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. Author manuscript; available in PMC: 2026 Oct 15.
Published in final edited form as: Development. 2026 Apr 17;153(8):dev204936. doi: 10.1242/dev.204936

Hyperactive GNAQ in endothelial cells causes blood vessel malformation in developing mouse skin

Jenna Meyers 1, Zach Tower 1, Olivia Leighton 1, Beth Drolet 1,*, Hao Chang 1,2,*
PMCID: PMC13667911  NIHMSID: NIHMS2214908  PMID: 41866987

Abstract

Vascular anomalies are a group of congenital disorders characterized by structural abnormalities in the blood vessels. Over the past 20 years, genetic investigations have identified mosaic mutations in multiple genes in individuals with vascular anomalies, including G protein alpha subunit q/11-encoding genes GNAQ/GNA11. Here, we report a tunable and reversible mouse tool for studying the frequent GNAQ mutant, GNAQ R183Q. We have generated a transgenic mouse line carrying GNAQ R183Q under the control of the tet-responsive elements. The transgene expression level is tunable and can be induced at physiologically relevant ranges. We have shown that endothelial expression of GNAQ R183Q is sufficient to drive blood vessel malformation in developing mouse embryos. We have further identified multiple transcriptional changes caused by GNAQ R183Q in the skin using RNA-seq. This work strengthens the causal link between GNA mutations and vascular anomalies, demonstrates that endothelial cell-specific expression of GNAQ R183Q is sufficient to cause vascular defects during development, and offers valuable insights into the mechanisms underlying GNA mutation-associated diseases.

Keywords: GNAQ, Endothelial cell, Vascular anomalies, Mouse model, tet-on

INTRODUCTION

Vascular anomalies are a group of congenital disorders characterized by structural abnormalities in the blood vessels. They encompass a set of diseases with devastating psychological, social and medical manifestations (Greene, 2011). These anomalies range from small birthmarks to extensive disfiguring malformations with multi-organ disease (Adams and Ricci, 2019; Cheraghlou et al., 2019; Steiner and Drolet, 2017). Despite rapid advancements in gene discovery, there remains a lack of comprehensive transcriptome and functional studies to identify treatment targets. There is an urgent need for a better understanding of the disease mechanisms. G-proteins and G protein-coupled receptors (GPCRs) are important transducers of signals from the extracellular space to the inside of the cell (Oldham and Hamm, 2008; Syrovatkina et al., 2016). Aberrant expression and activity of G proteins and GPCRs are frequently associated with tumorigenesis (O’Hayre et al., 2013). Several studies have shown that vascular anomalies harbor postzygotic somatic mutations in G protein alpha subunits (GNA proteins). For example, mutations in GNAQ (R183G, R183Q) have been found in affected tissue in individuals with Sturge-Weber syndrome (Nakashima et al., 2014; Shirley et al., 2013). Mutations in GNAQ (Q209L, Q209P) and GNA11 (Q209L, Q209P) have been found in congenital non-progressive hemangiomas (Ayturk et al., 2016; Funk et al., 2016). In addition, several GNAQ and GNA11 mutations (GNAQ R183Q/G, GNA11 R183C) have been found in isolated capillary malformation and in capillary malformations with overgrowth (Couto et al., 2017; Lian et al., 2014; Shirley et al., 2013; Siegel et al., 2018; Thomas et al., 2016; Van Raamsdonk et al., 2009). These human correlation studies identify associations between mutant GNA proteins and vascular anomalies but cannot establish causation, necessitating the use of animal models to investigate underlying mechanisms and potentially develop targeted therapies.

The first generation of mouse models is now emerging to investigate the underlying mechanisms of this condition, with a limited number of genetically engineered models demonstrating a causal relationship between GNA mutations and vascular anomalies. For example, a conditional overexpression allele of GNAQ Q209L has been generated by knocking the coding sequence of mutant GNAQ into the Rosa26 locus. When combined with endothelial cell-specific Cre divers, the expression of GNAQ Q209L causes abnormal angiogenesis and endothelial hyperproliferation in the skin and intestine (Schrenk et al., 2023). A conditional expression allele of GNAQ R183Q has also been generated by knocking a Cre-dependent swapping cassette into the endogenous GNAQ locus (Wetzel-Strong et al., 2023). In this allele, the existence of Cre recombinase deletes the coding sequence for a wild-type GNAQ and puts the coding sequence for GNAQ R183Q under the endogenous promoter. When combined with global Cre divers, the expression of GNAQ R183Q causes various degrees of embryonic lethality. Dilated vessels have been observed in some of the mutant embryos.

Here, we report a tunable and reversible mouse genetic tool for studying the common hyperactive GNAQ mutant, GNAQ R183Q, in vascular development and disease. In this transgenic line, GNAQ R183Q is under the control of the tet-responsive elements and its expression can be induced in mouse tissues when combined with the Cre-inducible rtTA allele and with doxycycline administration. We have shown that endothelial expression of GNAQ R183Q is sufficient to drive blood vessel phenotype in developing mouse embryos. We further identify multiple transcriptional changes caused by GNAQ R183Q in the skin using bulk RNA-seq, including high mobility group AT-hook 2 (HMGA2). This work supports the causal connection between GNA mutations and vascular anomalies, demonstrates that GNAQ R183Q expression in endothelial cells only is sufficient to cause vascular defects during development, and provides important insights for understanding the mechanism of GNA mutation-related disease.

