XB-ART-58611
Front Cell Dev Biol
2021 Jan 01;9:747969. doi: 10.3389/fcell.2021.747969.
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Retinoic Acid Fluctuation Activates an Uneven, Direction-Dependent Network-Wide Robustness Response in Early Embryogenesis.
Parihar M
,
Bendelac-Kapon L
,
Gur M
,
Abbou T
,
Belorkar A
,
Achanta S
,
Kinberg K
,
Vadigepalli R
,
Fainsod A
.
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Robustness is a feature of regulatory pathways to ensure signal consistency in light of environmental changes or genetic polymorphisms. The retinoic acid (RA) pathway, is a central developmental and tissue homeostasis regulatory signal, strongly dependent on nutritional sources of retinoids and affected by environmental chemicals. This pathway is characterized by multiple proteins or enzymes capable of performing each step and their integration into a self-regulating network. We studied RA network robustness by transient physiological RA signaling disturbances followed by kinetic transcriptomic analysis of the recovery during embryogenesis. The RA metabolic network was identified as the main regulated module to achieve signaling robustness using an unbiased pattern analysis. We describe the network-wide responses to RA signal manipulation and found the feedback autoregulation to be sensitive to the direction of the RA perturbation: RA knockdown exhibited an upper response limit, whereas RA addition had a minimal feedback-activation threshold. Surprisingly, our robustness response analysis suggests that the RA metabolic network regulation exhibits a multi-objective optimization, known as Pareto optimization, characterized by trade-offs between competing functionalities. We observe that efficient robustness to increasing RA is accompanied by worsening robustness to reduced RA levels and vice versa. This direction-dependent trade-off in the network-wide feedback response, results in an uneven robustness capacity of the RA network during early embryogenesis, likely a significant contributor to the manifestation of developmental defects.
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U01 EB023224 NIBIB NIH HHS
Species referenced: Xenopus laevis
Genes referenced: adhfe1 aldh1a2 aldh1a3 crabp2 cyp26a1 cyp26c1 dhrs3 gapdh hoxa1 hoxb1 hoxb4 hoxd1 hoxd4 rbp1 rdh10 rdh13 rdh14 sdr16c5 slc35b1
GO keywords: embryo development [+]
???displayArticle.gses??? GSE154399: NCBI
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FIGURE 1. Efficient RA signaling robustness following transient physiological manipulation of RA levels. (A) Schematic depiction of the experimental design to directly challenge and study the robustness of RA signaling during embryogenesis. Timing and developmental stages are shown. (B) Principal Components 1 and 2 (PC1, PC2) of all six biological replicates studied by time series RNAseq. (C) Heatmap of gene expression of the top-100 positive and top-100 negative loadings corresponding to PC1 and PC2. A subset of the genes is highlighted based on the relevance to early developmental processes. | |
FIGURE 2. Phenotypic and molecular robustness of the retinoic acid metabolic pathway. Retinoic acid levels were reduced in Xenopus laevis embryos by inhibition of the RALDH activity with DEAB or by CYP26A1 overexpression to render retinoic acid inactive. (A) Control embryo at st. 27. (B) Embryo injected with capped RNA (0.8 ng) encoding the CYP26A1 enzyme. (C) Embryo treated with DEAB (50 μM) from st. 8.5 until st. 27. (D) Embryo treated with DEAB and injected with cyp26a1 mRNA. To exemplify the induced developmental malformations, lines depicting the size of the head domain (blue) and the trunk (red) were drawn on the control embryo (A) and then copied unto the treated embryos (B–D). (E) Distribution of the developmental defect severity induced by the retinoic acid manipulations. Embryos were scored for the induction of moderate (B,C) or severe (D) phenotypes, or normal looking. (F–H) Gene expression changes as a result of retinoic acid manipulation. qPCR analysis of hoxa1.L (F), hoxa1.S (G), and dhrs3.L (H) relative expression levels as a result of the individual or combined retinoic acid manipulations. Statistical significance (Student’s t-test) was calculated compared to the combined treatment group. *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant. | |
FIGURE 3. Transient physiological manipulation of RA levels for robustness analysis. (A) Embryos were treated with increasing concentrations of all-trans RA from 1 nM to 1 μM. Treatments were initiated at st. 8.5 and RNA samples were collected at tailbud stages (st. 32–33) for phenotypic analysis. To exemplify the induced developmental malformations, lines depicting the size of the head domain (blue), the trunk (red), and the tail (purple), were drawn on the control embryo (-RA) and then copied unto the treated embryos (10–1,000 nM). (B) Distribution of the severity of the developmental defects induced by the retinoic acid manipulations. Treated embryos were scored for the induction of moderate (as in 25 or 50 nM) or severe phenotypes (as in 100 or 1,000 nM), or normal looking (as in -RA). (C–H) RA treated embryos were collected at early (st. 10.25) and late (st. 12) gastrula for analysis of the changes in expression level. The response of RA metabolic and target genes was studied by qPCR. (C) hoxb1 (D) cyp26a1 (E) dhrs3 (F) aldh1a2 (G) aldh1a3 (H) rdh10. Statistical significance (Student’s t-test) was calculated compared to the control group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant. | |
