Endothelial Arid1a deletion disrupts the balance among angiogenesis, neurogenesis and gliogenesis in the developing brain

Abstract The vascular system and the neural system processes occur simultaneously, the interaction among them is fundamental to the normal development of the central nervous system. Arid1a (AT‐rich interaction domain 1A), which encodes an epigenetic subunit of the SWI/SNF chromatin‐remodelling complex, is associated with promoter‐mediated gene regulation and histone modification. However, the molecular mechanism of the interaction between cerebrovascular and neural progenitor cells (NPCs) remains unclear. To generate Arid1a cKO‐Tie2 mice, Arid1a fl/fl mice were hybridized with Tie2‐Cre mice. The Angiogenesis, neurogenesis and gliogenesis were studied by immunofluorescence staining and Western blotting. RNA‐seq, RT‐PCR, Western blotting, CO‐IP and rescue experiments were performed to dissect the molecular mechanisms of Arid1a regulates fate determination of NPCs. We found that the absence of Arid1a results in increased the density of blood vessels, delayed neurogenesis and decreased gliogenesis, even after birth. Mechanistically, the deletion of Arid1a in endothelial cells causes a significant increase in H3k27ac and the secretion of maternal protein 2 (MATN2). In addition, matn2 alters the AKT/SMAD4 signalling pathway through its interaction with the NPCs receptor EGFR, leading to the decrease of SMAD4. SMAD complex further mediates the expression of downstream targets, thereby promoting neurogenesis and inhibiting gliogenesis. This study suggests that endothelial Arid1a tightly controls fate determination of NPCs by regulating the AKT‐SMAD signalling pathway.


| INTRODUCTION
Mammalian brain development is a complex process that depends on angiogenesis, neurogenesis and gliogenesis. [1][2][3] Blood vessels support brain function by delivering sufficient oxygen and nutrients. 4,5 The vascular system and the neural system processes occur simultaneously. [6][7][8] During embryonic E8.5 to E10.5, meningeal blood vessels gradually penetrate into the developing brain. 9 The periventricular vessels form a complex network within the telencephalon and gradually spread to the dorsal part of the telencephalon starting from E11.5. 10 During early embryonic development at E9.5-E11.5 (mouse), VZ is almost non-angiogenic. Periventricular vessels regulate progenitor cell behaviour, neurogenesis and gliogenesis. Subsequently, the development of blood vessels is accompanied by changes in gene expression and chromatin epigenetic inheritance. 11,12 The vascular system coordinates with the nervous system to maintain their homeostasis and participate in the fate of other cells around them. Radial glial progenitors (RGPs) expand the cell pool of the neural set mainly through symmetrical division at E9.5-11.5 (mouse). 13 Then, the RGP generates multiple types of neurons through asymmetric division, which migrate to the cerebral cortex. At E16.5-E17.5, RGP enters the gliogenesis stage and produces glial cells. The ordered mitotic behaviour of RGP is the basis of normal brain development. Due to the physical proximity of blood vessels to NPCs, NPCs located in ventricles and sub-ventricular regions (VZ/SVZ) communicate with blood vessels and receive signals that regulate cell fate determination. [14][15][16] PI3K/AKT signalling pathway plays a key role in cell proliferation, neuronal differentiation and glial cell differentiation during mammalian cortical development. [17][18][19] In this process, the vascular system form a homeostasis to guide neural development, for example, by secreting one or more cytokines to change the local microenvironment of NPCs. 20,21 Therefore, the maintenance of brain homeostasis is mainly achieved through the interaction and coordination between blood vessels and NPCs. In addition, cerebral vessels are also thought to play a role in regulating neuronal activity, glial cell differentiation and apoptotic cell phagocytosis. The development of blood vessels is essential for neurogenesis and gliogenesis. However, the molecular mechanism of cerebrovascular-NPCs interaction remains unclear.
Vascular homeostasis is also associated with chromatin epigenetic inheritance during development. At-rich interaction domain 1A(Arid1a), a non-catalytic subunit of the chromosomal remodelling complex, is one of the most commonly mutated genes in tumours and plays a critical role in enhancer mediated gene regulation and chromatin epigenetic inheritance. Arid1a relies on the energy provided by ATP hydrolysis to change the binding position of nucleosome, gene promoter, enhancer and other regions of DNA, remodel chromatin structure, then regulate the expression of genes closely related to multiple cell functions, such as cell replication, differentiation, proliferation and DNA repair. There is evidence showed that Arid1amediated gene regulation plays an important role in anti-tumour and damage regeneration. For example, inhibition of Arid1a epigenetic modification improves the anti-tumour activity of CD8T cells, 22 damages organ regeneration, and leads to organ disorders. 23,24 In addition, epigenetic factor Arid1a is essential for the establishment of chromatin spatial configuration, and is closely related to cell fate determination, pluripotency maintenance and body development. [25][26][27] Arid1a regulates the expression of downstream genes by altering the deposition of histone H3K27ac on its promoter/enhancer. 28,29 Studies have shown that Arid1a, as an epigenetic factor, is involved in vascular remodelling, indicating that Arid1a plays a crucial role in regulating vascular development. 30 Therefore, it is of great significance to study the function of Arid1a in embryonic cerebrovascular system. However, the underlying molecular mechanism remains unknown.
In this study, we found that the absence of Arid1a in blood vessels leads to changes in blood vessel density. Histone modifications alter the chromatin state of homeostasis genes. H3K27ac is a histone marker associated with fate determination. Absence of Arid1a leads to increased H3K27ac levels and induces cytokine secretion. Further analysis revealed that maternal protein 2 (Matn2), as a secretory factor, plays a crucial role in signal transduction between blood vessels and NPCs during embryonic brain development. MATN2 secreted by endothelial cells activates the downstream signalling cascade of PI3K by interacting with the neural progenitor receptor EGFR to regulate neurogenesis and gliogenesis during brain development. In summary, our study suggests that Arid1a alters the micro-environment around NPCs by secreting MATN2 which mediated PI3K/AKT signalling pathway NPCs, further leading to delayed neurogenesis and decreased gliogenesis.