RESULTS AND DISCUSSION

To study the role of GNAQ R183Q in vivo, we generated an inducible transgenic mouse allele that expresses GNAQ R183Q under the control of the tetracycline-responsive element (referred to as tet-GNAQ R183Q mice; Fig. 1A–C). Briefly, the R183Q mutation was introduced to the full-length cDNA of human GNAQ, and the GNAQ R183Q coding sequence was placed downstream of the tetracycline-responsive element. The construct was linearized and used to generate the tet-GNAQ R183Q mice on the C57Bl/6 background. The Cre-inducible rtTA allele, Rosa26-LSL-rtTA, allows the expression of the reverse tetracycline-activated transactivator in tissues that express the Cre recombinase (Belteki et al., 2005). We crossed the tet-GNAQ R183Q, Rosa26-LSL-rtTA and Cre alleles together to generate triple transgenic mice, which allowed us to express the mutant GNA proteins in a tissue- (by Cre) and time-specific (by doxycycline administration) manner. To test whether the tet-GNAQ R183Q transgenic mice function as designed, we first used the Krt14-Cre, which enables the expression of mutant GNA proteins in the ectoderm and its derivatives (Dassule et al., 2000). We crossed the tet-GNAQ R183Q; Krt14-Cre males with Rosa26-LSL-rtTA females and pregnant females were given a doxycycline diet (625 mg/kg) at embryonic day (E) 12.5 to induce the transgene expression. We harvested embryonic skin at E15.5 and detected the GNAQ R183Q expression using RT-PCR (Fig. 1D). No expression of GNAQ R183Q was detected in the triple transgenic embryos without doxycycline treatment. However, robust expression of the GNAQ R183Q was detected in the triple transgenic embryos treated with doxycycline, suggesting the tet-on system of GNAQ R183Q worked successfully as designed.

Fig. 1.

Fig. 1.

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Generation of a tet-on mouse line for expressing GNAQ R183Q in mice. (A) Diagram showing the domain structure of the GNAQ protein. R183 is in the 1st switch region (SR). N and C represent the amino and carboxy terminals of the protein, respectively. (B) Sequencing confirmation of the mutant construct of GNAQ R183Q. (C) Genotyping strategy for screening transgenic mice and the gel electrophoresis of PCR reactions from genomic DNA of mice derived from microinjections. Red asterisk indicates the positive founder mouse carrying the tet-GNAQ R183Q transgene. (D) Strategy for the tet-on system. Top, diagram of the Rosa26-LSL-rtTA and tet-GNAQ R183Q alleles. Without Cre, no expression of the rtTA and the GNAQ R183Q. Middle, rtTA expression in tissues after Cre-induced recombination. Bottom, GNAQ R183Q expression is enabled in the Cre lineages after doxycycline administration. Gel images showing the tet-on system with Krt14-Cre. RT-PCR showing the robust expression of GNAQ R183Q in the E15.5 skin after doxycycline treatment in the tet-GNAQ R183Q; Rosa26-LSL-rtTA; Cre triple transgenic mouse embryos. Gapdh was used as a control.

To express GNAQ R183Q in endothelial cells, we next used the Tie2-Cre (Kisanuki et al., 2001). We crossed the tet-GNAQ R183Q; Tie2-Cre males with Rosa26-LSL-rtTA females and pregnant females were given a doxycycline diet (625 mg/kg) at E12.5 to induce the transgene expression in endothelial cells. We harvested the embryonic skin at E14.5 and confirmed the GNAQ R183Q expression in the tet-GNAQ R183Q; Rosa26-LSL-rtTA; Tie2-Cre embryos treated with doxycycline by RT-PCR (Fig. 2A). Triple transgenic embryos without doxycycline treatment or control embryos (tet-GNAQ R183Q; Rosa26-LSL-rtTA and Rosa26-LSL-rtTA; Tie2-Cre) with or without doxycycline treatment showed no detectable GNAQ R183Q expression. The expression level of mutant GNAQ was much lower than the endogenous wild-type allele. This is not surprising since GNAQ might be ubiquitously expressed in all cell types in the skin and we only ectopically expressed the mutant GNAQ in endothelial cells. We confirmed this with real-time RT-PCR and found that the transgene was expressed at about 3–5% of the endogenous level (Fig. 2B). Given that endothelial cells only account for ~1–3% of total skin cells in E14.5 mouse skin (Joost et al., 2020), we estimated that the GNAQ R183Q transgene is expressed within physiological ranges in the endothelial cells with the doxycycline dose we selected (625 mg/kg). The small variation in transgene levels observed across four mutant embryos from different females suggests that the induction method is highly robust.

Fig. 2.

Fig. 2.

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Phenotypic analysis of the GNAQ R183Q Tie2-Cre endothelial mutants. (A) RT-PCR showing the expression of GNAQ R183Q in the E14.5 skin after doxycycline treatment in the tet-GNAQ R183Q; Rosa26-LSL-rtTA; Tie2-Cre triple transgenic mouse embryos. Gapdh was used as a control. (B) qRT-PCR showing the relative transgene expression level induced by doxycycline treatment. (C) Expression of GNAQ R183Q in endothelial cells with Tie2-Cre causes several blood vessel malformations in mouse embryos. Doxycycline treatment started at E12.5 and embryos were harvested at E15.5. (D) H&E staining on sections of E15.5 embryos showing blood-filled regions in multiple anatomical areas, including the craniofacial region, body trunk, liver and skin of mutant embryos (arrows). Scale bars: 1 mm (left); 0.5 mm (magnifications, right). (E) Whole-mount immunostaining of the E15.5 back skin with PECAM1 antibody. Although back skin from control embryos showed clear vascular networks (a), GNAQ R183Q mutant skin displayed many dilated vessels filled with blood cells (b). a’ and b’ show converted black and white images of a and b, respectively. Scale bar: 100 μm. Bar graphs showing the quantification of vein and capillary diameters in the control and mutant skin (three embryos per genotype). **P<0.01; ns, not significant. (F) Sagittal sections of E14.5 back skin stained with ZO-1 and collagen IV (green). PECAM1 (magenta) was used to highlight the endothelial cells. DAPI was used to highlight nuclei. Scale bar: 50 μm. Bar graphs showing the quantification of staining intensity of ZO-1 and collagen IV in endothelial cells (n=20 cells per genotype): no significant differences in the expression levels of these markers were found. Data are mean±s.e.m.

