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:Microarray analysis of chick embryo tissues: Hamburger Hamilton (HH) stage 3+/4 and HH6 Hensen’s 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 goal of the current NS23158 funding is to understand how neural fate-stabilizing (NFS) genes function in order to provide fundamental knowledge about their potential roles in neural stem cell development. Neural fate-stabilization is characterized by the expansion of the neural plate shortly after the presumptive neural ectoderm has been induced by the repression BMP signaling. During this time period a number of early-expressed NFS genes are expressed, each of which expands the neural plate in gain-of-function assays. But, we do not know how these genes are related to one another or whether they are arranged in a linear gene pathway or multi-path hierarchy. A key aim of this grant is to elucidate the relationship of foxD5, an early-expressed NFS gene that we cloned, to other NFS-genes. We propose to greatly enhance this analysis by utilizing DNA microarray analyses to identify unsuspected and novel target genes. What is the function of foxD5 in neural fate-stabilization, and how does it specifically relate to other genes in the early-expressed group? In the parent grant we proposed to study the relationship of early-expressed NFS genes to one that we cloned, FoxD5 (Sullivan et al 2001). FoxD5 expands the neural plate and holds it in an immature state. In the original grant, we proposed to study whether foxD5 activates or suppresses the expression of 6 known early-expressed NFS genes, using PCR and in situ hybridization of animal caps and whole embryos. Herein we propose to use DNA microarray technology to reveal a much broader spectrum of potential target genes. FoxD5, a fork-head transcription factor which is expressed in the early neuroectoderm, is a key regulator of neural plate fate during expansion of the neural plate. Being a newly cloned gene, we hypothesize that we will identify numerous downstream targets of FoxD5, by the proposed microarray analyses. This information will allow us to study their function by gain- and loss-of function studies in the whole embryo. Animal cap (AC) explants are a naïve embryonic ectoderm that is removed from embryonic signaling centers. They allow one to perform gene induction assays in the absence of confounding growth factors, and thus are ideal for identifying downstream targets of transcription factors. In this experiment we will take a gain-of-function approach to identify which genes are induced/repressed by foxD5. FoxD5 mRNA (200pg) will be injected at the 2-cell stage, and embryos cultured until stage 8, at which time the zygotic genome begins transcription. AC explants will be cut from the embryos and cultured in Normal Amphibian?s Medium. When ACs reach stage 10.5, an early step in neural ectoderm specification, they will be snap frozen in liquid nitrogen and RNA purified according to the Qiagen Rneasy Protect kit protocols. Our preliminary experiments indicate that 8-10ug of total RNA can be recovered with high purity from ~150 ACs with this method. As controls, RNA will be purified from uninjected animal caps derived from sibling embryos. If foxD5 represses NFS gene expression, it will not be detected in the above experiment because NFS genes are not expressed in AC explants in the absence of neural induction. Therefore, we will repeat the experiment but additionally treat the ACs (FoxD5-injected and sibling uninjected) at stage 8 with Noggin protein (R&D Systems) to induce neural ectoderm. For all four sample sets, RNA will be reverse transcribed, amplified and biotinylated using the NuGEN Ovation kit.
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:Nanog null neural stem (NS) cells were reprogrammed to naive pluripotency in 2i/LIF conditions with chick (c) and zebrafish (z) Nanog orthologs. Global gene expression was compared to iPS cells derived with mouse (m) Nanog. Murine iPS cells derived with zebrafish nanog, chick nanog, and mouse nanog orthologs (2 replicates each).
Project description:Cells were isolated from mouse embryonic neural crest stem cells at culture day 2 (NCSC), from day 7 in vitro differentiated progeny (NCP) and day 2 epidermal neural crest stem cells from bulge explants of adult whisker follicles (EPI-NCSC). Keywords: LongSAGE embryonic neural crest stem cells at culture day 2 (NCSC), from day 7 in vitro differentiated progeny (NCP) and day 2 epidermal neural crest stem cells from bulge explants of adult whisker follicles (EPI-NCSC).
Project description:Neural development requires crosstalk between signaling pathways and chromatin. In this study, we demonstrate that neurogenesis is promoted by an interplay between the TGFβ pathway and the H3K27me3 histone demethylase (HDM) JMJD3. Genome-wide analysis showed that JMJD3 is targeted to gene promoters by Smad3 in neural stem cells (NSCs) and is essential to activate TGFβ-responsive genes. In vivo experiments in chick spinal cord revealed that the generation of neurons promoted by Smad3 is dependent on JMJD3 HDM activity. Overall, these findings indicate that JMJD3 function is required for the TGFβ developmental program to proceed. We immunoprecipitate endogenous Smad3 or JMJD3 proteins from neural stem cells treated with TGFb for 30 minutes.
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:In order to isolate novel genes regulating neural induction, we utilized a DNA microarray approach. As neural induction is thought to occur via the inhibition of BMP signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant-negative receptor. RNAs were isolated from control animal cap explants and from dominant-negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high-density Xenopus DNA microarray chips. Keywords = neural induction Keywords = BMP Keywords = nervous system Keywords = Xenopus Keywords = microarray