Essential Roles of Exocyst Complex Component 3-like 2 on Cardiovascular Development in Mice

Abstract

Angiogenesis is a process to generate new blood vessels from pre-existing vessels and to maintain vessels, and plays critical roles in normal development and disease. However, the molecular mechanisms underlying angiogenesis are not fully understood. This study examined the roles of exocyst complex component (Exoc) 3-like 2 (Exoc3l2) during development in mice. We found that Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 are expressed abundantly in endothelial cells at embryonic day 8.5. The generation of Exoc3l2 knock-out (KO) mice showed that disruption of Exoc3l2 resulted in lethal in utero. Substantial numbers of Exoc3l2 KO embryos exhibited hemorrhaging. Deletion of Exoc3l2 using Tie2-Cre transgenic mice demonstrated that Exoc3l2 in hematopoietic and endothelial lineages was responsible for the phenotype. Taken together, these findings reveal that Exoc3l2 is essential for cardiovascular and brain development in mice.

1. Introduction

Angiogenesis is a dynamic process to generate new blood vessels via sprouting from pre-existing vessels, followed by the remodeling of generated lumenized endothelial tubes to mature into hierarchical networks. Subsequently, pericytes or vascular smooth muscle cells cover the lumenized endothelial tubes and provide stability. Angiogenesis plays critical roles in normal development and diseases such as cancer [1,2]. These processes are controlled by many genes, such as Vegf/Vegfr, Angiopoietin/Tie2, PDGF-B/PDGFR, TGF-β/ALK, Notch/Dll, Wnt/Frizzled and other signaling molecules [1,2].

The exocyst complex comprises eight subunits, including Exoc 1–8 [3,4,5]. Members of the exocyst complex were first found as genes implicated in polarized exocytosis in Saccharomyces cerevisiae during the genetic screening of secretory mutants [6]. The Exoc1 to Exoc8 subunits are conserved among mammalian species. Notably, Exoc3 has four homologous genes, called Exoc3-like 1 (Exoc3l1), Exoc3-like 2 (Exoc3l2), Exoc2-like 3 (Exoc3l3, also called tumor necrosis factor, alpha-induced protein 2, Tnfaip2) and Exoc3-like 4 (Exoc3l4) in vertebrates. Exoc3l2 is associated with human developmental anomalies [7,8,9], but its precise developmental functions remain unclear.

In previous work utilizing the fetal liver kinase 1 knock-out (Flk1 KO) fetus lacking endothelial cells, we showed that a class of genes were abundantly expressed in developing endothelial cells in mice. Some of the orthologous human genes, including EXOC3L1, have been shown to function during angiogenesis in human umbilical vein endothelial cells, although most of them have yet to be functionally characterized [10].

Here, we report the generation of KO animals for Exoc3l2, and initial phenotypic characterization. Most of the Exoc3l2 KO embryos died in utero and showed hemorrhage, abnormal heart and brain development. Conditional KO animals lacking Exoc3l2 in hematopoietic and endothelial lineages showed similar phenotypes, such as hemorrhage and heart defects, indicating that Exoc3l2 in hematopoietic and endothelial lineages is responsible for normal cardiovascular development. Inducible KO in endothelial cells during postnatal retinal development resulted in normal angiogenesis in the retina, indicating that Exoc3l2 is dispensable for postnatal angiogenesis in the retina. Taken together, these results indicate that Exoc3l2 is essential for normal cardiovascular development in mice.

2. Materials and Methods

2.1. Animals

Wild-type ICR (CD1) mice were purchased from Japan SLC Inc. (Hamamatsu, Shizuoka, Japan). Genetically modified mice targeting Exoc3l2 were created by the animal facility in the University of Tsukuba. In brief, 5 ng/ul pX330-mC plasmid DNA carrying Cas9-Cdt1 fusion and respective gRNA for Exoc3l2 together with 10 ng/ul donor DNA were injected into fertilized eggs from C57BL/6J mice according to a previous report [11]. Mice were transferred to Shiga University of Medical Science. Exoc3l2 KO mice were initially maintained in the C57BL/6J background and then outbred with the ICR background for further phenotypic analysis. Genotyping PCR analysis for Exoc3l2 mice was performed using a Gotaq G2 Green Master mix (Cat # M7822; Promega, Madison, WI, USA) with the primers listed in Supplementary Materials Table S1.