To determine the functional consequence of post-zygotic GNAQ R183Q in endothelial cells, we induced the transgene expression at E12.5 (by giving pregnant females a doxycycline diet at 625 mg/kg) and harvested embryos at E15.5. While tet-GNAQ R183Q; Rosa26-LSL-rtTA; Tie2-Cre embryos without doxycycline treatment or control embryos (tet-GNAQ R183Q; Rosa26-LSL-rtTA and Rosa26-LSL-rtTA; Tie2-Cre) with or without doxycycline treatment showed normal size and branching of the vasculatures, we observed dilated blood vessels all over the triple transgenic embryos treated with doxycycline (Fig. 2C). Hematoxylin and Eosin (H&E)-stained sections of E15.5 embryos show enlarged blood vessels and blood-filled regions in multiple anatomical areas, including the craniofacial region, body trunk, liver and skin (Fig. 2D). Extended treatment of doxycycline until E17.5 caused embryonic lethality and fetal resorption in mutant embryos. These results suggest that the expression of GNAQ R183Q causes severe blood vessel malformation in developing mouse embryos. We examined the blood vessel phenotype in the GNAQ R183Q Tie2-Cre mutants in more detail by whole-mount immunostaining of the back skin with PECAM1 antibody (Fig. 2E). E15.5 back skin from control embryos showed clear vascular networks of large veins, arteries and smaller capillaries. GNAQ R183Q mutant skin displayed many dilated vessels filled with numerous blood cells. It appears that only major blood vessels (veins by morphology), not capillaries, were affected. Immunostaining the skin sections with the tight junction marker ZO-1 (also known as TJP1) and the basement membrane marker collagen IV revealed no changes in the expression levels of both markers, suggesting that the mutant phenotype does not arise from gross disruptions in epithelial barrier function or basement membrane stability (Fig. 2F).

A recent study showed that endothelial cells expressing GNAQ R183Q (via viral transfection) form dilated vessels when xenografted in nude mice, and the expression of ANGPT2 and DSCR1 (RCAN1) is upregulated by GNAQ R183Q (Huang et al., 2022). To determine whether ANGPT2 and DSCR1 are involved in the blood vessel phenotype in the GNAQ R183Q mutant mice, we examined their expression by immunostaining the skin sections. ANGPT2 and DSCR1 staining were found in various regions of the skin, especially in some cells in the dermal layer. However, we did not see a significant increase in their expression levels in the mutant endothelial cells compared with the wild-type cells (Fig. 3A–D). This discrepancy is most likely due to the difference in the biological context (e.g. grafting in adult mice versus normal developing embryos) and particular cell types used (e.g. established cell line versus developing embryonic endothelial cells). GNAQ Q209L, a different hyperactive mutant of GNAQ, has been shown to cause vascular cell overproliferation and increased permeability in mice (Schrenk et al., 2023). To determine whether GNAQ R183Q endothelial cells in the mutant embryos are also hyperproliferative, we stained the skin sections with phospho-histone H3 (PH3) antibody. While proliferating cells were found in dermal keratinocytes and dermal cells, we did not observe increased PH3 and PECAM1 double-positive cells in the mutant skin. JNK activation positively regulates endothelial cell proliferation (Uchida et al., 2008). We stained the skin sections with pJNK antibody and saw no significant changes (Fig. 3E–H). YAP activation has been indicated as a downstream mechanism during mutant GNAQ/11-induced uveal melanomagenesis (Feng et al., 2014; Yu et al., 2014). We stained the skin sections with YAP antibody and did not observe significant changes in the YAP nuclear localization pattern, suggesting that YAP activation is not involved in the GNAQ R183Q Tie2-Cre endothelial phenotypes (Fig. 3I,J). These results suggest that different GNAQ variants might activate distinct downstream pathways, potentially leading to variant-specific cellular responses. This highlights the importance of characterizing each variant individually to better understand its unique contributions to vascular phenotypes, as the previous work performed in cell lines (Galeffi et al., 2022).

Fig. 3.

Fig. 3.

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Marker analysis of the GNAQ R183Q Tie2-Cre endothelial mutants. (A-D) Sagittal sections of E14.5 back skin stained with ANGPT2 and DSCR1. ANGPT2 and DSCR1 staining (green) were found in various regions of the skin, especially in some cells in the dermal layer. Arrows point to their low expression in endothelial cells and no significant difference was found when comparing the staining intensity in wild-type and mutant endothelial cells (n=20 cells per genotype). (E-J) Similar analyses were conducted for pJNK and PH3 (E-H) and YAP (I-J). No significant difference (ns) was found in staining intensity or pattern of these markers when comparing the mutant endothelial cells to wild-type cells. Data are mean±s.e.m. PECAM1 (magenta) was used to highlight the endothelial cells. DAPI was used to highlight nuclei. Arrows point to the endothelial cells. Scale bars: 50 μm (main images, A-J); 20 μm (magnifications, a-j′).

