Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Human notochordal cell transcriptome in the developing intervertebral disc

ABSTRACT: The adult nucleus pulposus originates from the embryonic notochord, but loss of notochordal cells with skeletal maturity in humans is believed to initiate intervertebral disc degeneration. Thus, defining the phenotype of human embryonic/fetal notochordal cells is essential for understanding their roles and for development of novel therapies. However, a detailed transcriptomic profiling of human notochordal cells has never been achieved. In this study, the notochord-specific marker CD24 was used to specifically label and isolate (using FACS) notochordal cells from human embryonic and fetal spines (7.5-14 weeks post-conception), which were studied by microarray analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Leo Zeef

PROVIDER: E-MTAB-6868 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Human notochordal cell transcriptome unveils potential regulators of cell function in the developing intervertebral disc.

Rodrigues-Pinto Ricardo R Ward Lizzy L Humphreys Matthew M Humphreys Matthew M Zeef Leo A H LAH Berry Andrew A Hanley Karen Piper KP Hanley Neil N Richardson Stephen M SM Hoyland Judith A JA

Scientific reports 20180827 1

The adult nucleus pulposus originates from the embryonic notochord, but loss of notochordal cells with skeletal maturity in humans is thought to contribute to the onset of intervertebral disc degeneration. Thus, defining the phenotype of human embryonic/fetal notochordal cells is essential for understanding their roles and for development of novel therapies. However, a detailed transcriptomic profiling of human notochordal cells has never been achieved. In this study, the notochord-specific mark ...[more]

PMID: 30150762

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Project description:Transcription factors play diverse roles during embryonic development, combinatorially controlling multiple cellular states in a spatially and temporally defined manner. Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level. Here we show how temporal, tissue-specific changes in embryonic transcription factor function can be discerned by integrating caged morpholinos (cMOs) with photoactivatable fluorophores, fluorescence-activated cell sorting (FACS), and microarray technologies. As a proof of principle, we have dynamically profiled No tail-a (Ntla)-dependent genes at different stages of axial mesoderm development in zebrafish, discovering and characterizing discrete sets of transcripts that are coincident with either notochord cell fate commitment or differentiation. Our studies demonstrate how optically controlled chemical tools can be use to probe developmental processes with spatiotemporal precision and reveal the sequential activation of distinct transcriptomes within a cell lineage by a single transcriptional factor. Zebrafish zygotes were injected with a mixture of ntla caged morpholino (cMO) and caged fluorescein dextran (cFD), and a 100 µm-diameter region of the posterior chordamesoderm 125 μm anterior to the center of Kupffer’s vesicle was UV-irradiated at 12 hours post fertilization (hpf). The UV irradiation generated an active morpholino targeting the ntla 5'UTR and simultaneously labeled the cells with green fluorescence. By 36 hpf, the irradiated, green-fluorescent cells contributed to both notochord and floor plate, but the notochord cells were partially vacuolated and disorganized. Combining these caged reagents with microarray analysis identified transcriptional changes coincident with loss of ntla at 16 hpf and subsequent notochord differentiation. Zebrafish zygotes were injected with a mixture of ntla cMO/cFD, and a 100 µm-diameter region of the posterior chordamesoderm 125 μm anterior to the center of Kupffer’s vesicle was UV-irradiated at 12 hpf. Control embryos were injected with cFD and similarly irradiated at 12 hpf. Experimental and control sets of embryos were enzymatically dissociated at 16 hpf, green-fluorescent cells were isolated by FACS, and collected in trizol. Thirty embryos were used for each experiment or controls, each yielding approximately 8,000 green fluorescent cells. Five biological replicates were performed along with five paired controls. Total RNA was isolated from each sample, reverse transcribed, and amplified using WTA2 TransPlex Complete Whole Transcriptome Amplification kit (Sigma) using a miniaturized procedure and manufacturer-recommended incubation steps. The synthesized cDNA was sent to Nimblegen for dye labeling and hybridization. Samples were hybridized to the Nimblegen 2007 (Zv 7) Danio rerio Gene Expression Array chip using one dye per chip. Raw probe data (.pair file) was subjected to RMA, normalization, background correction, and generation of gene expression summary (.calls file) by Nimblegen. Processed data was analyzed in ArrayStar software (DNAStar). Significantly affected genes (fold change > 2 and p-value < 0.10) were identified using a moderated t-test.

Dataset Information

Human notochordal cell transcriptome in the developing intervertebral disc

Publications

Human notochordal cell transcriptome unveils potential regulators of cell function in the developing intervertebral disc.

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