Molars and incisors: show your microarray IDs.

BACKGROUND: One of the key questions in developmental biology is how, from a relatively small number of conserved signaling pathways, is it possible to generate organs displaying a wide range of shapes, tissue organization, and function. The dentition and its distinct specific tooth types represent a valuable system to address the issues of differential molecular signatures. To identify such signatures, we performed a comparative transcriptomic analysis of developing murine lower incisors, mandibular molars and maxillary molars at the developmental cap stage (E14.5). RESULTS: 231 genes were identified as being differentially expressed between mandibular incisors and molars, with a fold change higher than 2 and a false discovery rate lower than 0.1, whereas only 96 genes were discovered as being differentially expressed between mandibular and maxillary molars. Numerous genes belonging to specific signaling pathways (the Hedgehog, Notch, Wnt, FGF, TGFβ/BMP, and retinoic acid pathways), and/or to the homeobox gene superfamily, were also uncovered when a less stringent fold change threshold was used. Differential expressions for 10 out of 12 (mandibular incisors versus molars) and 9 out of 10 selected genes were confirmed by quantitative reverse transcription-PCR (qRT-PCR). A bioinformatics tool (Ingenuity Pathway Analysis) used to analyze biological functions and pathways on the group of incisor versus molar differentially expressed genes revealed that 143 genes belonged to 9 networks with intermolecular connections. Networks with the highest significance scores were centered on the TNF/NFκB complex and the ERK1/2 kinases. Two networks ERK1/2 kinases and tretinoin were involved in differential molar morphogenesis. CONCLUSION: These data allowed us to build several regulatory networks that may distinguish incisor versus molar identity, and may be useful for further investigations of these tooth-specific ontogenetic programs. These programs may be dysregulated in transgenic animal models and related human diseases leading to dental anomalies.journal articleresearch support, non-u.s. gov't2013 Mar 262013 03 26importe

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Transcriptomic Analysis of Murine Embryos Lacking Endogenous Retinoic Acid Signaling

<div><p></p><p>Retinoic acid (RA), an active derivative of the liposoluble vitamin A (retinol), acts as an important signaling molecule during embryonic development, regulating phenomenons as diverse as anterior-posterior axial patterning, forebrain and optic vesicle development, specification of hindbrain rhombomeres, pharyngeal arches and second heart field, somitogenesis, and differentiation of spinal cord neurons. This small molecule directly triggers gene activation by binding to nuclear receptors (RARs), switching them from potential repressors to transcriptional activators. The repertoire of RA-regulated genes in embryonic tissues is poorly characterized. We performed a comparative analysis of the transcriptomes of murine wild-type and <i>Retinaldehyde Dehydrogenase 2</i> null-mutant (<i>Raldh2</i>^−/−) embryos — unable to synthesize RA from maternally-derived retinol — using Affymetrix DNA microarrays. Transcriptomic changes were analyzed in two embryonic regions: anterior tissues including forebrain and optic vesicle, and posterior (trunk) tissues, at early stages preceding the appearance of overt phenotypic abnormalities. Several genes expected to be downregulated under RA deficiency appeared in the transcriptome data (e.g. <i>Emx2</i>, <i>Foxg1</i> anteriorly, <i>Cdx1</i>, <i>Hoxa1</i>, <i>Rarb</i> posteriorly), whereas reverse-transcriptase-PCR and in situ hybridization performed for additional selected genes validated the changes identified through microarray analysis. Altogether, the affected genes belonged to numerous molecular pathways and cellular/organismal functions, demonstrating the pleiotropic nature of RA-dependent events. In both tissue samples, genes upregulated were more numerous than those downregulated, probably due to feedback regulatory loops. Bioinformatic analyses highlighted groups (clusters) of genes displaying similar behaviors in mutant tissues, and biological functions most significantly affected (e.g. mTOR, VEGF, ILK signaling in forebrain tissues; pyrimidine and purine metabolism, calcium signaling, one carbon metabolism in posterior tissues). Overall, these data give an overview of the gene expression changes resulting from embryonic RA deficiency, and provide new candidate genes and pathways that may help understanding retinoid-dependent molecular events.</p></div

Details of the gene expression profiles obtained after hierarchical clustering of the experimental samples by relative gene expression level analysis.

