Classic Tobacco” and its aerosol in comparison with the impact of 3R4F reference cigarettes. In the first layer, we evaluated the cytotoxicity profile of the MESH Classic Tobacco liquid (containing humectants, nicotine, and flavors) and its Base liquid (containing humectant and nicotine only) in comparison with total particulate matter (TPM) of 3R4F cigarette smoke (CS). In the second layer, we explored changes in specific markers using high content screening assays to identify potential toxicity-related mechanisms of the MESH Classic Tobacco and Base liquids beyond cell viability compared with the 3R4F TPM-induced effects. In the third layer, we compared the biological impact of exposure to the MESH Classic Tobacco aerosol with CS using human organotypic buccal and small airway epithelial cultures. The results showed that the cytotoxicity profile of the MESH Classic Tobacco liquid was similar to the Base liquid but lower than the toxicity of 3R4F TPM at comparable nicotine concentrations. When compared with CS exposure, MESH Classic Tobacco aerosol exposure did not cause tissue damage and elicited lower changes in the global mRNA, global microRNA, and protein markers. The global mRNA changes following Classic Tobacco aerosol exposure indicated perturbations in processes related to cell fate, cell stress, and inflammatory response that were less than 20% of the perturbations following CS exposure. In the context of tobacco-harm reduction strategy, the framework is suitable to assess the potential reduced impact of EC aerosol relative to CS.
Project description:The homeodomain transcription factor, Pdx-1, has important roles in pancreatic development and β-cell function and survival. In the present study, we demonstrate that adenovirus-mediated overexpression of Pdx-1 in rat or human islets also stimulates cell replication. Moreover, co-overexpression of Pdx-1 with another homeodomain transcription factor, Nkx6.1, has an additive effect on proliferation compared to either factor alone, implying discrete activating mechanisms. Consistent with this, Nkx6.1 stimulates mainly β-cell proliferation, whereas Pdx-1 stimulates both α- and β-cell proliferation. Furthermore, cyclins D1/D2 are upregulated by Pdx-1 but not by Nkx6.1, and inhibition of cdk4 blocks Pdx-1- but not Nkx6.1-stimulated islet cell proliferation. Genes regulated by Pdx-1 and not Nkx6.1 were identified by microarray analysis. Two members of the transient receptor potential cation (TRPC) channel family, TRPC3 and TRPC6, are upregulated by Pdx-1 overexpression, and siRNA-mediated knockdown of TRPC3/6 or TRPC6 alone inhibits Pdx-1-induced but not Nkx6.1-induced islet cell proliferation. Pdx-1 also stimulates ERK1/2 phosphorylation, an effect partially blocked by knockdown of TRPC3/6, and blockade of ERK1/2 activation with a MEK1/2 inhibitor partially impairs Pdx-1-stimulated proliferation. These studies define a pathway by which overexpression of Pdx-1 activates islet cell proliferation that is distinct from and additive to a pathway activated by Nkx6.1. We identified genes that were upregulated or downregulated at 48 h with Pdx-1 overexpression as compared to untreated and βgal controls.
Project description:Nacre, the iridescent material found in pearls and shells of molluscs, is formed through an extraordinary process of matrix-assisted biomineralization. Despite recent advances, many parts of the biomineralization process and its evolutionary origin remain a mystery. The pearl oyster Pinctada fucata martensii is a well-known master of biomineralization, but the molecular mechanisms underlie its production of remarkable shells and pearls is not fully understood. We sequenced the highly polymorphic genome of the pearl oyster and conducted multi-omic and biochemical studies to probe nacre formation. We identified a large set of novel proteins participating in matrix-framework formation, many in expanded families, including components similar to that found in vertebrate bones such as collagen-related VWA-containing proteins (VWAP), chondroitin sulfotransferases and regulatory elements.Considering that there are only collagen-based matrices in vertebrate bones and chitin-based matrices in most invertebrate skeletons, the presence of both chitin and elements of collagen-based matrices in nacre matrices suggests that elements of chitin- and collagen-based matrices are deeply rooted and might be part of an ancient biomineralizing matrix. Our results expand the current shell matrix-framework model and provide new insights into the evolution of diverse biomineralization systems.