2.2. Reverse Transcription (RT)-Quantitative (q) PCR

Total RNA was extracted using a RNeasy Micro kit (Cat # 74004; Qiagen, Hilden, Germany), followed by cDNA synthesis using the ReverTra Ace kit (Cat # TRT-101X5; Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Real-Time PCR was performed with the Thermal Cycler Dice Real Time System (TaKaRa Bio Inc., Otsu, Shiga, Japan) and Thunderbird SYBR qPCR Mix (Cat # QPS-201T; Toyobo). Data were normalized against the expression of Hprt. Primer sequences are listed in Supplementary Materials Table S2.

2.3. Single Cell (sc) RNA-Seq Analysis

scRNA-seq data for E8.5 mouse embryo (GSE186069) were downloaded from GEO database [12]. We performed conventional scRNA-seq processing using Seurat_v4.1.1 by R (https://www.r-project.org/, accessed on 10 October 2022). At first, we extracted “A2-” data set from whole scRNA-seq data for the analysis for Exoc3l2. Extracted scRNA-seq data were filtered to remove the dead cell population and empty droplets using cell annotation information described in C. Qiu et al. After the normalization of UMI counts by the total counts per cells, chose 2000 most highly variable genes for scaling the expression, then PCA was performed. To find the significant PCs, efficacious PC threshold was calculated by (1) cumulative %stdev of PCs > 90 and %variation associated with PCs < 5, and (2) last point of change of %PC variation > 0.1. We selected minimum value between (1) and (2) as the significant PC for this analysis. Finishing pre-processing of clustering, k-NN graph was generated by Louvain clustering using the top 14 of PCs (resolution = 0.4) and UMAP was drawn. Endothelial marker genes (Pecam1 and Cdh5) and Exoc3l2 expressions were shown on the same UMAP.

2.4. Immunohistochemistry

Mice were sacrificed by cervical dislocation. Embryos were then dissected and fixed in 4% paraformaldehyde for 2–4 h at 4 °C, as described previously [13] For whole-mount immunohistochemistry, after washing twice in PBS, embryos were permeabilized in 0.5% TritonX-100 (CAS: 9002-93-1; Sigma-Aldrich, St. Louis, MO, USA) in Phosphate-Buffered Saline (PBS) for 30 min at 4 °C. Permeabilized embryos were then blocked in blocking solution containing 10% donkey serum (Cat # IHR-8135, Immuno Bioscience, Mukilteo, WA, USA), 0.2% bovine serum albumin (A7906-50G; Sigma-Aldrich, St. Louis, MO, USA) and 0.01% Tween20 in PBS (0.01% PBT)(Code: 28353-85; Nacalai Tesque, Inc., Kyoto, Japan) for 1 h at 4 °C, followed by incubation overnight at 4 °C. The embryos were further treated with rat anti-Pecam1 antibody (1:100; Cat # 553370; BD Pharmingen, Franklin Lakes, NJ, USA) and anti-GFP rabbit polyclonal antibody Alexa Fluor 488 conjugate (1:200; Cat # A21311; Life Technologies, Inc., Carlsbad, CA, USA). After three washes with 0.2% PBT, embryos were incubated with Cy3-conjugated donkey anti-Rat IgG (H + L) secondary antibody (Cat # 712-165-150; Jackson Immunoresearch, West Grove, PA, USA) for 1–3 h at room temperature. Embryos were then washed three times with 0.2% PBT. Nuclei were stained with 10 µg/mL Hoechst33342 (Cat # H3570; Molecular Probes Inc., Eugene, OR, USA) for 20 min at 4 °C.

Brain sections were immunostained as described previously [14]. In brief, 200 µm thick coronal sections were prepared from telencephalon. Brains were fixed in 1% paraformaldehyde overnight at 4 °C, embedded in agarose gel, and cut using a vibrating microtome (Leica VT1200). Vibratome sections were treated with blocking buffer (10% donkey serum and 0.3% Triton X-100) overnight at 4 °C, followed by incubation with primary antibodies [anti-Pecam1 (# MAB1398Z; Millipore, Burlington, MA, USA), anti-platelet-derived growth factor-beta receptor (PDGFRβ) (# 14-1402-82; eBioscience), anti-phospho-histone H3 (# 9701; Cell Signaling)] diluted in the same buffer overnight at 4 °C. Immunolabeled sections were incubated with secondary antibodies overnight at 4 °C. After washing, sections were mounted under a cover glass with mounting medium (Fluoro-Keeper Antifade Reagent, Nacalai). Images were acquired on a confocal microscope (FV3000, Olympus) and processed using Adobe Photoshop.