To define the molecular mechanism of the phenotype in GNAQ R183Q Tie2-Cre embryos, we performed unbiased whole-transcriptome profiling using RNA-seq. Total RNA was extracted from the back skin at E14.5. Transcriptomic differences between GNAQ R183Q Tie2-Cre mutant and control skins were compared to reveal global changes in gene expression. We found 622 differentially expressed genes, and 491 exhibited a >1.5-fold change (with a P-value <0.05) (Fig. 4A; Table S1). Next, we performed Gene Ontology (GO) analysis on differentially expressed genes. Skin development was the most enriched GO term in the GNAQ R183Q mutant skin. Epidermal cell differentiation, wound healing and multiple biosynthetic and metabolic pathways were also enriched. RNA processing genes and genes involved in the detection of chemical stimulus were underrepresented in the GNAQ R183Q mutant skin (Fig. 4B). Given that our genetic manipulation (GNAQ R183Q expression) is in endothelial cells, we reasoned that genes endogenously expressed in endothelial cells are likely to be the targets of GNAQ R183Q. We took advantage of the existing database (single-cell RNA-seq on developing mouse embryonic skin) (Jacob et al., 2023) and selected 16 endothelial cell-expressing genes for follow-up studies (12 from the top 30 upregulated genes and four from the top 30 downregulated genes). We performed quantitative real-time PCR (qRT-PCR) on these 16 genes using E14.5 back skins and confirmed their expression changes in GNAQ R183Q mutants (Fig. 4C,D). These data revealed distinct transcriptional changes in both endothelial cells and non-endothelial cells caused by GNAQ R183Q.

Fig. 4.

Fig. 4.

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Downstream targets regulated by GNAQ R183Q in endothelial cells. (A) Transcriptome changes in response to GNAQ R183Q expression in endothelial cells at E14.5, revealed by RNA-seq. Lists of the top 10 genes with the highest fold change. Magenta, upregulated expression; blue, downregulated expression. (B) Enriched GO terms for the differentially expressed genes in GNAQ R183Q mutant skin compared to the control, analyzed by PANTHER. (C) Heatmap from E14.5 RNA-seq datasets showing the expression of 16 selected endothelial cell-expressing genes in the control and GNAQ R183Q mutant mice: 12 from the top 30 upregulated genes and four from the top 30 downregulated genes, compared to the control. (D) Validation of the RNA-seq data by qRT-PCR using RNA extracted from E14.5 back skins. All data represent mean±s.e.m. of four biological replicates. Gapdh was used as a control. (E) Sagittal sections of E14.5 back skin stained with HMGA2 (green) and PECAM1 (magenta). DAPI was used to highlight nuclei. In control skin, HMGA2 staining was found only in non-endothelial dermal cells (arrowheads). Strong HMGA2 staining was found in endothelial cells of mutant embryos (arrows). Scale bar: 50 μm. (F) Tubule formation assay in vitro showing GNAQ R183Q expression in HMEC-1 cells significantly impairs the tubule formation capability of endothelial cells. GNAQ R183Q is sufficient to drive the expression of HMGA2 in HMEC-1 cells. (G) HMGA2 knockdown can partially rescue the phenotype caused by GNAQ R183Q. GAPDH was used as a control. Scale bar: 200 μm (F,G). **P<0.01; ns, not significant. Data are mean±s.e.m.

Interestingly, HMGA2, one of the genes upregulated by GNAQ R183Q, has been shown to have a broad pro-angiogenesis role in zebrafish development (Wang et al., 2021) and several mouse cancer models (Li et al., 2020; Sakata et al., 2019; Zhu et al., 2013). We examined the HMGA2 expression in the GNAQ R183Q Tie2-Cre mutant endothelial cells by immunostaining on the skin sections and observed a significant increase in its expression in the mutant endothelial cells compared with control cells (Fig. 4E). A recent study has shown that GNAQ R183Q impairs tubule formation in TIME endothelial cells in vitro (Zecchin et al., 2024). We used a similar in vitro angiogenesis model with HMEC-1 cells and determined the functional involvement of HMGA2 in GNAQ R183Q-induced vascular phenotype (Fig. 4F,G). Control HMEC-1 cells formed a well-organized network 4 h after seeding on Matrigel. GNAQ R183Q-expressing HMEC-1 cells clustered together and formed limited connections, while overexpression of wild-type GNAQ had no effect on tubule formation. Expression of HMGA2 was significantly upregulated by GNAQ R183Q, but not the wild-type counterpart. HMGA2 knockdown using siRNA significantly improved the tubule formation capability of GNAQ R183Q-overexpressing HMEC-1 cells. These results suggest that the upregulation of HMGA2 contributes to the vascular malformation phenotype we observed in the GNAQ R183Q mutants. Calb1 (encoding calcium-binding protein calbindin 1) is another top-upregulated gene identified from our RNA-seq experiments. It has been shown that calbindin 1 can modulate calcium levels and flux to regulate cellular senescence (Raynard et al., 2022). A recent study showed that GNAQ/11 mutant variants (especially GNAQ R183Q) cause prolonged calcium influx in endothelial cells, and calcium-release-activated channel inhibitors can rescue the tubule formation defects in endothelial cells expressing GNAQ R183Q. It will be particularly compelling to pursue further investigation into whether the blood vessel malformation phenotype observed in GNAQ R183Q mutant skin involves HMGA2 signaling (using Hmg2 conditional knockout mice) or dysregulation of the calcium signaling pathway (using pharmacological inhibitions), both of which could represent unique and targetable mechanisms downstream of GNAQ mutation.