Project description:The definitive endoderm germ layer is the provenance of multiple internal organs, including the lungs, liver, pancreas and intestines. Molecular events driving initial endoderm germ layer specification and subsequent anterior-posterior patterning of endoderm into distinct organ primordia remain largely cryptic. Through microarray analyses, we captured genome-wide transcriptional dynamics driving successive stages of endoderm development with the intent of identifying novel regulatory genes or diagnostic markers that respectively drive or mark endoderm committment. HES3 human embryonic stem cells (hESCs) were differentiated into highly homogeneous endodermal progenitor populations, and microarray analyses were conducted of six different populations at different tiers of the endodermal lineage hierarchy: undifferentiated hESCs, anterior primitive streak (day 1 of in vitro differentiation), definitive endoderm (day 3) and anterior foregut, posterior foregut or midgut/hindgut patterned endoderm populations (day 7). Additionally, we compared hESCs differentiated using two alternative endoderm induction protocols, serum-based or AFBLy-based differentiation (both day 3 of differentiation).
Project description:The N6-methyladenosine (m6A) has recently emerged as an important layer of the gene expression regulatory network with critical implications in vertebrate and insect development. However, despite an m6A-RNA pathway is present in the pacific oyster Crassostrea gigas, the developmental significance of epitranscriptomes in lophotrochozoan organisms remains unknown. We performed RNA sequencing (RNA-seq) and m6A-RNA immunoprecipitation sequencing (MeRIP-seq) on 10 developmental stages, on two distinct developments, to covering the whole oyster developement. The m6A-RNA methylomes show the conservation of m6A consensus motif RRACH, a m6A peak around the stop codon and highlight specific m6A signatures according to RNA species between mRNAs, lncRNAs and transposable elements. The differentially methylated RNAs constitute developmental clusters that correspond to chronological steps of oyster development (cleavage, gastrulation, tissues differentiation and metamorphosis). They are marked with an overall drop of mRNA and lncRNA methylation at the morula stage followed by a global increase up to pre-metamorphosis. Messenger RNA m6A levels are correlated to transcript content and shifts in methyladenine profiles correspond to expression kinetics. The m6A of TE transcripts is also regulated and peaks during the gastrulation. Altogether our results indicate that m6A epitranscriptomes are an important regulator of oyster development. This first epitranscriptome profiling across the development of a lophotrochozoan model brings new insights into the epigenetic control of developmental processes and their evolution.
Project description:We explored the potential for small RNA biology in organisms that diverged basal to Bilateria, identifying both miRNAs and piRNAs in Nematsotella and Amphimedon Employ HTS of endogenous small RNAs
Project description:We explored the potential for small RNA biology in organisms that diverged basal to Bilateria, identifying both miRNAs and piRNAs in Nematsotella and Amphimedon
Project description:Originating from Northeast Asia, the Pacific oyster Crassostrea gigas has been introduced into a large number of countries for aquaculture purpose. Following introduction, the Pacific oyster has turned into an invasive species in an increasing number of coastal areas, notably in Northern Europe. To explore adaptation on reproductive traits of population considered as invasive, we set up a common garden experiment based on the comparison of progenies from two populations of Pacific oyster sampled in France and Denmark. A female-biased sex-ratio and a higher condition index were observed in the Danish progeny, possibly reflecting an evolutionary reproductive strategy to increase the potential success of natural recruitment in recently settled population. Using multifarious statistical approaches and accounting for sex differences we identified several genes differentially expressed between the Danish and French progenies, and with an intermediate expression level in hybrids (additive behavior). Candidate transcripts included mRNA coding for sperm quality and insulin metabolism known to be implicated in coordinated control of reproduction. Our results suggest adaptation of invasive populations during expansion acting on reproductive traits, and in particular on a female-biased sex-ratio, fertility and gamete quality. A common garden experiment was performed in order to compare progenies from two populations of Pacific oyster sampled in France and Denmark and their hybrids. Progenies were reared under standard hatchery and nursery conditions until gonadal maturation. The employed arrays were Agilent 60-mer 4x44K custom microarrays, containing 31,918 C. gigas ESTs, designed by Dheilly et al. (2011).