Project description:Notochord sheath cells were FACS isolated from 13 days post fertilization (dpf) transgenic zebrafish. Three biological replicates were used for each population, and expression profiles were determined using Illumina HiSeq. Comparison of the sample groups allowed for identification of unique candidates. The sequence reads that passed quality filters were analyzed using HISAT2, and gene counts were analyzed using HTSeq.
Project description:The segmental organization of the vertebral column is established early in embryogenesis when pairs of somites are rhythmically produced by the presomitic mesoderm (PSM). The tempo of somite formation is controlled by a molecular oscillator known as the segmentation clock. While this oscillator has been well-characterized in model organisms, whether a similar oscillator exists in humans remains unknown. Genetic analysis of patients with severe spine segmentation defects have implicated several human orthologs of cyclic genes associated with the mouse segmentation clock, suggesting that this oscillator might be conserved in humans. Here we show that in vitro-derived human as well as mouse PSM cells recapitulate oscillations of the segmentation clock. Human PSM cells oscillate twice slower than mouse cells (5-hours vs. 2.5 hours), but are similarly regulated by FGF, Wnt, Notch and YAP. Single cell RNA-sequencing reveals that mouse and human PSM cells in vitro follow a similar developmental trajectory to mouse PSM in vivo. Furthermore, we demonstrate that FGF signaling controls the phase and period of oscillations, expanding the role of this pathway beyond its classical interpretation in 'Clock and Wavefront' models. Overall, our work identifying the human segmentation clock represents an important breakthrough for human developmental biology.
Project description:The notochord is the eponymous feature of the chordates and an essential organ in chordate development. The notochord of the invertebrate chordate Ciona consists of only 40 cells, and is a longstanding model for studying differentiation and morphogenesis in a small, simple embryo. Here we perform RNAseq analysis on flow-sorted notochord cells from multiple stages of development to define a comprehensive Ciona notochord transcriptome. We identify 1364 genes with enriched expression in the notochord and extensively validate the results by in situ hybridization. This notochord gene set is highly enriched for Gene Ontology codes related to the extracellular matrix, cell adhesion and cytoskeleton, and contains numerous genes with intriguing potential functions in morphogenesis. Orthologs of 112 of the Ciona notochord genes have known notochord expression in vertebrates, more than twice as many as would be predicted by chance alone. This set of putative effector genes with notochord expression conserved from tunicates to vertebrates will be invaluable for testing hypotheses about the evolution of the notochord.
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:Chordoma is a rare malignant tumor thought to originate from embryonic notochord. However, no molecular comparison of chordoma and notochord has been performed to date, leaving the identities of dysregulated pathways unclear. Absence of a molecular description of a control tissue clouds our understanding of chordoma. Thus, we conducted an unbiased comparison of chordoma and notochord using gene expression profiling to clarify chordoma’s tissue of origin and identify novel drug targets
Project description:Chordoma is a rare malignant tumor thought to originate from embryonic notochord. However, no molecular comparison of chordoma and notochord has been performed to date, leaving the identities of dysregulated pathways unclear. Absence of a molecular description of a control tissue clouds our understanding of chordoma. Thus, we conducted an unbiased comparison of chordoma and notochord using gene expression profiling to clarify chordoma’s tissue of origin and identify novel drug targets
Project description:Liver tissue from three-spine stickleback individuals from 3 populations were tested on arrays to determine array suitability for transcriptomics experiments.