Whole transcriptome sequencing of mouse notochord-derived cells at developmental stages E12.5 and P0
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ABSTRACT: Recapitulation of developmental signals represents a promising strategy for treating intervertebral disc degeneration. During development, embryonic notochord-derived cells (NDCs) are the direct progenitors of cells that populate the adult nucleus pulposus (NP) and are an important source of secreted signaling molecules. The objective of this study was to define global gene expression profiles of NDCs at key stages of embryonic disc formation. NDCs were isolated from Shh-cre;ROSA:YFP mice at embryonic day 12.5 and postnatal day 0, representing opposite ends of the notochord to NP transformation. Differences in global mRNA abundance across this developmental window were established using RNA-Seq. Principal component analysis revealed clustering of gene expression at each developmental stage with more than 5000 genes significantly differentially expressed between E12.5 and P0. This study represents the first transcriptome-wide analysis of embryonic NDCs. Results suggest signaling and biosynthesis of NDCs change dramatically as a function of developmental stage.
Project description:During embryonic development, the notochord is the precursor to the nucleus pulposus (NP) in the intervertebral disc (IVD), where the notochord cells differentiate to become notochordal-like cells in the NP, and are considered as potential progenitor cells to support the function of the NP. With aging and degeneration, there is a loss of these cells from the NP which ultimately affects IVD function. The characterisation of the molecular signature of the 8 week-old (gestational age) human embryonic notochord is a valuable resource to better understand the development from the notochord to the NP progenitors and as a benchmark for comparison to other datasets.
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: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:Using a combination of cell sorting and microarray analysis, we identified almost 200 genes as having a high level of expression in the notochord. After whole mount in situ hybridization screening, we confirmed approximately one third of these as having a novel notochord expression pattern. Keywords: cell type comparison - embryonic Noto-GFP+ notochord progenitors versus surrounding GFP- comparator cells
Project description:Using a combination of cell sorting and microarray analysis, we identified almost 200 genes as having a high level of expression in the notochord. After whole mount in situ hybridization screening, we confirmed approximately one third of these as having a novel notochord expression pattern. Keywords: cell type comparison - embryonic Noto-GFP+ notochord progenitors versus surrounding GFP- comparator cells 3 biological replicates - each replicate includes an experiment cell population (Noto-GFP positive cell sort) and a comparator population (GFP negative cell sort)
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:Fertilized eggs are plulipotent and during early embryogenesis, embryonic cells gradually restrict their developmental potential and are eventually destined to give rise to one type of cells. Molecular mechanism underlying developmental fate restriction is one of the major research subjects of developmental biology. Here we address this question by combining a classical technique of blastomere isolation with a modern technique of microarray analysis. During the 6th cleavage of the Ciona intestinalis embryo, from the 32-cell to the 64-cell stage, four mother cells divide into daughter cells with two distinct fates, one giving rise to notochord and the other to nerve-cord precursor cells. Nearly 500 notochord or nerve-cord precursor cells each were isolated, and difference in the quality of mRNAs between them was compared by microarray. This analysis identified 111 and 69 genes that are differentially expressed in the notochord and nerve-cord precursor cells, respectively. These included not only genes for transcription factors and signalling molecules but also those with functions that many kinds of cells used. In addition, whole-mount in situ hybridization showed complex and dynamic profiles of spatial expression of these genes during the segregation of the two fates. The process is accomplished by that transcripts that have been already present in the mother cell are properly partitioned into either type of cells and that new and preferential expression of genes in either type of cells. Two kinds of sample, Dye Swap design with 2 arrays
Project description:Fertilized eggs are plulipotent and during early embryogenesis, embryonic cells gradually restrict their developmental potential and are eventually destined to give rise to one type of cells. Molecular mechanism underlying developmental fate restriction is one of the major research subjects of developmental biology. Here we address this question by combining a classical technique of blastomere isolation with a modern technique of microarray analysis. During the 6th cleavage of the Ciona intestinalis embryo, from the 32-cell to the 64-cell stage, four mother cells divide into daughter cells with two distinct fates, one giving rise to notochord and the other to nerve-cord precursor cells. Nearly 500 notochord or nerve-cord precursor cells each were isolated, and difference in the quality of mRNAs between them was compared by microarray. This analysis identified 111 and 69 genes that are differentially expressed in the notochord and nerve-cord precursor cells, respectively. These included not only genes for transcription factors and signalling molecules but also those with functions that many kinds of cells used. In addition, whole-mount in situ hybridization showed complex and dynamic profiles of spatial expression of these genes during the segregation of the two fates. The process is accomplished by that transcripts that have been already present in the mother cell are properly partitioned into either type of cells and that new and preferential expression of genes in either type of cells.
Project description:Failure of the nucleus pulposus (NP) causes intervertebral disc (IVD) disease and associated low-back pain, which are highly prevalent among the aged populations. Molecular and cellular changes underpinning the structural failures in age-associated IVD diseases (IDDs) remain poorly elucidated. Here, we first identified that TAGLN, which encodes the cytoskeleton regulator transgelin, was transcribed in healthy NPs of both human and mouse, but diminished with ageing. Immunostaining showed that TAGLN were expressed on the peripheral of mouse NP (periNP). Lineage analyses in Tagln-CreERT2 mice showed that NP cells were derived from Tagln+ cells in the periNP. The PeriNP cells were proliferative and can differentiate into the inner NP (innerNP). These Tagln+ cells and their descendants diminish with ageing or puncture-induced degeneration. Single-cell transcriptomics from neonatal and adult Tagln-CreERT2 IVDs confirmed that Tagln descendants uniquely populate the NP, wherein four sub-populations were identified: chondrocyte-like, Cd228+, Tagln+ and Car3+. Immunostaining confirmed Car3 expressed in innerNP. Computational analysis indicated the lineage trajectory from Tagln+ to Car3+ sub-population, along with the involvement of Fos/Jun/TGFβ cascades and partial epithelial-to-mesenchymal transition process. Removal of TGFβ mediator Smad4 by notochord specific Foxa2mNE-Cre resulted in decreased Tagln+ cells and abnormal disc morphology, resembling disc degeneration. Our study generates a single-cell transcriptomic atlas for healthy IVD, identifies Tagln+ PeriNP cells as a progenitor pool crucial for the IVD homeostasis, and provides potential targets for regenerative cell therapy against IVD degeneration.