| Animals
All experimental mice were conducted in accordance with the requirements and specifications of Laboratory Animal Center, Institute of Zoology, Chinese Academy of Sciences. Arid1a floxed (Arid1a fl/fl ) 26 mouse line was a gift from Hui Lijian Laboratory (Shanghai Institute of Biochemistry and Cell Biology). Tie2-Cre (TEK-Cre) mice were purchased from Shanghai model organism. To generate Arid1a cKO-Tie2 mice, Arid1a fl/fl mice were hybridized with Tie2-Cre mice. It was confirmed by tail genotype identification and PCR sequencing. All mice were housed at 22-25 C, with a 12-h light/dark cycle. They were provided adequate food and drinking water.
2.2 | Isolation and culture of primary mouse endothelial E15 embryonic brains were rapidly dissected and placed in PBS solution. The meninges are carefully removed from the embryonic brain.
The ventral and dorsal sides of the telencephalon were chopped into pieces with a medical blade, transferred into 1.5 mL EP tubes and digested with 1 mL papain at 37 C for 5 min. The sample was shaken regularly every 2 min until digestion is complete. After digestion, the sample was repeatedly blown and ground with a 200 μL pipette gun.
The single-cell suspension was filtered with a sterile 70-μm nylon mesh (Falcon). The filtered cells were collected and resuspended with 1-mL erythrocyte lysate at room temperature for 2 min. The lysate was terminated with DMEM (2%FBS) medium. The cells were collected and washed twice with DPBS (Gibco) and labelled by anti-

| Isolation and culture of primary mouse NPC
Minor modifications were made to the isolation and culture of primary mouse NPCs. E14 or E15 embryonic brains were rapidly dissected and placed in PBS solution. The meninges were carefully removed with tweezers and transferred into 1.5-mL tubes. Digest with 1-mL Papain at 37 C for 5 min. Shake every 2 min until digestion is complete. Then, the sample was repeatedly blown with a 200-μL pipette gun and the single-cell suspension was filtered with a sterile 70-μm nylon mesh (Falcon). Finally, primary neural precursor cells were cultured on 6-well or 24-well plates coated with laminin and poly (D-lysine).

| Three-dimensional reconstruction
Confocal imaging of 40 μm brain sections was performed on a Zeiss LSM880 microscope. The vessels were scanned from top to bottom at 0.83 μm intervals in the Z direction. Images were analysed using Imaris 9.0 software. Subsequently, Surface module in Imaris 9.0 software was used to measure the surface area of blood vessels, and Filament module was used to measure the volume of blood vessels. The results were used for vascular density analysis and three-dimensional (3D) reconstruction.

| RNA-sequencing and data analysis
Total RNA of E15 cortex was extracted from primary endothelial cells isolated by FACS using the RNAeasy Mini kit (QIAGEN). RNA quality was analysed by Agilent 2100 Bioanalyzer. The sequencing was performed by Anoroad using Illumina HiSeq 2500 platform. All sequencing data reported in this paper were submitted to NCBI's GEO with accession number GSE221176.