For cryosections, embryos were mounted in OCT (Surgipath FSC 22 Blue Frozen section compound; Leica, Wetzlar, Germany). Tissue blocks were sectioned using a cryostat (CM1860 UV; Leica) and then immunostained with rabbit anti-mouse Type-IV collagen (1:500; Cat # 2150-1470; AbD Serotec, Hercules, CA, USA), rabbit anti-mouse Lyve1 antibody (Cat# ab14917, abcam, Cambridge, UK) and rat anti-mouse Ter119 antibody (Cat # 14-5921-82; Thermo Fisher Scientific, Waltham, MA, USA) as previously described [15].

2.5. Immunostaining of Whole-Mount Retinas

VE-cad-CreER mice [16] were mated with floxed Exoc3l2 mice to obtain conditional Exoc3l2 KO mice. Forty μg 4-hydroxytamoxifen was subcutaneously injected at postnatal day 2, 3.5 and 5. Littermates were used as controls.

Whole-mounted retinas were prepared according to a previous report [17]. Briefly, enucleated eyes were fixed for 20 min in 4% paraformaldehyde at room temperature. A small hole was made in the cornea using a 27-gauge needle, and a circular incision was made using fine scissors. Then, retinal cups were dissected from the eyes and postfixed for 30 min in 4% paraformaldehyde at 4 °C. The primary antibodies used were rabbit anti-GFP (Alexa 488-conjugated; Molecular Probes), biotinylated isolectin B4 (1:500; B-1205; Vector Laboratories), mouse anti-Ter119 (1:250; MAB1125; R&D Systems, Inc., Minneapolis, MN, USA), rabbit anti-neuron-glial antigen-2 (NG2) (1:200; AB5320; Millipore-Sigma, Burlington, MA, USA) and hamster anti-Pecam1 (1:1000; ab119341; Abcam, Cambridge, UK). The secondary antibodies were suitable species-specific secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or streptavidin coupled to Alexa Fluor dyes (Invitrogen, Waltham, MA, USA).

2.6. Image Acquisition

Confocal images were acquired on a Leica TCS-SP8 (Leica). Embryos were mounted in PBS on glass-bottom dishes (Cat # 3971-035; IWAKI, Tokyo, Japan). Fluorescence was excited with a 405 nm UV laser to detect Hoechst 33342, a 552 nm laser to detect Cy3, and a 488 nm laser to detect Alexa Fluor 488. The hybrid detector HyD (Leica) was used for signal amplification. Projection images were created by 3D viewer software (Leica) from z-stack images every 2–4 µm.

Whole retinas were mounted using ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific). Fluorescence was excited with a 647 nm laser for Cy5, a 552 nm laser for Cy3, a 488 nm laser for Alexa Fluor 488, and a 405 nm UV laser for Hoechst 33342 and DAPI. Z-stack images were obtained every 5 µm.

2.7. Flowcytometry Analysis

Embryos were incubated with 0.3% collagenase (Cat # 038-22361; Wako, Osaka, Japan) at 37 °C for 15 min. After washing, single cells were exposed to allophycocyanin-conjugated anti-mouse Pecam1 antibody (MEC13.3, Cat #102510; BioLegend, San Diego, CA, USA). Cells were analyzed by FACS calibur (Becton Dickinson, San Jose, CA, USA) and the data were processed by the FlowJo software (TreeStar, Ashland, OR, USA).

2.8. CT imaging Analyses

Mouse embryos were fixed with Bouin’s fixative solution, a 15:5:1 mixture of water-saturated picric acid, concentrated formalin and concentrated acetic acid. Samples were washed with 70% EtOH and soaked in 1% phosphotungstic acid in 70% EtOH as contrast agent. Then, samples were scanned with Scanxmate-E090S (Comscantechno, Yokohama, Kanagawa, Japan), and rotated 360° in steps of 0.3°, generating 1200 projection images of 992 × 992 pixels. Reconstruction of the CT images was carried out using the filtered back projection. The cardiovascular system and soft tissues were visualized using maximum intensity projection and multi-planar reconstruction, respectively, in OsiriX software (www.osirix-viewer.com, accessed on 5 June 2021).