FIGURE 4. Comparative pattern analysis to uncover genes responding to opposing RA manipulations. (A) Genes were grouped into discretized expression patterns based on up-regulation (yellow), down-regulation (blue), or no change (gray) compared to the control sample at the same time point. The numbers below the patterns indicate the number of genes that show the corresponding expression pattern in RA vs. control or DEAB vs. control. Only 32 out of 81 theoretically possible (four time points, 3*3*3*3 = 81) dynamic patterns were exhibited by at least one gene and are included in the figure. (B) COMPACT matrix comparing gene expression changes due to RA and DEAB relative to control. The subset of 32 × 32 patterns with non-zero number of genes in either perturbation group are shown. The gene counts were grouped within related patterns based on the time of initial up- or down-regulation. Supplementary Data 1 contains a version of the COMPACT matrix shown with the gene identifiers corresponding to the counts. The different quadrants are labeled a to d. (C) Dynamic expression patterns of genes showing opposite changes in response to RA and DEAB treatments (quadrant b). | |
FIGURE 6. RA responsiveness and differential expression dynamics of select RA network genes and targets in different clutches. (A–F) Embryos were treated at midblastula (st.8.5) with increasing concentrations of RA (1–1,000 nM) or DEAB 1–50 μM) to generate a gradient of RA responses. At mid gastrula (st. 11) the changes in RA network genes was determined by qPCR. (A) hoxa1.L, hoxa1.S; (B) dhrs3.L, dhrs3.S; (C) cyp26a1.L, cyp26a1.S; (D) rdh10.L, rdh10.S; (E) aldh1a2.L, aldh1a2.S; (F) aldh1a3.L, aldh1a3.S. (G) Dynamic expression patterns of multiple RA network genes across the different clutches. The data was combined from RNAseq (clutches A-F) and HT-qPCR (clutches G-L). The clutches are ordered left to right based on the earliest time at which hoxa1 expression returned to the baseline levels. | |
FIGURE 7. Trajectory analysis to compare the extent of individual clutch robustness based on hox expression. (A) 3-dimensional principal curve for the hox genes, showing projections of the sample points on the curve for (A, top) RA and (A, bottom) DEAB treatments. Principal curves for clutches C, J, and E are shown. The black star indicates the beginning of the curve for the distance measurement along the trajectory. Ranking of clutches is based on the net (absolute) normalized distance of treatment samples from the corresponding control sample for each time point. (B) Normalized expression shift profile calculated from the principal curve as the arc distance between the treatment and the corresponding control. Clutches are rank-ordered from lowest to highest net expression shift for hox genes in the RA group. Clutches A-F data from RNA-seq, clutches G-L data from HT-qPCR. (C) Distribution of clutches based on the trajectory determined robustness to RA and DEAB treatments relative to each other based on hox expression. The letters indicate the distinct clutches. (D) Heat map representation of gene expression changes of five additional clutches transiently treated with RA or DEAB. Samples were analyzed at the end of the pulse (t0) and 2 h in the chase (t2). Asterisks denote clutches with abnormal responses. | |
FIGURE 8. Differential robustness to direction of RA change is related to the effectiveness of feedback regulatory action. (A,B) Comparative distribution of clutches based on the trajectory determined robustness to RA (A) and DEAB (B). The change of the hox genes as a function of the change in the RA network genes is plotted for each RA manipulation. The letters indicate the distinct clutches. (C) Schematic diagram of the robustness efficiency-efficacy matrix in which different quadrants indicate the relative level of feedback observed and robustness achieved. (D,E) Mapping the differential expression data of clutches A, E, and F onto the RA network shown in Figure 5A to highlight the heterogeneity of differential regulation of RA network components for clutches closely situated in the robustness efficiency matrix (A,B). | |
FIGURE 9. Proposed model of the alternative regulatory schemes to achieve RA robustness. Schematic representation of two alternative scenarios in the decision tree to achieve robustness. These two scenarios are representative of the multiple permutations possible to achieve similar robustness outcomes. Each clutch follows a decision trajectory based on the direction of RA fluctuation and clutch-specific composition of RA network variants. In this scheme, uneven robustness arises when the regulatory feedback response of a clutch is optimized for one scenario over another. Our results revealed such a trade-off over distinct objectives of counteracting RA increase versus decrease. |
References [+] :
Adams,
The retinaldehyde reductase activity of DHRS3 is reciprocally activated by retinol dehydrogenase 10 to control retinoid homeostasis.