| Co-immunoprecipitation
The sample was cleaved in RIPA (Solarbio) with a 1% protease inhibitor cocktail and 1%PMSF. The supernatant was collected by centrifugation at 4 C and incubated overnight with 25-μL anti-Flag or anti-HA magnetic beads (MBL) at 4 C. After washing for three times and boiling for 10 min, the protein was analysed by western blotting.

| Co-culture
NPCs were laid on the coverslips coated with poly-D-Lysine and Laminin were placed in the bottom chamber of a transwell plate, endothelial cells were then seeded in the top chamber. The virus was added by equal volume after 24 h. Cells were cultured for 3 days and processed for immunocytochemistry as described earlier.

| Statistical analysis
All immuno-stained sections were imaged using Zeiss LSM880. During 3D vascular reconstruction, 40-μm brain sections were used and Imaris 9.0 software was used to generate images for statistical purposes. The Filament module in Imaris 9.0 software analyses the density of blood vessels. All statistical analyses were performed using GraphPad Prism software 6.0. Unpaired two-tailed Student's t-test (L) Quantification of the density of blood vessel showing a persistent disruption in Arid1a cKO-Tie2 brain cortex at E18, n = 4 each group. The data are represented as the mean ± SEM. Unpaired two-tailed Student's t-test, one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001.
was used between the two groups, and one-way ANOVAs were used for multiple comparisons. The results are expressed as mean ± SEM.

| RESULTS
3.1 | The developing blood vessels and neural progenitor cells are well-positioned to interact in vivo In the early stage of brain development, neural development and vascular development occur simultaneously. 6,7,31 Blood vessels act as niches and scaffolds for the migration of neurons during development.
The establishment of vascularization in the central nervous system (CNS) is essential to maintain the homeostasis of neural networks. 32 We first examined the developmental patterns of blood vessel and neural progenitor cells (NPCs) during the embryonic period (E13-P0), which is a critical period for the fate determination of NPCs. We first examined the position relationships of blood vessels and neural precursor cells at E13 embryo. The results shows that the vascular colonization was in the VZ/SVZ region and was very close to the physical location of SOX2 + neural stem cells ( Figure 1A,B). We also found that the physical positions of blood vessels and glial progenitor cells (GLAST and BLBP) at E16 and E18 were very close ( Figure 1C,D; Figure S1A). Similar phenomena appeared at P0 ( Figure S1B). These data suggest that developing blood vessels are closely related to neural precursor cells and may play a crucial role in regulating the fate determination of cortical NPCs. In addition, we assessed the expression of Arid1a in endothelial cells and found that Arid1a was highly expressed in endothelial cell ( Figure S1C). In order to further explore the expression of Arid1a during cerebrovascular development, we also performed immunostaining on E13.5 blood vessels. Results showed that Arid1a was highly expressed at E13.5, suggesting that Arid1a may play an important role in early brain development ( Figure 1E). In addition, epigenetic inheritance is related to vascular development.
Therefore, it will be important to determine whether Arid1a also plays a role in vascular development. To answer this question, we purified ECs by FACS and performed RT-PCR using Arid1a-specific primers.
The results showed that the expression of Arid1a gradually changed from day E13 to P2 ( Figure 1F), indicating that the expression of Arid1a in ECs are correlated with the fate determination of neural precursor cells during embryonic development.

| Loss of epigenetic Arid1a increases the density of blood vessels
To investigate whether the loss of Arid1a in endothelial alters vascular remodelling, we crossed the Tie2-Cre mouse lines with mice carrying the Arid1a conditional allele (Arid1a fl/fl ) to generate endothelial conditional knockout mice (Arid1a cKO-Tie2 ) ( Figure 1G). Arid1a cKO-Tie2 mice exhibit a specific deletion of endothelial cells Arid1a during cortical development. First, endothelial cells purified by FACS were cultured to identify the absence of Arid1a expression in the vessels of the mutated cerebral cortex ( Figure S1D). Western blotting results showed that Arid1a was specifically knocked out in blood vessels ( Figure S1E). In addition, RT-PCR analysis of endothelial cells confirmed a decrease in the abundance of Arid1a mRNA in the cerebral cortex of Arid1a cKO-Tie2 ( Figure 1H).
Next, we detected changes in vascular homeostasis at different stages of cortical neurogenesis and gliogenesis in Arid1a fl/fl and Ari-d1a cKO-Tie2 by immunostaining IB4. These results suggest that epigenetic Arid1a loss impairs vascular homeostasis at E16 and E18, at which point neurogenesis and gliogenesis have begun. The Arid1a cKO-Tie2 had significantly higher vascular density than Arid1a fl/fl at E16 (Figure 1I,J). In addition, the immunostaining results of E18 were similar ( Figure 1K,L). Taken together, these results suggest that epigenetic Arid1a loss increase cerebral vascular density and disrupt vascular homeostasis in developing cortex. In addition, immunofluorescence staining showed that endothelial Arid1a depletion did not affect perivascular recruitment and tight connections ( Figure S2A-D). We used the fluorescent tracer Alexa Fluor 555 cadaver amine (Cad-A555) to detect blood-brain integrity and found no significant blood-brain barrier leakage in the cortex of Arid1a cKO-Tie2 mice ( Figure S2E). These results suggest that the loss of endothelial Arid1a has no effect on the blood-brain barrier.