2.9. Statistical Analysis

The differences in Mendelian ratio were analyzed by the chi-square test, with p < 0.05 considered significant.

Student’s t-test was used for statistical analysis. Data were presented by means and standard errors. Statistical significance was determined at p < 0.05.

3. Results

3.1. Expression of Exoc3l Members and Generation of Exoc3l2 KO Mice

Our previous work showed that several exocyst complex component 3-like members were abundantly expressed in developing endothelial cells [10], but it is not known whether they play any physiological role during development. To address this issue, the gene expression patterns of Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 were determined by RT-qPCR analysis using cDNA from a wild-type (WT) fetus at embryonic day 8.5 (E8.5), Flk1 KO fetus at E8.5 (which lacks endothelial cells), endothelial cells purified from a Flk1^+/GFP knock-in fetus at E8.5, and peripheral blood cells isolated at E9.5 (Figure 1a). RT-qPCR analysis showed that Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 were abundantly expressed in WT but Flk1 KO fetuses at E8.5 were not. They were also expressed more abundantly in endothelial cells sorted from a WT fetus at E8.5, but not in peripheral blood cells at E9.5 (Figure 1a). These results indicated that Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 are expressed abundantly in endothelial cells at E8.5, in a sharp contrast with the ubiquitous pattern of other exocyst complex members, such as Exoc3 (Figure 1a). Analysis with publicly available scRNA-seq data on E8.5 embryo also confirmed the endothelial expression of Exoc3l2 (Figure S1).

To investigate whether Exoc3l2 plays roles in developing endothelial cells, KO mice for Exoc3l2 were created by deleting most of the protein coding exons (Figure 1b). For the GFP knock-in allele, a linker sequence, GSG, a self-cleaving peptide sequence, P2A, and GFP were introduced into exon2 (Figure 1b).

Whole-mount immunohistochemistry of an Exoc3l2^+/GFP embryo at E9.5 showed GFP expression considerably overlapped with that of Pecam1, an endothelial marker, and GFP expression was stronger in microcapillary rather than in moderately sized blood vessels (Figure 1c). GFP expression was also observed in non-endothelial cells, which are most likely cranial neural crest cells (Figure 1c). Since GFP expression is low or negative in large blood vessels, GFP expression was evaluated in Pecam1-positive endothelial cells by FACS. This analysis found that 15% of Pecam1-positive cells expressed GFP (Figure 1d), suggesting that subpopulation of Pecam1-positive endothelial cells express Exoc3l2, although the biological roles of the subpopulation remain unclear.

3.2. Phenotypes of Exoc3l2 KO Embryo

When Exoc3l2 heterozygotes were crossed with each other, Exoc3l2 KO pups were never born, suggesting an important role for Exoc3l2 during embryonic development (Figure 2a). Exoc3l2 KO embryos were present at a Mendelian ratio until E15.5 (p(X²) = 0.9131 by the chi-square test), although a substantial number (9 out of 17) were dead (Figure 2a). Exoc3l2 KO but not WT embryos exhibited extensive hemorrhages at E15.5 (9 out of 17) (Figure 2a,b). Histological analysis with hematoxylin and eosin indicated that WT embryos had most, if not all, peripheral red blood cells inside blood vessels, and many red blood cells were seen beneath the skin in Exoc3l2 KO embryo (Figure 2b). Immunohistochemistry to highlight hemorrhaged red blood cells showed that Ter119-positive peripheral red blood cells were distributed outside blood vessels as well as inside, while most red blood cells were located inside in WT embryo (Figure 2c). Micro-CT analysis was used to inspect the location of hemorrhaging throughout body. The µ-CT contrast agent (CECT) is a powerful tool for high-throughput, high-resolution embryonic phenotyping, and is used for embryonic lethal phenotyping at the International Mouse Phenotyping Consortium [18]. In addition, CTEC can visualize the cardiovascular system via the possible detection of blood components such as clots [19]. The CECT indicated that hemorrhaging occurred in internal organs, including the liver as well as under the skin (Figure 2d).

