DRS transcriptomics of differentiating neural tube tissue in HH10 chick embryos
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ABSTRACT: Confirmation of microarray transcriptomic profiling during differentiation of the neural tube in HH stage 10 chick embryos with Helico Biosciences DRS data.
Project description:Microarray analysis of chick embryo tissues: Hamburger Hamilton (HH) stage 3+/4 and HH6 Hensenâs node, HH 3+/4 posterior primitive streak, notochord with ventral neural tube at HH10-11, dorsal neural tube at HH10-11 and anterior and posterior thirds of the wing bud at stages HH20-21 and HH24.
Project description:The spinal cord is generated progressively as cells leave the caudal region of the elongating body axis such that the temporal steps of neural differentiation become spatially separated along the head to tail axis. At key stages, it is therefore possible to isolate near-adjacent cell populations from the same embryo in distinct differentiation states. Cells in the caudal lateral epiblast adjacent to the primitive streak (also known as the stem zone, SZ, in the chick) express both early neural and mesodermal genes. Other cells in the stem zone will gastrulate to form the paraxial mesoderm or remain in the epiblast cell sheet and become neural progenitors. These latter cells form a new region called the preneural tube (PNT), which is flanked by unsegmented presomitic mesoderm and represents an early neural progenitor state that can be induced by FGF signalling to revert back to a multi-potent SZ state. Rostral to this, the closed caudal neural tube (CNT) is flanked by somites and is an early site of co-expression of genes characteristic of neural progenitors, and of ventral patterning genes (Diez del Corral et al., 2003). The CNT contains the first few neurons and exposure to FGF cannot revert this tissue to a multi-potent SZ state (Diez del Corral et al., 2002). The transition from the PNT to the CNT thus involves commitment to a neural fate that this is regulated by a switch from FGF to retinoid signalling. More advanced neuroepithelium is then located in more rostral neural tube (RNT), in which neuronal differentiation is ongoing and dorsoventral pattern is refined. This experiment uses the Affymetrix GeneChip chicken genome microarray to compare the transcriptomes of microdissections of these spatially distinct cell populations from the elongating neural axis of HH stage 10 chick embryos. Dissections were carried out in L15 medium at 4°C and explants pooled in TRIzol reagent (Gibco) for RNA extraction. Notochord was removed by controlled trypsin digestion that aimed to keep the neural ventral midline. For the microarrays, at least five tissue samples for each region were pooled to make each of three biological replicates for each (n>15 for each region).
Project description:Illumina sequencing was used to characterize the transcriptome of somites dissected from chick embryos HH stage 14-16. Somites were treated as follows: uninjected, injected with antagomiR-206 or with scrambled antagomiR. Transcripts were mapped to the chicken genome.
Project description:A systematic survey of the transcriptional status of individual segments of the developing chick hindbrain (r1-5) and the adjacent region of the embryonic midbrain (m) during the HH11 stage of chick development Affymetrix Chicken GeneChip Expression Study Paralell comparison of defined regions of the neural tube during early chick development
Project description:Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. We investigated vascular development in vivo by determining global gene regulation throughout the formation of the chick chorio-allantoic membrane (CAM). Our study provides a comprehensive molecular map of vascular maturation during developmental angiogenesis and might thus be a valuable resource to streamline further research of candidates susceptible to mediate pathological angiogenesis. Experiment Overall Design: The developmental stage of the embryos was determined after isolation of the CAM according to Hamburger & Hamilton (HH) (1992). CAMs were isolated from embryos at developmental day E5 (HH26), E7 (HH30), E10 (HH>35) and E14 (HH40), (n=3 embryos/day). mRNA was isolated and hybridized to Affymetrix chicken GeneChips using the Affymetrix standard protocol. We compared one embryo at a lower HH stage to three embryos at a more advanced HH stage and repeated the comparison with two more embryos. Overall, we compared expression between E7 and E5, E10 and E5, E14 and E5, E10 and E7 and E14 and E10.
Project description:Developing tissues rely on the coordinated differentiation of stem cell populations in dynamically changing environments. The formation of the vertebrate neural tube is a well characterized example, where stem cells in the caudal epiblast differentiate to neural tissue while transitioning through a changing landscape of signals: rostro-caudally from Retinoic Acid (RA) to Wnt/FGF and dorsal-ventrally from sonic hedgehog to BMP. Despite an understanding of the signaling pathways involved in neural specification, the precise signal interpretation and gene regulatory mechanisms within an embryonic context remain poorly defined. To address this, we first developed an in vivo CRISPR screening approach in chick embryos. We then performed a multiplexed in vivo single-cell perturbation screen of transition genes from caudal epiblast to the neural tube. Our screen revealed a role for MLLT3, a component of the super elongation complex, in the specification of neural identity. MLLT3 is expressed in the epiblast and not the neural tube. Perturbation of MLLT3 disrupted caudal epiblast morphology, reduced neural tube identities, and resulted in the misregulation of genes involved in WNT and RA signaling. Neural specification was also disrupted by expressing mutant forms of Retinoic Acid Receptor A (RARα) lacking the MLLT3 binding domain or consisting only the MLLT3 binding region. Together, these findings validate an in vivo CRISPR screen in chick embryos for the first time and identify a previously unreported role for MLLT3 in caudal neural cell specification, mediated through an interaction with RARα.
Project description:Type IV collagen is the main component of the basement membrane which gives strength to the blood-gas barrier. In avians the formation of the blood-gas barrier happens rapidly and before hatching. We have performed a microarray expression analysis in late chick lung development and found that COL4A1 and COL4A2 were among the most significantly upregulated genes during the formation of the avian blood-gas barrier. Our study showed that type IV collagen and therefore the basement membrane play fundamental roles in coordinating alveolar morphogenesis. Four developmental stages of chick lung maturation (E14, E15, E16, E18). Three biological replicates per time point.
Project description:Objective: We developed an unbiased strategy to identify genes important in endocardial epithelial-to-mesenchymal transformation (EMT) using a spatial transcriptional profile. Methods and Results: Endocardial cells overlaying the cushions of the atrioventricular canal (AVC) and outflow tract (OFT) undergo an EMT to yield VICs. RNA sequencing (RNA-seq) analysis of gene expression between AVC, OFT, and ventricles (VEN) isolated from chick and mouse embryos at comparable stages of development (chick HH18; mouse E11.0) was performed. EMT occurs in the AVC and OFT cushions, but not VEN at this time. 210 genes in the chick (n=1) and 105 genes in the mouse (n=2) were enriched 2-fold in the cushions. Gene regulatory networks (GRN) generated from cushion-enriched gene lists confirmed TGFβ as a nodal point and identified NF-κB as a potential node. To reveal previously unrecognized regulators of EMT four candidate genes, Hapln1, Id1, Foxp2, and Meis2, and a candidate pathway, NF-κB, were selected. In vivo spatial expression of each gene was confirmed by in situ hybridization and a functional role for each in endocardial EMT was determined by siRNA knockdown in a collagen gel assay. Conclusions: Our spatial-transcriptional profiling strategy yielded gene lists which reflected the known biology of the system. Further analysis accurately identified and validated previously unrecognized novel candidate genes and the NF-κB pathway as regulators of endocardial cell EMT in vitro. 3 separate regions of the developing heart tube were dissected and processed for RNA sequencing analysis in both HH18 chick and E11.0 ICR mouse (done in duplicate)