Transcriptomic analysis of murine embryos lacking endogenous retinoic acid signaling
ABSTRACT: 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 Retinaldehyde Dehydrogenase 2 null-mutant (Raldh2-/-) 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. Emx2, Foxg1 anteriorly, Cdx1, Hoxa1, Rarb 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 clustering analysis allowed us to extract groups of genes displaying similar behaviors in mutant tissue samples. These data give an overview of the gene expression changes occurring under a state of embryonic RA deficiency, and provide new candidate genes and pathways for a better understanding of retinoid-dependent molecular events. Two sets of samples were used for analyzing transcriptome changes in Raldh2-/- embryos. The rostral part of the head (including the anterior forebrain, optic vesicles, and overlying tissues), was collected from wild-type and mutant embryos at the 14 somite stage.The posterior tissues were analyzed at the 4 somite stage, and samples were collected from a transverse section plane excluding all tissues from the level of the first branchial arch.
Project description:Embryonic stem cells (ESC) are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass in their developmental potential. ESCs pluripotency is maintained through a complex interplay of different signaling pathways and a network of transcription factors, which is centered around Oct3/4, Sox2 and Nanog. Although, in general, much is known about this pluripotency self-renewal circuitry, the molecular events that lead ESC to exit from pluripotency and begin differentiation are currently less known. Retinoic acid, an active metabolite of the vitamin A (retinol), plays important and pleiotropic roles in vertebrate embryonic development and ESC differentiation. Here we demonstrate that RA promotes early steps of ESC differentiation, and that ESC increase their capacity to synthesize RA during spontaneous differentiation as embryoid bodies, up-regulating the RA biosynthetic pathway components RDH1, RDH10, ADH3, RALDH2, and CRABP2. Microarray derived from total RNA of mESC not treated or treated with all-trans retinoic acid (ATRA) for 2 hours.
Project description:Somites form during embryonic development and give rise to unique cell and tissue types, such as skeletal muscles and bones and cartilages of the vertebrae. Using somitogenesis stage human embryos, we performed the first ever transcriptomic profiling of human presomitic mesoderm as well as nascent and developed somites. Using this approach we uncovered novel regulators unique duing human somitogensis, and efficiently guided human pluripotent stem cells to differentiate to somite cells and downstream progeny. This work improves our understanding of human somite development and may enhance our ability to model and treat diseases affecting somite derivatives. Overall design: Human embryos of 4.5-5 weeks of gestation were obtained from electively aborted fetuses following informed consent and de-identification. After procurement, tissues were immediately washed in sterile PBS, placed in PBS supplemented with 5% fetal bovine serum, 1% Penicillin-Streptomycin and 2.5 µg/mL Amphotericin B, and shipped on ice. Within 48 hours, targeted tissues were micro-dissected from the embryos. Developed somites (SM Dev) included the two somite pairs at the forelimb bud level, nascent somites (SM) included the two somite pairs just anterior to the most caudal segmentation border that was visible, and presomitic mesoderm (PSM) included the tail region mesoderm posterior to SM. Duplicates were used for each sample type for RNA-seq.
Project description:The vertebrate embryo undergoes a series of dramatic morphological changes as the body extends to form the complete anterior-posterior axis during the somite-forming stages. The molecular mechanisms regulating these complex processes are still largely unknown. We show that the Hippo pathway transcriptional coactivators Yap1 and Wwtr1 are specifically localized to the ectoderm and notochord, and play a critical and unexpected role in posterior body extension by regulating the assembly of Fibronectin underneath the ectoderm and surrounding the notochord. We also find that Yap1/Wwtr1, also acting through Fibronectin, have an essential role in the ectodermal morphogenesis necessary to form the initial dorsal and ventral fins, a process that had been thought to involve bending of an epithelial sheet, but which we now show involves active cell migration. Our results reveal how the Hippo pathway transcriptional program, localized to two specific tissues, acts to control essential morphological events in the vertebrate embryo. Overall design: two biological replicates of tails of yap1/wwtr1 double homozygous mutants and siblings (24 each at 16-18 somite stage) were collected for RNAseq. Tails are tissues of the posterior end until the third newest somite (S-III).