3.3. Phenotypes of Tie2-Cre: Exoc3l2 Conditional Knock-Out Embryos

Although conventional KO of Exoc3l2 in mice showed that the lack of Exoc3l2 resulted in embryonic lethality, possibly due to hemorrhaging, Exoc3l2 is expressed in non-endothelial cells in the head region, likely to be cranial neural crest cells, and potentially non-endothelial cells (Figure 1c). To investigate whether endothelial Exoc3l2 is critical for proper embryonic development, a floxed allele for Exoc3l2 was generated by introducing loxP sequences at both ends of exon 3 (Figure 3a). Exon3 was floxed, because deletion of exon3 leads to a frameshift and production of a truncated protein (209 amino acids) lacking entire Sec6 domain which is conserved among Exoc genes. Crossing Exoc3l2^flox/flox mice with Tie2-Cre: Exoc3l2^+/Δ mice (expressing Cre in hematopoietic and endothelial lineages) generated Tie2-Cre:: Exoc3l2^flox/Δ embryos (hereafter denoted Exoc3l2^ΔHEC), which lack Exoc3l2 in hematopoietic and endothelial lineages (Figure 3a,b). Gross observation indicated that a significant number of Exoc3l2^ΔHEC embryos showed embryonic lethality around E17.5 and exhibited hemorrhaging under the skin, similar to the phenotype of Exoc3l2 KO embryos (Figure 3b,d). Previous work reported that blood cells could be observed inside lymphatic vessels because of improper fusion of the vascular system and lymphatic vessels [20,21]. However, this scenario is not the case in the current study because there were no blood cells inside lymphatic vessels in Exoc3l2^ΔHEC embryos (Figure 3e).

CECT analysis was used to inspect the hemorrhage phenotype more globally in deeper tissues of the embryos. Although WT embryos showed no detectable hemorrhaging (Figure 4a–c), Exoc3l2^ΔHEC embryo showed hemorrhaging from various blood vessels throughout the body, including large vessels (Figure 4d–h). These data suggest that Exoc3l2 function in hematopoietic and endothelial is essential for vessel development and/or homeostasis.

3.4. Postnatal Roles of Exoc3l2 in Retinal Vascular Development

Since Exoc3l2 plays a critical role in vascular stability during embryonic development, we predicted that Exoc3l2 may participate in vascular stability during the postnatal stage. To investigate a role of Exoc3l2 on angiogenesis during postnatal development, we have used an angiogenesis model in retina, because retinal angiogenesis starts from the birth and often used to measure the angiogenic ability of endothelial cells. Exoc3l2-GFP was weakly expressed in retinal endothelial cells (Figure 5a). Therefore, VE-cadherin-CreER: Exoc3l2^+/GFP mice and Exoc3l2 ^flox/flox mice were crossed to conditionally delete Exoc3l2 in the postnatal stage by treatment with tamoxifen (Figure 5b). This strategy generated VE-cadherin-CreER::Exoc3l2 conditional KO mice (hereafter denoted Exoc3l2^iΔEC), which lack Exoc3l2 in the postnatal endothelial lineage (Figure 5b). Immunohistochemistry for Pecam1 and NG2, a pericyte marker in the retina showed that radial growth, pericyte coverage and filopodia number were similar in WT and Exoc3l2^iΔEC mice, indicating that Exoc3l2 is dispensable for postnatal angiogenesis in retina (Figure 5c).

3.5. Roles of Exoc3l2 in Heart and Brain Development

Since Exoc3l2 is associated with Dandy–Walker malformation in Meckel–Gruber syndrome in human [7,8,9], Exoc3l2 may play other physiological roles in other cell types than endothelial cells. To address this issue, CECT was used to examine the phenotype of Exoc3l2^ΔHEC KO to determine potential Exoc3l2 functions during embryogenesis. This analysis used three and six Exoc3l2^ΔHEC KO embryos at E12.5 and E15.5, respectively (Figure 6). Some Exoc3l2^ΔHEC KO embryos showed ventricular septal defects (2 out of 3 examined) (Figure 6b, arrows) and hypoplasia of myocardium (2 out of 3 examined) (Figure 6b,c, arrowheads) in the heart at E12.5. Furthermore, Exoc3l2^ΔHEC KO embryos had morphological brain anomalies in the telencephalon and cerebellum at E15.5 (arrowheads in Figure 6e,f). These results indicated that Exoc3l2 in hematopoietic and endothelial lineages play a role in normal heart and brain development.