| Loss of endothelial cells Arid1a disrupts neurogenesis and gliogenesis during brain development
During brain development, the orderly generation of neurons and glial cells is essential for the structure and function of the cerebral cortex.
This process requires the micro-environment to precisely coordinate the fate of the NPCs. 33 Given the increased vascular density in the mutated cortex, we evaluated whether neuronal generation in the cerebral cortex was impaired when vascular homeostasis was dis-

| Loss of epigenetic
Arid1a leads to persistent decrease of astrocyte production and increase of neuron production during the developing cortex We examined long-term astrocyte production after birth. At P2, the number of GFAP + astrocytes in Arid1a cKO-Tie2 mice were significantly lower than in Arid1a fl/fl mice ( Figure 4A,B). In addition, we observed that the number of GS + and S100β + astrocytes in Ari-d1a cKO-Tie2 mice were also obviously lower than in Arid1a fl/fl mice ( Figure S4A-D).
At P8, we observed a decrease in the number of S100β + and GFAP + positive astrocytes ( Figure 4C-F). At P30, we detected a sustained decrease in the number of GFAP + positive astrocytes ( Figure 4G,H). These results showed a sustained decrease in astrocyte production in the cortex of endothelial Arid1a knockout mice.
On the other hand, we examined postnatal neurogenesis after birth. At P8, the number of NEUN + neurons in Arid1a cKO-Tie2 mice were significantly higher than in Arid1a fl/fl mice ( Figure S4E,F). These  Figure 5D). Down-regulated genes are involved in gliogenesis, phospholipid metabolism and fatty acid oxidation ( Figure 5E).
Next, we measured differences in gene expression in endothelial cells.
The maternal protein Matn2 gene was significantly increased, which was consistent with the volcanic map and RT-PCR results signalling pathways which regulate stem cell pluripotency in brain development ( Figure S5A).
In addition, key pathways involved in PI3K/AKT signalling induced changes in gene expression also play a number of key roles in the fate determination of NPCs. Therefore, NPCs of VZ/SVZ regions were isolated from the Arid1a fl/fl and Arid1a cKO-Tie2 cerebral cortex to detect whether AKT signalling was affected in the NPCs. Levels of proteins associated with signalling pathways were detected and showed increased phosphorylation of AKT and decreased binding partner SMAD4( Figure 6A,B) Figure S5C). In addition, we introduced Matn2-shRNA into an in vitro co-culture system of NPCs and endothelial cells. Primary endothelial cells were co-cultured with NPCs in vitro and treated with exogenous Matn2-shRNA virus for 3 days. Immunostaining showed that the proportion of MAP2 + neurons in the Arid1a cKO-Tie2 group decreased after Matn2-shRNA stimulation ( Figure 6D,E), which is similar to that in the  Figures 6F and S5D,E).

| DISCUSSION
The complexity of embryonic brain development not only involves the ability of NPC to generate neurons and glial cells, but also involves the interaction between multiple systems to ensure the precise regulation of tissue homeostasis function.  transduction. 51,52 The interaction between MATN2 and the EGFR receptor is critical for AKT phosphorylation. SMAD complex is a transcriptional activator that induces the expression of downstream target proteins. There is a variety of evidence supporting the interaction between MATN2 and EGFR to regulate BMP signalling pathways. 53 MATN2 interaction with EGFR leads to activation of PI3K/AKT signalling and reduced synthesis of co-binding partner SMAD4. SMAD complex acts as a transcriptional activator to induce downstream target expression and fate determination in NPCs. 38 Therefore, exploring vascular homeostasis during embryonic brain development will help to understand the balance between neurogenesis and gliogenesis.