2014, Pubmed
Adams, The retinaldehyde reductase activity of DHRS3 is reciprocally activated by retinol dehydrogenase 10 to control retinoid homeostasis. 2014, Pubmed
Begemann, The zebrafish neckless mutation reveals a requirement for raldh2 in mesodermal signals that pattern the hindbrain. 2001, Pubmed
Belyaeva, The antagonistically bifunctional retinoid oxidoreductase complex is required for maintenance of all-trans-retinoic acid homeostasis. 2017, Pubmed
Belyaeva, Human retinol dehydrogenase 13 (RDH13) is a mitochondrial short-chain dehydrogenase/reductase with a retinaldehyde reductase activity. 2008, Pubmed
Billings, The retinaldehyde reductase DHRS3 is essential for preventing the formation of excess retinoic acid during embryonic development. 2013, Pubmed
Blaner, Vitamin A signaling and homeostasis in obesity, diabetes, and metabolic disorders. 2019, Pubmed
Blaner, Vitamin A Absorption, Storage and Mobilization. 2016, Pubmed
Blentic, Retinoic acid signalling centres in the avian embryo identified by sites of expression of synthesising and catabolising enzymes. 2003, Pubmed
Blumberg, An essential role for retinoid signaling in anteroposterior neural patterning. 1997, Pubmed , Xenbase
Chen, A concentration gradient of retinoids in the early Xenopus laevis embryo. 1994, Pubmed , Xenbase
Chen, Increased XRALDH2 activity has a posteriorizing effect on the central nervous system of Xenopus embryos. 2001, Pubmed , Xenbase
Corcoran, Absence of retinoids can induce motoneuron disease in the adult rat and a retinoid defect is present in motoneuron disease patients. 2002, Pubmed
Creech Kraft, Xenopus laevis: a model system for the study of embryonic retinoid metabolism. II. Embryonic metabolism of all-trans-3,4-didehydroretinol to all-trans-3,4-didehydroretinoic acid. 1995, Pubmed , Xenbase
Creech Kraft, Temporal distribution, localization and metabolism of all-trans-retinol, didehydroretinol and all-trans-retinal during Xenopus development. 1994, Pubmed , Xenbase
Cui, Retinoid receptors and vitamin A deficiency: differential patterns of transcription during early avian development and the rapid induction of RARs by retinoic acid. 2003, Pubmed
D'Aniello, Depletion of retinoic acid receptors initiates a novel positive feedback mechanism that promotes teratogenic increases in retinoic acid. 2013, Pubmed
D'Aniello, Input overload: Contributions of retinoic acid signaling feedback mechanisms to heart development and teratogenesis. 2015, Pubmed
Dobbs-McAuliffe, Feedback mechanisms regulate retinoic acid production and degradation in the zebrafish embryo. 2004, Pubmed
Durston, Retinoic acid causes an anteroposterior transformation in the developing central nervous system. 1989, Pubmed , Xenbase
Eldar, Elucidating mechanisms underlying robustness of morphogen gradients. 2004, Pubmed
Epstein, Patterning of the embryo along the anterior-posterior axis: the role of the caudal genes. 1997, Pubmed , Xenbase
Fainsod, Fetal Alcohol Spectrum Disorder: Embryogenesis Under Reduced Retinoic Acid Signaling Conditions. 2020, Pubmed , Xenbase
Fainsod, Xenopus embryos to study fetal alcohol syndrome, a model for environmental teratogenesis. 2018, Pubmed , Xenbase
Feng, Dhrs3a regulates retinoic acid biosynthesis through a feedback inhibition mechanism. 2010, Pubmed
Fujii, Metabolic inactivation of retinoic acid by a novel P450 differentially expressed in developing mouse embryos. 1997, Pubmed
Ghyselinck, Retinoic acid signaling pathways. 2019, Pubmed
Godsave, Expression patterns of Hoxb genes in the Xenopus embryo suggest roles in anteroposterior specification of the hindbrain and in dorsoventral patterning of the mesoderm. 1994, Pubmed , Xenbase
Grandel, Retinoic acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud. 2002, Pubmed
Hartomo, Involvement of aldehyde dehydrogenase 1A2 in the regulation of cancer stem cell properties in neuroblastoma. 2015, Pubmed
Hollemann, Regionalized metabolic activity establishes boundaries of retinoic acid signalling. 1998, Pubmed , Xenbase