Project description:We screened for differentially expressed genes in the developing notochord using the Affymetrix microarray system in Xenopus laevis. At late gastrula, we dissected four regions from the embryo, anterior mesoderm, posterior mesoderm, notochord and presomitic mesoderm. Three types of comparison were carried out to generate a list of predominantly notochord expressed genes: (1) Posterior mesoderm vs. anterior mesoderm; notochord genes are expected to be increased since the notochord is located in the posterior mesoderm. (2) Posterior mesoderm vs. whole embryos; notochord genes are expected to be increased. (3) Notochord vs. somite. This comparison sub-divided the group of posterior mesodermal genes identified in (1) and (2). All tissues are dissected using tungsten needles. We first dissected dorsal tissue above the archenteron from late gastrula to early neurula. To loosen tissue, we treated the dissected dorsal explant in a 1% cysteine solution (pH 7.4) and removed the neuroectodermal layer. Anterior mesoderm was dissected corresponding to about the anterior one-third of the archenteron roof, and the rest was collected as posterior mesoderm. The posterior mesodermal explant was dissected into notochord and somites, following a clearly visible border between the two tissues. The accuracy of all dissection was confirmed by RT-PCR of marker genes.
Project description:Somitogenesis is the segmentation of the developing embryonic body axis into somites and is guided by oscillating genes, which create waves of expression that travel across the presomitic mesoderm (PSM) from posterior to anterior. Upon arrival of a wave at the PSM's anterior end, a new somite is formed. To identify genes that are expressed in a wave-like pattern we dissected the PSM of four different mouse embryos (pre-turned), separated the left and right sides, and divided each into five segments, from posterior to anterior (sampling sites 1 to 5). Each segment was used to construct libraries for high-throughput RNA-sequencing. For one embryo, we also sequenced two somites.
Project description:In this series the ocular primordium was microdissected from normal CD-1 (outbred) mouse embryos harvested days 8-10 post-coitus, corresponding to human development during the 3rd-4th weeks post-fertilization. The precise embryological age was ascertained by counting somite pairs to an accuracy of +/- 2 pairs per litter. Samples were pooled as follows: optic pit pooled from 48-50 embryos (6 litters) on 8 d.p.c. (4-8 somite pairs), optic vesicle pooled from 36-40 embryos (5 litters) on 9 d.p.c. (16-20 somite pairs), and optic cip pooled from 27-30 embryos (3 litters) on 10 d.p.c. (28-32 somite pairs). The common reference for this series was RNA collected from embryonic headfold microdissected on 8 d.p.c. RNA samples were collected from CD-1 rat embryos at equivalent somite stages for the headfold, optic vesicle, and opic cup. Keywords = embryo Keywords = eye development Keywords = mouse Keywords = rat Keywords: time-course
Project description:In this series the ocular primordium was microdissected from normal CD-1 (outbred) mouse embryos harvested days 8-10 post-coitus, corresponding to human development during the 3rd-4th weeks post-fertilization. The precise embryological age was ascertained by counting somite pairs to an accuracy of +/- 2 pairs per litter. Samples were pooled as follows: optic pit pooled from 48-50 embryos (6 litters) on 8 d.p.c. (4-8 somite pairs), optic vesicle pooled from 36-40 embryos (5 litters) on 9 d.p.c. (16-20 somite pairs), and optic cip pooled from 27-30 embryos (3 litters) on 10 d.p.c. (28-32 somite pairs). The common reference for this series was RNA collected from embryonic headfold microdissected on 8 d.p.c. RNA samples were collected from CD-1 rat embryos at equivalent somite stages for the headfold, optic vesicle, and opic cup. Keywords = embryo Keywords = eye development Keywords = mouse Keywords = rat
