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Sox1a:eGFP transgenic line and single-cell transcriptomics reveal the origin of zebrafish intraspinal serotonergic neurons

ABSTRACT: sox1a:eGFP transgenic line and single-cell transcriptomics reveal the origin of zebrafish intraspinal serotonergic neurons

PROVIDER: PRJNA940435 | ENA |

REPOSITORIES: ENA

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	SRR23690175_1.fastq.gz	Fastqsanger.gz
	SRR23690175_2.fastq.gz	Fastqsanger.gz

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<i>sox1a:eGFP</i> transgenic line and single-cell transcriptomics reveal the origin of zebrafish intraspinal serotonergic neurons.

Project description:Sox transcription factors are crucial for vertebrate nervous system development. In zebrafish embryo, sox1 genes are expressed in neural progenitor cells and neurons of ventral spinal cord. Our recent study revealed that the loss of sox1a and sox1b function results in a significant decline of V2 subtype neurons (V2s). Using single-cell RNA sequencing, we analyzed the transcriptome of sox1a lineage progenitors and neurons in the zebrafish spinal cord at four time points during embryonic development, employing the Tg(sox1a:eGFP) line. In addition to previously characterized sox1a-expressing neurons, we discovered the expression of sox1a in late-developing intraspinal serotonergic neurons (ISNs). Developmental trajectory analysis suggests that ISNs arise from lateral floor plate (LFP) progenitor cells. Pharmacological inhibition of the Notch signaling pathway revealed its role in negatively regulating LFP progenitor cell differentiation into ISNs. Our findings highlight the zebrafish LFP as a progenitor domain for ISNs, alongside known Kolmer-Agduhr (KA) and V3 interneurons.

| S-EPMC10387610 | biostudies-literature

sox1a:eGFP transgenic line and single-cell transcriptomics reveal the origin of zebrafish intraspinal serotonergic neurons

Project description:The Sox family of transcription factors plays a crucial role in the development of the vertebrate nervous system. In the zebrafish embryo, sox1 genes are expressed in neural progenitor cells and neurons of the ventral spinal cord. We recently reported that the loss of function of sox1a and sox1b results in a significant decrease in a subtype of V2 neurons, called V2s, in zebrafish. Here a single-cell RNA sequencing (scRNA-Seq) approach was used to analyse the transcriptome of sox1a lineage progenitors and neurons in the zebrafish spinal cord at four different time points during the development of an earlier established Tg(sox1a:EGFP) line. In addition to the sox1a-expressing neurons described previously, we found that this gene is also expressed in late-developing intraspinal serotonin neurons (ISNs). Developmental trajectory analysis of single-cell data and morpholino knockdown of lateral floor plate (LFP) cells expressing the nkx2.9 gene suggest that ISNs are generated from LFP progenitor cells. Pharmacological inhibition of the Notch signalling pathway demonstrates the necessity of this pathway for the development of LFP progenitor cells into ISN populations. Our results confirm that the zebrafish LFP is a progenitor domain from which ISN neurons are generated in addition to the previously described KA" and V3.

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Identification of the interaction proteins of PRSS37 by GFP-IP/MS using testis proteins from PRSS37-EGFP knock-in mice and pAcr-EGFP transgenic mice

Project description:To identify the potential interaction partners of PRSS37, three testis lysates of Prss37E/+ mice (PRSS37-EGFP knock-in heterozygous mice) and three testis lysates of pAcr-EGFP transgenic mice were immunoprecipitated using anti-GFP mAb magnetic beads. The GFP-IP products of six samples from two groups were subsequently analyzed by mass spectrometry (MS) to identify differentially immmunoprecipitated proteins (DIPs) in the PRSS37-EGFP group. Proteins detected in pAcr-EGFP group were used as negative controls to exclude the endogenous proteins and polypeptides that interact nonspecifically with the anti-GFP mAb magnetic beads.

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Project description:We generated a transgenic mouse line which express EGFP in the retina driven by the Crx promoter using BAC transgenesis. We sorted EGFP-positive photoreceptor precursors at E17.5 using FACS, and subsequently performed microarray analysis of the FACS-sorted cells. In order to clarify a molecular role of Crx in developing cone photoreceptor precursors, we investigated the expression profile of the BAC-Crx-EGFP-positive cells compared with that of the BAC-Crx-EGFP-negative cells at E17.5.

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2020-10-20 | E-MTAB-9698 | biostudies-arrayexpress

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Project description:Human serotonergic neurons are derived using published transdifferentiation protocols. Human Fibroblasts correspond to human fibroblasts (from Line#1, Coriell bought (AG08498)). Samples labeled human neurons or induced neurons (iN) correspond to neurons transdifferentiated from fibroblasts using two transcription factors, as previously described (#1-AG08498 or #2:ERF-1, Erlangen Germany, a line given to us by collaborators) into primarily glutamatergic neruons. Samples labled 5-HT neurons or serotonergic neurons or iSN correspond to serotonergic neurons derived from the stated fibroblast lines, using an additional four transcription factors. For transdifferentiation of iN and iSN, fibroblasts were made to overexpress the stated transcription factors in a doxycycline inducible manner for up to 3 weeks, and then neurons are sorted out and collected directly into Trizol for RNA preparation and sequencing. The non-transdifferentiated fibroblast lines were collected in bulk withtout differentiation into neurons. The line number corresponds to the same fibroblast line either being transdifferentiated into iN or iSN, as labeled - for direct and groupwise comparison.

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Next Generation Sequencing of zebrafish intraspinal serotonergic neurons in the injury segment and distal segments after spinal cord injury

Project description:The goals of this study is to compare transcriptome profiles (RNA-seq) of zebrafish intraspinal serotonergic neurons in the injury segment and distal segments after spinal cord injury. Bulk RNA-Seq samples of ISNs from the injury area and residual segments respectively were FAC-sorted from Tg(tph2:GFP) line. Total RNA was isolated using SMART-SeqTM v4 UltraTM Low Input RNA Kit for Sequencing (Clontech). Sequencing libraries (N=5-6) were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina following the manufacturer’s instructions (NEB). We mapped about 50-60 million sequence reads per sample to the zebrafish genome and identified 39,714 transcripts in the zebrafish intraspinal serotonergic neurons. Our study represents the detailed analysis of transcriptomes of zebrafish intraspinal serotonergic neurons in the injury segment and distal segments after spinal cord injury.

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