In summary, we reported a novel mouse allele that can be used to express the hyperactive GNAQ allele (R183Q mutant) in developing mouse embryos in a spatial- and temporal-controlled manner. This is particularly useful for mimicking postzygotic pathogenesis affecting only a subset of cells during development. We used Tie2-Cre to drive the rtTA expression in endothelial cells and controlled the GNAQ R183Q expression by doxycycline diet administration to pregnant females. We showed that GNAQ R183Q expression can be robustly induced in endothelial cells, which causes massive blood vessel dilation in mouse embryos. Our study supports the causal relationship between GNA mutations and vascular phenotypes established by earlier studies. Our study also demonstrates that induction of GNAQ R183Q expression late in development in endothelial cells only is sufficient to drive vascular defects and provides important insights for understanding the mechanism of GNA mutation-related disease. It is important to acknowledge that our transgenic approach has an inherent limitation compared to the knock-in method – the absence of a wild-type GNAQ overexpression control – which prevents us from fully distinguishing mutant-specific effects from those that may arise due solely to increased GNAQ dosage. However, our in vitro data show that overexpression of wild-type GNAQ does not lead to upregulation of HMGA2 expression and does not cause tubule malformation in HMEC-1 cells, suggesting that the observed phenotypes in mice are not simply due to elevated GNAQ expression. Previous work from the Marchuk and Boscolo groups and a recent paper published during the revision of this manuscript demonstrate that pathogenic vascular phenotypes can be driven by GNAQ mutants, also supporting our view and data interpretation (Galeffi et al., 2022; Schrenk et al., 2023; Smits et al., 2025; Wetzel-Strong et al., 2023). The tet-on system used here offers unique advantages, including reversible and titratable expression, making it a valuable resource for future mechanistic studies. Our endothelial-specific GNAQ R183Q model, together with the accompanying transcriptomic profiling, establishes a platform to identify and functionally test candidate genes that contribute to vascular malformations. Integrating this model with genetic or pharmacologic perturbations will enable systematic dissection of the disease mechanisms and facilitate the development of preclinical therapeutic strategies. Future work employing cell type-specific approaches, such as RNA in situ hybridization to spatially resolve transgene expression and downstream target regulation, will enable more precise mechanistic dissection of direct alterations in endothelial cells and secondary effects in surrounding non-endothelial cells.

MATERIALS AND METHODS

Mouse lines and husbandry

Full-length cDNA clone of GNAQ was purchased from Dharmacon (MHS6278–202806296). Point mutation was introduced by PCR mutagenesis to change Arg183 to Glu183 (CGA to CAA). The GNAQ R183Q coding sequence was cloned into pTREtight2 vector (Addgene plasmid #19407) and the 1.7 kb XhoI-digested DNA fragment was used for generating transgenic mice using standard pronuclear injection techniques. Transgenic mice were genotyped using the following primers: (Forward) 5′-ACGCAACAA-GATGTGCTTAGAGTTCAA; (Reverse) 5′-TGTGATAATTGTTCTAAA-GAGAGCCTTGCTT. Rosa26-LSL-rtTA (Belteki et al., 2005), K14-Cre (Dassule et al., 2000) and Tie2-Cre mice (Kisanuki et al., 2001) were purchased from The Jackson Laboratory (stock numbers 005670, 004782 and 008863, respectively) and genotyped as instructed. Doxycycline feed was purchased from Envigo (625 mg/kg). Mice were handled and housed according to the approved Institutional Animal Care and Use Committee (IACUC) protocol M006259 of the University of Wisconsin-Madison.

Immunostaining on sections and skin whole mounts

For immunostaining of sagittal sections, embryos were embedded in OCT, fresh frozen, cryosectioned (10 μm) and fixed for 10 min in neutral formalin. Sections were washed with PBST (0.3% Triton in PBS) for 10 min and blocked for 1 h with 5% normal donkey serum in PBST. Primary antibodies were incubated overnight at 4°C. The following primary antibodies were used: rabbit anti-ZO-1 (61–7300, Life Technologies; 1:200), rabbit anti-collagen IV (ab6586, Abcam; 1:400); rat anti-PECAM1 (550274, BD Pharmingen; 1:200); goat anti-ANGPT2 (AF623, R&D Systems; 1:50); rabbit anti-DSCR1 (D6694, Sigma-Aldrich; 1:100); rabbit anti-pJNK (4668, Cell Signaling Technology; 1:1000); rabbit anti-PH3 (9701, Cell Signaling Technology; 1:800); rabbit anti-YAP (8418, Cell Signaling Technology; 1:1000); and rabbit anti-HMGA2 (8179, Cell Signaling Technology; 1:500). Secondary antibodies in PBST were incubated for 1 h at room temperature. Secondary antibodies were Alexa Fluor 488- or 594-conjugated donkey anti-goat, donkey anti-rat or donkey anti-rabbit IgG antibodies (A-11055, A-21209, A-21206, respectively; Invitrogen; 1:500). Finally, sections were washed three times in PBST and mounted with aqueous mounting media. For whole-mount immunostaining of embryonic skins, embryos were fixed overnight in neutral formalin at 4°C. Back skins were dissected, rinsed with PBS, washed with 0.3% PBST for 30 min and incubated with primary antibodies in 0.3% PBST containing 5% normal donkey serum overnight at 4°C. Skins were then washed in 0.3% PBST for 30 min three times, incubated in secondary antibodies in 0.3% PBST at room temperature for 2 h, washed in 0.3% PBST and flat-mounted with aqueous mounting media. Immunostained samples were imaged using the Nikon A1R confocal microscope with NIS-Elements software. Capillaries and veins were identified based on their anatomical location and morphology within the same vascular plexus. Diameters were measured at the widest point perpendicular to the longitudinal axis of the vessel using the line measurement tool in ImageJ (Fiji). For each embryo, 8–10 vessels were measured in three independent fields. Data from three embryos per genotype were included for quantification.