Project description:Retinoic acid (RA), the bioactive derivative of vitamin A, is essential for vertebrate heart development. Both excess and reduced RA signaling lead to cardiovascular malformations, particularly affecting the cardiac outflow tract (OFT). The cellular mechanisms underlying the effects of RA signaling during OFT morphogenesis are not fully understood. To address this question, we used transient maternal RA supplementation to rescue the early lethality resulting from inactivation of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene. By embryonic day 13.5, Raldh2-/- hearts exhibited OFT septation defects. Although cardiac neural crest cells (cNCC) were present in OFT cushions of Raldh2-/- mutant embryos, we observed that cNCC were ectopically located in the peripheral region of the endocardial cushions, closer to the myocardium rather than immediately subjacent to the endocardium. Mislocated cNCC were aligned in parallel instead of perpendicular arrays to the endocardial surface within the proximal OFT. Supernumerary mesenchyme was generated both in and ex vivo within the cushions by Raldh2-/- mutant endocardium and found in place of the cNCC in a subendocardial, medial position. Although mislocated and misoriented, mutant cNCC resembled wildtype cells in their compaction density and individually elongated shape. Our data show that RA signaling acts on cNCC orientation relative to the septation plane and position within OFT cushions during septation process. Transcriptomic analysis and pathway-specific readout suggest that up-regulation of the Bmp pathway may be responsible for increased endothelial-to-mesenchymal transition, leading thereby to displacement of cNCC. E10.5 stage embryonic mouse outflow tracts were microdissected. Four pools of four outflow tracts were established for wildtype (WT) and mutant (Raldh2-/-) embryos derived from dams whose food had been supplemented with all-trans-RA (Sigma) directly mixed into powdered food at 0.1 mg/g food and offered between E7.5 and E8.5. Total RNA was extracted using Macherey-Nagel NucleoSpin® RNA columns without on-column DNase digestion.
Project description:Bipotent neuromesodermal progenitors (NMPs) residing in the caudal epiblast drive coordinated body axis extension by generating both posterior neuroectoderm and presomitic mesoderm. Retinoic acid (RA) is required for body axis extension, however the early molecular response to RA signaling is poorly defined, as is its relationship to NMP biology. As endogenous RA is first seen near the time when NMPs appear, we used WNT/FGF agonists to differentiate embryonic stem cells to NMPs which were then treated with a short 2-h pulse of 25 nM RA or 1 µM RA followed by RNA-seq transcriptome analysis. Differential expression analysis of this dataset indicated that treatment with 25 nM RA, but not 1 µM RA, provided physiologically relevant findings. The 25 nM RA dataset yielded a cohort of previously known caudal RA target genes including Fgf8 (repressed) and Sox2 (activated), plus novel early RA signaling targets with nearby conserved RA response elements. Importantly, validation of top-ranked genes in vivo using RA-deficient Raldh2-/- embryos identified novel examples of RA activation (Nkx1-2, Zfp503, Zfp703, Gbx2, Fgf15, Nt5e) or RA repression (Id1) of genes expressed in the NMP niche or progeny. These findings provide evidence for early instructive and permissive roles of RA in controlling differentiation of NMPs to neural and mesodermal lineages. Overall design: 3 samples were analyzed in duplicate: DMSO control (2 hours), 25 nM retinoic acid (2 hours), 1 µM retinoic acid (2 hours)
Project description:This experiment was performed in order to identify transcriptional differences between the anterior- and posterior-halves of mouse sclerotome. Cells-derived from the anterior- and posterior-sclerotome-halves from maturing mouse somites were compared to identify transcripts that are differentially expressed between these two halves of the somite. c57b6-/- female mice were time-mated, and at 9.5-dpc embryos were harvested. Theiler stage 17 embryos were selected for dissection. A stripe of segmented paraxial mesoderm corresponding to somites SX-SXVIII (standard somite nomenclature) was dissected from one-side of an embryo. The most anterior-third of up to 7 sclerotomes within each sample were dissected and pooled in RNALater. RNA was isolated using RNeasy Micro RNA extraction spin columns (Qiagen). Reverse transcription and initial cDNA amplification was performed using 18 PCR cycles with the SMART cDNA system (Clontech). Amplified cDNA was then re-amplified and labeled using the GeneChip® IVT Labeling Kit (Affymetrix).