<p>Eight clusters (panels B–I) have been extracted from the overall clustering analysis (available as additional online information, Figure S1), showing differential gene expression behaviors according to the experimental samples (ANT, RNA from anterior tissues; POST, RNA from posterior tissues; WT, wild-type embryos; KO, <i>Raldh2</i>^−/− embryos). Gene expression profiles are illustrated as a heat map (green: weak expression; red: strong expression – see scale below). The expression profile of the <i>Rarb</i> gene is also shown (panel J), which did not cluster with any other gene. This analysis further validated the experimental samples, as WT and KO samples segregated into fully distinct clusters, both for the ANT and POST tissue samples (panel A above).</p

VENN diagrams summarizing a cross-comparison of the genes downregulated in <i>Raldh2</i>^−/− embryos (A, anterior tissues; B, posterior tissues) with those identified as RAR-bound by ChIP-seq analysis of embryonic stem (ES) cells differentiating as embryoid bodies (ref.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062274#pone.0062274-Moutier1" target="_blank">[<b>72</b>]</a><b>), and those induced after RA treatment of embryoid bodies.</b> This analysis distinguished early RA-responsive genes (2 h after RA exposure: upper numbers; ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062274#pone.0062274-Moutier1" target="_blank">[72]</a>), from those identified at a later stage of differentiation (4 days post-RA treatment: lower numbers; ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062274#pone.0062274-Kim1" target="_blank">[73]</a>). Some selected genes are highlighted in the intersecting data sets.</p

Volcano plots representing corrected p-values (ordinates, −log10 scale) from Student t-test of the mRNA levels compared in wild-type and <i>Raldh2</i>^−/− samples anterior (A) and posterior (B) embryonic tissue samples.

<p>Genes with a fold change (abscissae) higher than 1.5 and a false discovery rate lower than 0.1 are shown in red.</p

Genes acting in selected signaling pathways and misregulated in <i>Raldh2</i>^−/− tissue samples (ANT: anterior samples; POST: posterior samples).

<p>Key: <b>Bold</b> indicates a fold change exceeding ±1.5.</p><p><i>Italics</i> indicate a p-value >0.05 (ANT) or >0.03 (POST).</p

Overview of the main biological and physiological functions correlating with the affected genes in <b><i>Raldh2</i></b><b>^−/− embryos.</b>

<p>The functions are listed by decreasing order of statistical significance [−log(p-values)] of differentially expressed and misregulated genes as calculated by the Ingenuity software. Left side (black bars): ANT microarray data; right side (gray bars): POST microarray data.</p

Bioinformatic analysis of putative transcription factor binding sites (oPOSSUM3 single site analysis method) in genes showing the highest downregulation in posterior or anterior tissues of <i>Raldh2</i>^−/− embryos.