Huang, Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. 2009, Pubmed
Janesick, ERF and ETV3L are retinoic acid-inducible repressors required for primary neurogenesis. 2013, Pubmed , Xenbase
Janesick, Active repression by RARγ signaling is required for vertebrate axial elongation. 2014, Pubmed , Xenbase
Janesick, Retinoic acid signaling and neuronal differentiation. 2015, Pubmed
Johnson, Adjusting batch effects in microarray expression data using empirical Bayes methods. 2007, Pubmed
Kam, Dhrs3 protein attenuates retinoic acid signaling and is required for early embryonic patterning. 2013, Pubmed , Xenbase
Karimi, Xenbase: a genomic, epigenomic and transcriptomic model organism database. 2018, Pubmed , Xenbase
Kedishvili, Enzymology of retinoic acid biosynthesis and degradation. 2013, Pubmed
Kedishvili, Retinoic Acid Synthesis and Degradation. 2016, Pubmed
Kessel, Respecification of vertebral identities by retinoic acid. 1992, Pubmed
Kessel, Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. 1991, Pubmed
Kim, The retinoic acid synthesis gene ALDH1a2 is a candidate tumor suppressor in prostate cancer. 2005, Pubmed
Koide, Active repression of RAR signaling is required for head formation. 2001, Pubmed , Xenbase
Kot-Leibovich, Ethanol induces embryonic malformations by competing for retinaldehyde dehydrogenase activity during vertebrate gastrulation. 2009, Pubmed , Xenbase
Kraft, Xenopus laevis: a model system for the study of embryonic retinoid metabolism. I. Embryonic metabolism of 9-cis- and all-trans-retinals and retinols to their corresponding acid forms. 1995, Pubmed , Xenbase
Kraft, The retinoid X receptor ligand, 9-cis-retinoic acid, is a potential regulator of early Xenopus development. 1994, Pubmed , Xenbase
Kuttippurathu, A novel comparative pattern analysis approach identifies chronic alcohol mediated dysregulation of transcriptomic dynamics during liver regeneration. 2016, Pubmed
Langfelder, WGCNA: an R package for weighted correlation network analysis. 2008, Pubmed
Lee, A paradoxical teratogenic mechanism for retinoic acid. 2012, Pubmed
Livak, Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. 2001, Pubmed
Lohnes, Developmental roles of the retinoic acid receptors. 1995, Pubmed
Love, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. 2014, Pubmed
Lupo, Dorsoventral patterning of the Xenopus eye: a collaboration of Retinoid, Hedgehog and FGF receptor signaling. 2005, Pubmed , Xenbase
Marshall, Retinoic acid alters hindbrain Hox code and induces transformation of rhombomeres 2/3 into a 4/5 identity. , Pubmed
Metzler, Enzymatic Metabolism of Vitamin A in Developing Vertebrate Embryos. 2016, Pubmed
Moss, Dynamic patterns of retinoic acid synthesis and response in the developing mammalian heart. 1998, Pubmed
Napoli, Cellular retinoid binding-proteins, CRBP, CRABP, FABP5: Effects on retinoid metabolism, function and related diseases. 2017, Pubmed
Niederreither, Restricted expression and retinoic acid-induced downregulation of the retinaldehyde dehydrogenase type 2 (RALDH-2) gene during mouse development. 1997, Pubmed
Niederreither, Embryonic retinoic acid synthesis is essential for early mouse post-implantation development. 1999, Pubmed
Nijhout, Systems biology of robustness and homeostatic mechanisms. 2019, Pubmed
Paganelli, Glyphosate-based herbicides produce teratogenic effects on vertebrates by impairing retinoic acid signaling. 2010, Pubmed , Xenbase
Pangilinan, Replication and exploratory analysis of 24 candidate risk polymorphisms for neural tube defects. 2014, Pubmed
Papalopulu, Retinoic acid causes abnormal development and segmental patterning of the anterior hindbrain in Xenopus embryos. 1991, Pubmed , Xenbase
Pavez Loriè, Both all-trans retinoic acid and cytochrome P450 (CYP26) inhibitors affect the expression of vitamin A metabolizing enzymes and retinoid biomarkers in organotypic epidermis. 2009, Pubmed
Porté, Aldo-keto reductases in retinoid metabolism: search for substrate specificity and inhibitor selectivity. 2013, Pubmed