Cell culture

HMEC-1 cells were obtained from ATCC (CRL-3243) and maintained in MCDB131 media supplemented with 10 ng/ml epidermal growth factor, 1 μg/ml hydrocortisone, 10 mM glutamine, and 10% fetal bovine serum in a humidified chamber with 5% CO2 at 37°C. GNAQ wild-type and R183Q expression plasmids were generated by cloning the coding sequence into the pRK5 vector and verified by sequencing. For each well of cells to be transfected in a six-well plate, 1.5 μg of plasmid DNA was diluted in 150 μl of serum-free media. Then 5.4 μl of 1 mg/ml polyethylenimine (PEI) was added to the diluted DNA solution, mixed gently, and incubated for 10 min at room temperature before adding to the cells. Cells were incubated for 48 h post-transfection before assaying for gene expression unless otherwise specified. ON-TARGETplus siRNA against HMGA2 was purchased from Dharmacon (L-013495-00-0005). ON-TARGETplus Nontargeting Pool (D-001810-10-05) was used as a control. For cells used in the tubule formation assay, a neomycin-resistant plasmid (pCI-neo, Promega) was included in the transfection mix to select transfected cells with the antibiotic G-418 (0.5 mg/ml for 48 h). We then seeded 15,000 cells into individual wells of a 96-well plate filled with 100 μl Matrigel and monitored them for tubule formation. Pictures were taken 4 h after cell seeding using the BioTek Lionheart FL microscope and Gen5 software. Tubule length was quantified using the ImageJ software.

RNA isolation, RNA-seq, RT-PCR and qRT-PCR

RNA was extracted from cells or tissues using Trizol (Invitrogen) and RNeasy (Qiagen) kits. cDNA synthesis was performed using the SuperScript IV First-Strand Synthesis System (#18091150, Invitrogen) following the manufacturer’s instructions. Quantitative real-time RT-PCR was performed in triplicate in 20 μl reactions with SYBR Premix Ex Taq II ROX plus (RR82LR, Takara) with 10 ng first-strand cDNA and 0.2 μg each forward and reverse primers. Samples were cycled once at 95°C for 2 min, followed by 40 cycles of 95°C, 58°C and 72°C for 30 s each. Relative mRNA level was calculated using the ΔΔCT method with Gapdh/GAPDH as an endogenous control. Primers used for semi-quantitative RT-PCR and qRT-PCR were designed or selected from the PrimerBank database (listed in Table S2). For RNA-seq experiments, E14.5 mouse back skins were dissected from mutant and control mice. Four biologically independent samples were collected for each genotype. RNA-seq analysis was performed using Pluto (https://pluto.bio). GO analysis was performed using PANTHER (Thomas et al., 2022).

Statistical analysis

Statistical analyses were performed with two-tailed unpaired Student’s t-test between two experimental groups and one-way analysis of variance (ANOVA) for more than two experimental groups followed by Dunnett’s or Tukey’s test for multiple comparisons using the GraphPad Prism 10 software. A P-value less than 0.05 was considered significant.

Supplementary Material

Table S2
Table S1

Acknowledgements

We acknowledge Kathy Krentz and the Animal Models Core at the UW Biotechnology Center for mouse pronuclear injections, and Sandra Splinter BonDurant and Mark Berres and the UW Gene Expression Center for RNA-seq service.

Funding

The work was supported by grants from the National Institutes of Health (the Skin Diseases Research Center Core Grant P30 AR066524 and the University of Wisconsin Carbone Cancer Center Support Grant P30 CA014520). H.C. was also supported in part by the Cripps/Ratcliff Professor for Skin and Cancer Research Endowed Professorship (University of Wisconsin-Madison). B.D. was supported by Pediatric Dermatology Research Alliance Research Fellowship Program (X01HL145695) and Geneva F. and Sture Johnson Distinguished Chair Endowment (University of Wisconsin-Madison).

Footnotes

Competing interests

B.D. is cofounder and owner of Arkayli Biosciences.

Data and resource availability

The RNA-seq data reported in this paper have been deposited in GEO under accession number GSE325430. All other relevant data and details of resources can be found within the article and its supplementary information.