(a)<p>59 genes analyzed from 63 genes downregulated with FC <−1.5.</p>(b)<p>137 genes analyzed from 146 genes downregulated with FC <−1.5.</p>(c)<p>The searched region encompassed 5 Kb upstream and 5 Kb downstream of the transcription start site. The top ten factors (highest Z-scores) appear in bold. Additional transcription factors (below) are the authors' seelection.</p>(d)<p>Genes with conserved RAR:RXR (DR5) binding sites are: <i>Csn3, Dhrs3, Fap, Meox1, Metrn, Rarb, Stra6, 5730596B20Rik</i> (NB: <i>5730596B20Rik</i> is an antisense EST found between the <i>Hoxa3</i> and <i>Hoxa4</i> genes).</p>(e)<p>Genes with conserved Pax5 binding sites are: <i>Apba2, Crabp2, Lgr5, Mettl1, NKx2-9, Pmm2, Ripply3, Rfx6</i>.</p>(f)<p>Genes with conserved NR2F1 binding sites are: <i>Apba2, Crabp2, Dhrs3, Dusp9, Fap, Gcsh, Hoxa1, Maob, NKx2-9, Pax6, Prdm13, Rarb, Sfrp5, Stra6, Timm8a1, Tmem56, 1700011H14Rik.</i></p>(g)<p>Genes with conserved STAT1 binding sites are: <i>Apba2, Dbx1, Dhrs3, Fap, Hoxa1, Gcsh, Kynu, Lgr5, Meox1, Pax6, Prdm13, Ptprz1, Rarb, Ripply3, Slc25a10, Snora34, Stra6, Timm8a1, 5730596B20Rik.</i></p>(h)<p>Genes with conserved PBX1 binding sites are: <i>Ccne1, Dbx1, Fap, Hoxa1, Kynu, Lgr5, Nepn, Nkx2-9, Nkx3-1, Pax6, Prdm13, Rarb, Rfx6, Ripply3, Tmem56.</i></p>(i)<p>Genes with conserved PPARG:RXRA binding sites are: <i>Apba2, Cpn1, Crabp2, Dbx1, Dhrs3, Dusp9, Gcsh, Hoxa1, Lhx1, Meox1, Mtap7d2, Nkx3-1, Pax6, Prdm13, Ptprz1, Rarb, Sh3bgrl2, Snord118, Stra6, Trmt61a, 1700011H14Rik, 5730596B20Rik.</i></p>(j)<p>Genes with conserved T binding sites are: <i>Lhx1, NKx2-9, Pmm2, Prdm13, Stra6, Timm8a1, Trmt61a, 5730596B20Rik.</i></p>(k)<p>Genes with conserved Pax6 binding sites are: <i>Dbx1, Pax6, Rarb, Stra6, 5730596B20Rik.</i></p>(l)<p>Genes with conserved REST binding sites are: <i>Acsl6, Ank1, Cntnap2, Lhx2, Mpped1.</i></p>(m)<p>Genes with conserved Pou5fI binding sites are: <i>Abcb10, Acsl6, Ank1, Apba2, Arrdc4, Aven, Cdh20, Cntnap2, Dhrs11, Gas5, Htr3b, Ikzf1, Lhx2, Mrpl18, Muc1, Neto2, Nova1, Nr2e1, Pak3, Pcdh19, Prdm16, Rragb, Shox2, Slc25a21, Taf1d, Wnt7a.</i></p>(n)<p>Genes with conserved NR3C1 binding sites are: <i>Cntnap2, Eef1d, Fez1, Hsd17b7, Htr3b, Itgb8, Klf1, Lrcc4, Lhx2, Mpped1, Mrpl20, Nova1, Nsdhl, Pcdh19, Pdss1, Phyhipl, Pou3f3, Rnf144b, Shox2, Syt11, AI504432.</i></p><p><b>Transcription factor binding site (TFBS) analysis: genes downregulated in POSTERIOR tissues.^(a)</b></p><p><b>TFBS analysis: genes downregulated in ANTERIOR tissues.^(b)</b></p

Summary diagram of the major molecular pathways emerging from Ingenuity analysis of the <i>Raldh2</i>^−/− transcriptome.

<p>The molecular pathways are listed by decreasing order of statistical significance as they appear through analysis of the ANTERIOR (left-side list) and POSTERIOR (right-side list) data sets. The most significant pathways identified for each data set are highlighted in gray. Additional pathways relevant for developmental processes are also listed. A graphic representation of the numbers of genes downregulated (green) or upregulated (red) in anterior (upper bars) or posterior (lower bars) <i>Raldh2</i>^−/− embryonic tissue samples (fold change ±1.2, filtered for FDR <10%) is shown. The total number of genes comprising each pathway (middle line, gray shaded), and the percentages of genes misregulated in each experiment, are also given.</p