Reijntjes, The control of morphogen signalling: regulation of the synthesis and catabolism of retinoic acid in the developing embryo. 2005, Pubmed
Reijntjes, The expression of Stra6 and Rdh10 in the avian embryo and their contribution to the generation of retinoid signatures. 2010, Pubmed
Ritchie, limma powers differential expression analyses for RNA-sequencing and microarray studies. 2015, Pubmed
Romand, Complementary expression patterns of retinoid acid-synthesizing and -metabolizing enzymes in pre-natal mouse inner ear structures. 2004, Pubmed
Rydeen, Excessive feedback of Cyp26a1 promotes cell non-autonomous loss of retinoic acid signaling. 2015, Pubmed
Saili, Molecular characterization of a toxicological tipping point during human stem cell differentiation. 2020, Pubmed
Sakai, The retinoic acid-inactivating enzyme CYP26 is essential for establishing an uneven distribution of retinoic acid along the anterio-posterior axis within the mouse embryo. 2001, Pubmed
Sandell, RDH10 oxidation of Vitamin A is a critical control step in synthesis of retinoic acid during mouse embryogenesis. 2012, Pubmed
Savova, Transcriptomic insights into genetic diversity of protein-coding genes in X. laevis. 2017, Pubmed , Xenbase
Schuetz, Multidimensional optimality of microbial metabolism. 2012, Pubmed
Schuh, v-erbA and citral reduce the teratogenic effects of all-trans retinoic acid and retinol, respectively, in Xenopus embryogenesis. 1993, Pubmed , Xenbase
See, A nutritional model of late embryonic vitamin A deficiency produces defects in organogenesis at a high penetrance and reveals new roles for the vitamin in skeletal development. 2008, Pubmed
Session, Genome evolution in the allotetraploid frog Xenopus laevis. 2016, Pubmed , Xenbase
Shabtai, Acetaldehyde inhibits retinoic acid biosynthesis to mediate alcohol teratogenicity. 2018, Pubmed , Xenbase
Shabtai, Competition between ethanol clearance and retinoic acid biosynthesis in the induction of fetal alcohol syndrome. 2018, Pubmed
Shabtai, Kinetic characterization and regulation of the human retinaldehyde dehydrogenase 2 enzyme during production of retinoic acid. 2016, Pubmed
Shabtai, ADHFe1: a novel enzyme involved in retinoic acid-dependent Hox activation. 2017, Pubmed , Xenbase
Sharpe, Retinoid receptors promote primary neurogenesis in Xenopus. 1997, Pubmed , Xenbase
Shoval, Evolutionary trade-offs, Pareto optimality, and the geometry of phenotype space. 2012, Pubmed
Sive, Identification of a retinoic acid-sensitive period during primary axis formation in Xenopus laevis. 1990, Pubmed , Xenbase
Sonneveld, Human retinoic acid (RA) 4-hydroxylase (CYP26) is highly specific for all-trans-RA and can be induced through RA receptors in human breast and colon carcinoma cells. 1998, Pubmed
Strate, Retinol dehydrogenase 10 is a feedback regulator of retinoic acid signalling during axis formation and patterning of the central nervous system. 2009, Pubmed , Xenbase
Taira, Expression of the LIM class homeobox gene Xlim-1 in pronephros and CNS cell lineages of Xenopus embryos is affected by retinoic acid and exogastrulation. 1994, Pubmed , Xenbase
Tendler, Evolutionary tradeoffs, Pareto optimality and the morphology of ammonite shells. 2015, Pubmed
Topletz, Induction of CYP26A1 by metabolites of retinoic acid: evidence that CYP26A1 is an important enzyme in the elimination of active retinoids. 2015, Pubmed
Urbizu, Chiari malformation type I: a case-control association study of 58 developmental genes. 2013, Pubmed
Yanai, Mapping gene expression in two Xenopus species: evolutionary constraints and developmental flexibility. 2011, Pubmed , Xenbase
Yelin, Ethanol exposure affects gene expression in the embryonic organizer and reduces retinoic acid levels. 2005, Pubmed , Xenbase
Yu, clusterProfiler: an R package for comparing biological themes among gene clusters. 2012, Pubmed
le Maire, Retinoic acid receptors: structural basis for coregulator interaction and exchange. 2014, Pubmed