References

  1. Adams DM and Ricci KW. (2019). Vascular anomalies: diagnosis of complicated anomalies and new medical treatment options. Hematol. Oncol. Clin. North Am. 33, 455–470. doi: 10.1016/j.hoc.2019.01.011 [DOI] [PubMed] [Google Scholar]
  2. Ayturk UM, Couto JA, Hann S, Mulliken JB, Williams KL, Huang AY, Fishman SJ, Boyd TK, Kozakewich HP, Bischoff J et al. (2016). Somatic activating mutations in GNAQ and GNA11 are associated with congenital hemangioma. Am. J. Hum. Genet. 98, 789–795. doi: 10.1016/j.ajhg.2016.03.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Belteki G, Haigh J, Kabacs N, Haigh K, Sison K, Costantini F, Whitsett J, Quaggin SE and Nagy A. (2005). Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction. Nucleic Acids Res. 33, e51. doi: 10.1093/nar/gni051 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cheraghlou S, Lim Y and Choate K. (2019). Genetic investigation of childhood vascular tumor biology reveals pathways for therapeutic intervention. F1000Res 8, F1000. doi: 10.12688/f1000research.16160.1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Couto JA, Ayturk UM, Konczyk DJ, Goss JA, Huang AY, Hann S, Reeve JL, Liang MG, Bischoff J, Warman ML et al. (2017). A somatic GNA11 mutation is associated with extremity capillary malformation and overgrowth. Angiogenesis 20, 303–306. doi: 10.1007/s10456-016-9538-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dassule HR, Lewis P, Bei M, Maas R and McMahon AP. (2000). Sonic hedgehog regulates growth and morphogenesis of the tooth. Development 127, 4775–4785. doi: 10.1242/dev.127.22.4775 [DOI] [PubMed] [Google Scholar]
  7. Feng X, Degese MS, Iglesias-Bartolome R, Vaque JP, Molinolo AA, Rodrigues M, Zaidi MR, Ksander BR, Merlino G, Sodhi A et al. (2014). Hippo-independent activation of YAP by the GNAQ uveal melanoma oncogene through a trio-regulated rho GTPase signaling circuitry. Cancer Cell 25, 831–845. doi: 10.1016/j.ccr.2014.04.016 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Funk T, Lim Y, Kulungowski AM, Prok L, Crombleholme TM, Choate K and Bruckner AL. (2016). Symptomatic congenital hemangioma and congenital hemangiomatosis associated with a somatic activating mutation in GNA11. JAMA Dermatol 152, 1015–1020. doi: 10.1001/jamadermatol.2016.2365 [DOI] [PubMed] [Google Scholar]
  9. Galeffi F, Snellings DA, Wetzel-Strong SE, Kastelic N, Bullock J, Gallione CJ, North PE and Marchuk DA. (2022). A novel somatic mutation in GNAQ in a capillary malformation provides insight into molecular pathogenesis. Angiogenesis 25, 493–502. doi: 10.1007/s10456-022-09841-w [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Greene AK. (2011). Vascular anomalies: current overview of the field. Clin. Plast. Surg. 38, 1–5. doi: 10.1016/j.cps.2010.08.004 [DOI] [PubMed] [Google Scholar]
  11. Huang L, Bichsel C, Norris AL, Thorpe J, Pevsner J, Alexandrescu S, Pinto A, Zurakowski D, Kleiman RJ, Sahin M et al. (2022). Endothelial GNAQ p.R183Q increases ANGPT2 (Angiopoietin-2) and drives formation of enlarged blood vessels. Arterioscler. Thromb. Vasc. Biol. 42, e27–e43. doi: 10.1161/ATVBAHA.121.316651 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacob T, Annusver K, Czarnewski P, Dalessandri T, Kalk C, Levra Levron C, Campamà Sanz N, Kastriti ME, Mikkola ML, Rendl M et al. (2023). Molecular and spatial landmarks of early mouse skin development. Dev. Cell 58, 2140–2162.e2145. doi: 10.1016/j.devcel.2023.07.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Joost S, Annusver K, Jacob T, Sun X, Dalessandri T, Sivan U, Sequeira I, Sandberg R and Kasper M. (2020). The molecular anatomy of mouse skin during hair growth and rest. Cell Stem Cell 26, 441–457.e447. doi: 10.1016/j.stem.2020.01.012 [DOI] [PubMed] [Google Scholar]
  14. Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA and Yanagisawa M. (2001). Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev. Biol. 230, 230–242. doi: 10.1006/dbio.2000.0106 [DOI] [PubMed] [Google Scholar]
  15. Li Y, Qiang W, Griffin BB, Gao T, Chakravarti D, Bulun S, Kim JJ and Wei JJ. (2020). HMGA2-mediated tumorigenesis through angiogenesis in leiomyoma. Fertil. Steril. 114, 1085–1096. doi: 10.1016/j.fertnstert.2020.05.036 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lian CG, Sholl LM, Zakka LR, Teresa MO, Liu C, Xu S, Stanek E, Garcia E, Jia Y, MacConaill LE et al. (2014). Novel genetic mutations in a sporadic port-wine stain. JAMA Dermatol 150, 1336–1340. doi: 10.1001/jamadermatol.2014.1244 [DOI] [PubMed] [Google Scholar]
  17. Nakashima M, Miyajima M, Sugano H, Iimura Y, Kato M, Tsurusaki Y, Miyake N, Saitsu H, Arai H and Matsumoto N. (2014). The somatic GNAQ mutation c.548G>A (p.R183Q) is consistently found in Sturge-Weber syndrome. J. Hum. Genet. 59, 691–693. doi: 10.1038/jhg.2014.95 [DOI] [PubMed] [Google Scholar]
  18. O’Hayre M, Vázquez-Prado J, Kufareva I, Stawiski EW, Handel TM, Seshagiri S and Gutkind JS. (2013). The emerging mutational landscape of G proteins and G-protein-coupled receptors in cancer. Nat. Rev. Cancer 13, 412–424. doi: 10.1038/nrc3521 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Oldham WM and Hamm HE. (2008). Heterotrimeric G protein activation by G-protein-coupled receptors. Nat. Rev. Mol. Cell Biol. 9, 60–71. doi: 10.1038/nrm2299 [DOI] [PubMed] [Google Scholar]
