DDX24 Spatiotemporally Orchestrates VEGF and Wnt Signaling During Developmental Angiogenesis

Cell Biology Genetics Biochemistry Life Sciences Medicine Immunology
angiogenesis endothelial cells spatial transcriptomics VEGF DDX24

Research Background

Vascular system development is a highly precise regulatory process involving two key stages: vasculogenesis and angiogenesis. Although the VEGF (vascular endothelial growth factor) and Wnt signaling pathways have been confirmed to regulate vascular development in the peripheral and central nervous systems (CNS), respectively, the spatiotemporal coordination of these pathways remains unclear. Previous studies found that dysfunction of DEAD-box RNA helicase DDX24 leads to multiorgan vascular malformations (MOVLD syndrome), but its molecular mechanism was unknown. This study aims to reveal how DDX24 achieves spatiotemporal specificity in brain and trunk vascular development by differentially regulating VEGF and Wnt signaling.

Source of the Paper

This research was conducted by a collaborative team including Fangbin Chen, Zhaohua Deng, and others from the Fifth Affiliated Hospital of Sun Yat-sen University, Wuhan University, Huazhong Agricultural University, and the University of Macau. The findings were published on May 8, 2025, in PNAS (Proceedings of the National Academy of Sciences) under the title “DDX24 spatiotemporally orchestrates VEGF and Wnt signaling during developmental angiogenesis.”

Research Process and Findings

1. Analysis of DDX24 Expression Patterns

Experimental Design:
- Analyzed dynamic DDX24 expression from 3.3 to 60 hpf (hours post-fertilization) using zebrafish embryonic spatial transcriptomics (Stereo-seq)
- Validated spatiotemporal expression via whole-mount in situ hybridization (WISH) and fluorescence in situ hybridization
- Isolated vascular endothelial cells (ECs) via fluorescence-activated cell sorting (FACS) for qRT-PCR validation

Key Findings:
- DDX24 was widely expressed in early embryos, enriched in trunk vasculature at 24 hpf, and restricted to the brain after 36 hpf (Fig. 1g-i)
- Fluorescence colocalization confirmed DDX24 co-expression with vascular marker fli1a in intersegmental vessels (ISVs) and central arteries (CTAs) (Fig. 1l-m)
- Endothelial-specific analysis showed DDX24 expression in GFP+ cells was 3.2-fold higher than in GFP- cells (p < 0.001)

2. Phenotypic Analysis of DDX24 Deficiency

Model Construction:
- Used two morpholinos (MOs) for knockdown and CRISPR/Cas9 gene editing (16-bp deletion mutant)
- Generated transgenic zebrafish line tg(fli1a:egfp) for live imaging

Phenotypic Results:
- Trunk Vasculature: DDX24 deficiency caused ectopic ISV branching (82.6% in mutants vs. 0% in wild-type at 54 hpf) (Fig. 2b-c)
- Brain Vasculature: Delayed CTA sprouting (54.3% reduction in mutant CTA count at 3 dpf) (Fig. 2h-i)
- Time-lapse imaging showed mutant ISV tip cells had 2.1-fold more filopodia, while CTA tip cells had 67% fewer (Fig. 2e-m)

3. Validation of Cell-Autonomous and Non-Autonomous Mechanisms

Methods:
- Embryonic transplantation: MO-treated donor cells transplanted into wild-type hosts
- In vitro models: Functional assays in human umbilical vein ECs (HUVECs) and brain microvascular ECs (HCMECs)

Mechanistic Evidence:
- Transplanted DDX24-deficient ECs in wild-type hosts still exhibited excessive ISV branching (p < 0.0001), confirming cell-autonomous effects (Fig. 3b-c)
- Reverse transplantation showed wild-type ECs in mutants also developed abnormalities, suggesting non-cell-autonomous regulation (Fig. 3d)
- In vitro: DDX24 knockdown increased HUVEC migration by 1.8-fold but reduced HCMEC tube formation by 62% (Fig. 3e-j)

4. Molecular Mechanism Elucidation

Multi-Omics Analysis:
- RNA-seq revealed upregulated VEGF pathway genes (e.g., KDR/VEGFR2) in HUVECs and downregulated Wnt pathway genes in HCMECs (Fig. 4a-b)
- RIP-qPCR confirmed DDX24 directly binds three conserved sites on KDR mRNA (Fig. 4h-i)
- Reporter assay: DDX24 deficiency reduced TOPFlash activity in HCMECs by 71% (Fig. 4l)

Key Pathways:
- Trunk Vasculature: DDX24 suppresses VEGFR2 expression by promoting KDR mRNA decay (2.3-fold protein increase) (Fig. 4c)
- Brain Vasculature: DDX24 activates Wnt7a/b signaling via the GPR124/RECK complex (83% CTA rescue in recovery experiments) (Fig. 4o)

5. Spatial Transcriptomic Network Analysis

Technical Innovation:
- Applied Stereo-seq with 15×15 DNB (DNA nanoball) resolution
- Defined EC “microenvironment units” (8 nearest neighboring spots)

Interaction Networks:
- Trunk ECs interact with muscle cells via Shh-Agrn ligand-receptor pairs (Fig. 5i)
- Brain ECs communicate with neural precursors via Rgma-Nfkbia axis to regulate Wnt signaling (Fig. 5k-l)
- Spatiotemporal analysis showed sustained VEGF pathway activation in 24 hpf trunk ECs and Wnt pathway suppression in 60 hpf brain ECs (Fig. 5f)

6. Therapeutic Strategy Validation

Sequential Intervention Experiments:
- VEGFR inhibitor Regorafenib (50 nM, 20–48 hpf) fully suppressed ectopic ISV branching (Fig. 6a-b)
- Wnt activator CHIR-99021 (5–72 hpf) restored CTA counts to wild-type levels (p < 0.001) (Fig. 6d-f)
- Critical Finding: Only sequential treatment (“VEGF inhibition first, then Wnt activation”) rescued both phenotypes (Fig. 6j-m)

Research Value and Highlights

Scientific Significance

  1. Mechanistic Innovation: First demonstration of RNA helicase regulating spatiotemporal vascular development via mRNA stability control
  2. Technical Breakthrough: Integrated CRISPR editing, spatial transcriptomics, and live imaging for multidimensional analysis
  3. Theoretical Contribution: Proposed “EC-microenvironment crosstalk” model for organ-specific angiogenesis (see Supplementary Fig. S14)

Application Prospects

  • Clinical Translation: Provides a time-specific treatment strategy for MOVLD syndrome (Patent No. CNP0005537)
  • Technology Extension: The 15×15 DNB spatial analysis method has been applied to other developmental biology studies

Research Features

  • Spatiotemporal Precision: First dynamic regulatory network of vascular development at single-cell resolution
  • Cross-Species Validation: Zebrafish model and human primary cell data mutually corroborated
  • Translational Medicine: Phenotypic rescue via timed drug intervention directly informs clinical practice

Supplementary Information

Raw data are deposited in NCBI GEO (GSE185261) and CNGBdb (CNP0005537). Supported by the National Natural Science Foundation of China (82322008) and the National Key R&D Program (2024YFA0919700).