Our previous report indicated endothelial cells regulate the proliferation and differentiation of neural stems [14]. Therefore, the numbers of apical and basal progenitors (phospho-histone H3-positive neural stem and progenitors) were quantified in the E16.5 neocortex (Figure 7). There were no significant differences between apical and basal progenitor numbers in WT and Exoc3l2^ΔHEC KO embryos, suggesting that endothelial Exoc3l2 regulates brain development through other pathways.

4. Discussion

In this paper, we report for the first time that Exoc3l2 is required for normal angiogenesis, heart and brain development, and the lack of Exoc3l2 resulted in hemorrhages, abnormal heart and brain development. By using conditional knock-out approaches, we demonstrated that Exoc3l2 function in hematopoietic and endothelial lineages is indispensable for normal angiogenesis, heart and brain development. In contrast with essential roles of Exoc3l2 during embryonic development, Exoc3l2 is not required for postnatal retinal angiogenesis. However, role of Exoc3l2 on pathological angiogenesis such as tumour angiogenesis remains unclear.

Since Exoc3l2 is deleted in both hematopoietic and endothelial lineages of Exoc3l2^ΔHEC embryos, it is not clear which lineage is responsible for the hemorrhages and abnormal heart development. Further analysis with specific Cre-expressing mice for hematopoietic or endothelial cells will be required for the clarification.

Currently, the molecular and cellular mechanism of how Exoc3l2 regulates angiogenesis, heart and brain development is not known. Previous reports show that proper interaction between vascular endothelial cells is needed for normal angiogenesis, and disrupted interaction results in hemorrhage [22]. Normal pericyte recruitment on endothelial cells mediated by smooth muscle cells or pericytes is also required for vascular stabilization [1,2]. Exocyst complex members regulate E-cadherin trafficking from recycling endosomes to plasma membranes, E-cadherin clustering [23], epithelial polarization [24], Notch signaling and primary ciliogenesis [25]. Therefore, Exoc3l2 may function as a regulator for cadherin trafficking and Notch signaling in endothelial cells, to control vascular stabilization. Since Exoc genes are implicated in vesicle trafficking, Exoc3l2 may affect various organ development other than cardiovascular and brain development via paracrine manner.

Qu and coworkers showed that endocardial Tie2 regulates myocardial trabeculation through the retinoic acid pathway [26]. Therefore, heart development may be regulated by Exoc3l2 through Tie2. Barkefors and coworkers reported that Exoc3l2 is expressed in vascular endothelial cells and forms a complex with Exoc4, a component of exocyst, and Exoc3l2 knock-down resulted in reduced phosphorylation of Flk1 and decreased sprouting of endothelial cells in vitro [27]. It will be interesting to investigate whether the VEGF-Flk1 pathway is affected in Exoc3l2 KO endothelial cells in future research.

Shaheen and coworkers reported that a patient with a biallelic EXOC3L2 (human ortholog of murine Exoc3l2) mutation had renal and neurological phenotypes called the Dandy–Walker malformation in Meckel–Gruber syndrome [7]. There are two other reports indicating that biallelic mutations of EXOC3L2 causes developmental anomalies, such as peritrigonal and cerebellar abnormalities, with enlargement of ventricular trigones, pituitary hypoplasia, severe renal dysplasia and bone marrow failure [7]. Since we have shown that conditional Exoc3l2 KO embryos exhibited developmental anomalies in the blood vessels, brain and heart, Exoc3l2 KO mice provide a valuable animal model for the Dandy–Walker malformation in Meckel–Gruber syndrome, although further analysis on other organs and tissues of Exoc3l2 mutant mice will be needed.

Recent genome-wide association studies revealed an association between the EXOC3L2 rs597668 variant and Alzheimer’s disease in European and Asian populations [28,29,30,31]. Since Exoc3l2 was shown to contribute to brain development by our study, it will be interesting to examine whether Exoc3l2 also plays a role in human brain disorders, such Alzheimer’s disease.

Taken together, we have shown that Exoc3l2 is essential for cardiovascular and brain development. It will be interesting to investigate the molecular mechanism by Exoc3l2 and apply the knowledge to therapy for human diseases.