  20. Raynard C, Tessier N, Huna A, Warnier M, Flaman JM, Van Coppenolle F, Ducreux S, Martin N and Bernard D. (2022). Expression of the calcium-binding protein CALB1 is induced and controls intracellular ca. Int. J. Mol. Sci. 23, 9376. doi: 10.3390/ijms23169376 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sakata J, Hirosue A, Yoshida R, Kawahara K, Matsuoka Y, Yamamoto T, Nakamoto M, Hirayama M, Takahashi N, Nakamura T et al. (2019). HMGA2 Contributes to distant metastasis and poor prognosis by promoting angiogenesis in oral squamous cell carcinoma. Int. J. Mol. Sci. 20, 2473. doi: 10.3390/ijms20102473 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schrenk S, Bischoff LJ, Goines J, Cai Y, Vemaraju S, Odaka Y, Good SR, Palumbo JS, Szabo S, Reynaud D et al. (2023). MEK inhibition reduced vascular tumor growth and coagulopathy in a mouse model with hyperactive GNAQ. Nat. Commun. 14, 1929. doi: 10.1038/s41467-023-37516-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Shirley MD, Tang H, Gallione CJ, Baugher JD, Frelin LP, Cohen B, North PE, Marchuk DA, Comi AM and Pevsner J. (2013). Sturge-Weber syndrome and port-wine stains caused by somatic mutation in GNAQ. N. Engl. J. Med. 368, 1971–1979. doi: 10.1056/NEJMoa1213507 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Siegel DH, Cottrell CE, Streicher JL, Schilter KF, Basel DG, Baselga E, Burrows PE, Ciliberto HM, Vigh-Conrad KA, Eichenfield LF et al. (2018). Analyzing the genetic spectrum of vascular anomalies with overgrowth via cancer genomics. J. Invest. Dermatol. 138, 957–967. doi: 10.1016/j.jid.2017.10.033 [DOI] [PubMed] [Google Scholar]
  25. Smits P, Marrs L, Cheng YS, Ad M, Nasim S, Zurakowski D, Bischoff J and Greene AK. (2025). An endothelial specific mouse model for the capillary malformation mutation Gnaq p.R183Q. Angiogenesis 28, 33. doi: 10.1007/s10456-025-09986-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Steiner JE and Drolet BA. (2017). Classification of vascular anomalies: an update. Semin. Intervent. Radiol. 34, 225–232. doi: 10.1055/s-0037-1604295 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Syrovatkina V, Alegre KO, Dey R and Huang XY. (2016). Regulation, signaling, and physiological functions of G-proteins. J. Mol. Biol. 428, 3850–3868. doi: 10.1016/j.jmb.2016.08.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Thomas AC, Zeng Z, Rivière JB, O’Shaughnessy R, Al-Olabi L, St-Onge J, Atherton DJ, Aubert H, Bagazgoitia L, Barbarot S et al. (2016). Mosaic activating mutations in GNA11 and GNAQ are associated with phakomatosis pigmentovascularis and extensive dermal melanocytosis. J. Invest. Dermatol. 136, 770–778. doi: 10.1016/j.jid.2015.11.027 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thomas PD, Ebert D, Muruganujan A, Mushayahama T, Albou LP and Mi H. (2022). PANTHER: Making genome-scale phylogenetics accessible to all. Protein Sci. 31, 8–22. doi: 10.1002/pro.4218 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Uchida C, Gee E, Ispanovic E and Haas TL. (2008). JNK as a positive regulator of angiogenic potential in endothelial cells. Cell Biol. Int. 32, 769–776. doi: 10.1016/j.cellbi.2008.03.005 [DOI] [PubMed] [Google Scholar]
  31. Van Raamsdonk CD, Bezrookove V, Green G, Bauer J, Gaugler L, O’Brien JM, Simpson EM, Barsh GS and Bastian BC. (2009). Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 457, 599–602. doi: 10.1038/nature07586 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wang J, Chen Y, Zeng Z, Feng R, Wang Q, Zhang Q, Sun K, Chen AF, Lu Y and Yu Y. (2021). HMGA2 contributes to vascular development and sprouting angiogenesis by promoting IGFBP2 production. Exp. Cell Res. 408, 112831. doi: 10.1016/j.yexcr.2021.112831 [DOI] [PubMed] [Google Scholar]
  33. Wetzel-Strong SE, Galeffi F, Benavides C, Patrucco M, Bullock JL, Gallione CJ, Lee HK and Marchuk DA. (2023). Developmental expression of the Sturge-Weber syndrome-associated genetic mutation in Gnaq: a formal test of Happle’s paradominant inheritance hypothesis. Genetics 224, iyad077. doi: 10.1093/genetics/iyad077 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yu FX, Luo J, Mo JS, Liu G, Kim YC, Meng Z, Zhao L, Peyman G, Ouyang H, Jiang W et al. (2014). Mutant Gq/11 promote uveal melanoma tumorigenesis by activating YAP. Cancer Cell 25, 822–830. doi: 10.1016/j.ccr.2014.04.017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zecchin D, Knopfel N, Gluck AK, Stevenson M, Sauvadet A, Polubothu S, Barberan-Martin S, Michailidis F, Bryant D, Inoue A et al. (2024). GNAQ/GNA11 Mosaicism causes aberrant calcium signaling susceptible to targeted therapeutics. J. Invest. Dermatol. 144, 811–819.e814. doi: 10.1016/j.jid.2023.08.028 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhu S, Deng S, Ma Q, Zhang T, Jia C, Zhuo D, Yang F, Wei J, Wang L, Dykxhoorn DM et al. (2013). MicroRNA-10A* and MicroRNA-21 modulate endothelial progenitor cell senescence via suppressing high-mobility group A2. Circ. Res. 112, 152–164. doi: 10.1161/CIRCRESAHA.112.280016 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S2
NIHMS2214908-supplement-Table_S2.xlsx (10.6KB, xlsx)
Table S1
NIHMS2214908-supplement-Table_S1.xlsx (2.4MB, xlsx)

Data Availability Statement

The RNA-seq data reported in this paper have been deposited in GEO under accession number GSE325430. All other relevant data and details of resources can be found within the article and its supplementary information.

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