DOI: 10.5061/dryad.tb2rbp0g3
title: Single-cell RNA-seq of the embryonic zebrafish heads from wild-type siblings and betaPix CRISPR mutants at 1 dpf and 2 dpf

description: Proper establishment of the blood–brain barrier and maintenance of
 cerebrovascular integrity require precise coordination among neural and
 vascular cell types during early vertebrate development. To define the
 transcriptional programs underlying these processes and the role of glial
 betaPix, we generated a comprehensive single-cell RNA-seq dataset from
 embryonic heads of wild-type siblings and betaPix CRISPR mutants at 1 and
 2 days post-fertilization (dpf). Comparative analyses revealed that loss
 of betaPix selectively disrupted transcriptional programs in glial and
 neuronal progenitors, including down-regulation of microtubule-associated
 stathmin signaling and pro-angiogenic transcription factors Zfhx3/4. These
 changes highlight betaPix as an upstream regulator of PAK1–Stathmin and
 Zfhx3/4–Vegfaa pathways required for coordinated glial migration and
 angiogenesis. This single-cell resource thus provides a developmental
 framework for understanding how glial betaPix contributes to blood–brain
 barrier formation and vascular integrity. To support broad community use,
 we provide our raw datasets, enabling the discovery of candidate
 regulators of cerebrovascular stability in vivo.
description: # Single-cell RNA-seq of the embryonic zebrafish heads from wild-type
 siblings and betaPix CRISPR mutants at 1 dpf and 2 dpf Dataset DOI:
 [10.5061/dryad.tb2rbp0g3](https://doi.org/10.5061/dryad.tb2rbp0g3) ##
 Description of the data and file structure This dataset is associated with
 Chiu et al. 2025, Glial betaPix is essential for blood vessel development
 in the zebrafish brain. In this study, single-cell transcriptome libraries
 were obtained from embryonic zebrafish heads of wild-type siblings and
 betaPix CRISPR mutants at 1 dpf and 2 dpf. 300 ng/µL Cas9 mRNA and a
 mixture of number 1 to 4 *betaPix* gRNAs with 50 ng/µL each were
 co-injected into wildtype zebrafish embryos at one-cell stage. Injection
 with equivalent concentration of Cas9 mRNA with PBS served as siblings.
 After microinjection, embryos were collected and maintained at 28.5°C. At
 24 or 48 hpf, zebrafish were dechorionated, paralyzed and transferred onto
 agarose plate. Heads were harvested with dissecting scissors in cold
 sterile 1X PBS with pooling 200 heads for each group. Heads were then
 dissociated in 900 µL Accutase cell detachment solution (Sigma-Aldrich,
 A6964) at 28.5°C for 3 hours and re-suspended by pipetting every 30 min.
 Once digestion was complete, 100 µL FBS was added to cell suspension and
 centrifuged at 4°C, 500 g for 3 min. The cells were gently re-suspended in
 cold 500 µL 2.5% FBS in IX PBS and filtered through a 40 µm strainer. PI
 and Hoechst33342 were stained for distinguishing living cells. The single,
 living cells were sorted by Aria SORP (BD biosciences) into 1.5 ml tubes.
 The cell counts and vitality were verified by AOPI staining coupled with
 an Automated Cell Counter (Countstar BioTech). Around 10,000-12,000 cells
 were loaded for each group. Single cells were barcoded with Chromium Next
 GEM Single Cell 3 ‘Reagent Kits V3.1 kit (10x Genomics, 1000269) in 10×
 Chromium Controller (10× Genomics). After qualified by peak shapes,
 fragment size and tailing with Fragment Analyzer System kit (Agilent
 Technologies, DNF-915), single-cell transcriptome libraries were sequenced
 via Illumina High Throughput Sequencing PE150 (Novogene, Beijing). ###
 Files and variables The raw data files are named using a composite
 identifier that provides essential information about the sample type,
 developmental stage, library, and sequencing run. All files are raw
 single-cell RNA sequencing data in compressed FastQ format. The file names
 follow the general structure:
 `[Treatment]-[Developmental_Stage]-[Novogene_Accession]-[Sample_ID]_[Read_Pair].fq.gz` | **Abbreviation / Segment** | **Description** | **Example Value & Explanation** | | :---------------------------- | :---------------------------------------------------------------------------------------------- | :-------------------------------------------------------------------------------------------- | | **Treatment** | The genetic status of the sample. | **`ctrl`**: Wild-type sibling (Control). **`ko`**: betaPix CRISPR mutant (Knockout/Crispant). | | **Developmental Stage** | The age of the zebrafish embryo at the time of collection. | **`1d`**: 1 day post-fertilization (dpf). **`2d`**: 2 days post-fertilization (dpf). | | **Novogene Accession number** | The unique library accession number assigned by the sequencing provider (Novogene). | **`FKDL210125450-1a`**, **`FKDL210231122-1a`**, etc. | | **Sample ID (AK-number)** | The specific index code assigned to the sample during library preparation. | **`AK4317`**, **`AK3562`**, etc. | | **Read Pair** | Denotes whether the file contains the forward or reverse read of the paired-end sequencing run. | **`1`**: Read 1 (R1, Forward). **`2`**: Read 2 (R2, Reverse). | 1 dpf samples are associated with Novogene Library Accession Numbers FKDL210231122-1a (Control) and FKDL210231123-1a (Knockout). 2 dpf samples are associated with Novogene Library Accession Numbers FKDL210125450-1a (Control) and FKDL210125451-1a (Knockout). Example File Interpretation: ko-2d-ko-2d_FKDL210125451-1a-AK3562_1.fq.gzDescription: Raw scRNA-seq data from a $betaPix$ crispant (ko) collected at 2 days post-fertilization (2d). It belongs to Novogene Library FKDL210125451-a, uses Index AK3562, and contains the first read (1) of the paired-end sequence. ## Code/software The sequencing data were analyzed using the CellRanger-6.1.1 (10x Genomics) and mapped to reference genome GRCz1l-GRCz11.103. Low-quality cells were excluded from subsequent analyses under the following conditions: when the number of expressed genes was less than 500, when there were abnormally high counts of UMIs or genes (outliers of a normal distribution), or when the mitochondrial content exceeded 9%. A total of 38,670 cells were qualified for subsequent analyses. 5’ adapter (5'-3'): AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACAC GACGCTCTTCCGATCT 3’ adapter (5'-3'): AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC（INDEX） ATCTCGTATGCCGTCTTCTGCTTG ctrl-1d index (5'-3'): GCATCTCC; TGTAAGGT; CTGCGATG; AACGTCAA ctrl-2d index (5'-3'): GTCTCTCG; AATCTCTC; CGGAGGGA; TCAGAAAT ko-1d index (5'-3'): AGCTATCA; CATATAAC; TCAGGGTG; GTGCCCGT ko-2d index (5'-3'): AAACCTCA; GCCTTGGT; CTGGACTC; TGTAGAAG