Angiopoietin1 (Angpt1) is a secreted protein that activates the endothelial Tie2 receptor. Angpt1 plays a critical role in cardiac development and vascular remodeling in response to disease or injury and shows cell type-restricted expression in the lung, eye, and hematopoietic system. However, the expression of Angpt1 in the developing and adult brain is not known. Here, we employ Angpt1-GFP knock-in reporter mice and a systematic analysis of multiple single-cell RNA sequencing datasets to map the expression of Angpt1 during brain development and adulthood. In the developing brain, Angpt1 displays specific spatiotemporal patterns, with strong expression in cerebellar GABA interneuron progenitors and, to a lower level, in glial progenitor and astrocyte lineages. In the adult brain, on the other hand, we show that neurons are the main source of Angpt1 in the cerebrum, while in the cerebellum, expression is mostly restricted to astrocytes. Together, our data provide clarity on the cell types that express Angpt1 in the developing and adult brain and can be utilized to guide future studies, examining Angpt1 function in brain development, homeostasis, and pathological conditions.

Angiopoietin-1 (Angpt1) is a secreted protein that is part of the larger angiopoietin growth factor family. Angpt1 is a potent activator of Tie2 (also known as Tek), a receptor tyrosine kinase expressed by most endothelial cells. Angpt1-mediated Tie2 activation promotes vascular stability, playing important roles in both normal development and vascular remodeling in response to injury or disease [1, 2]. This is highlighted by embryonic lethality due to cardiac defects in germ line Angpt1 knockout mice [3] and accelerated fibrosis and angiogenesis in Angpt1 conditional knockout models following injury [2].

Despite a lack of overt vascular phenotypes when Angpt1 is globally deleted after embryonic day 13.5 [2], recent studies have identified more subtle regional effects of Angpt1 deficiency. Within the developing lung, pericyte-derived Angpt1 is necessary for proper alveologenesis by promoting hepatocyte growth factor expression in an autocrine manner [4]. Angpt1 is expressed by hematopoietic stem cells, c-kit+ hematopoietic progenitors, megakaryocytes, and leptin receptor+ stromal cells, where it regulates niche regeneration following irradiation by reducing vascular permeability [5]. In the eye, Angpt1 is expressed by mesenchymal cells of the trabecular meshwork (TM) and cells surrounding Schlemm’s canal (SC) within the cornea, and in subsets of neurons in the ganglion cell and inner nuclear layer of the retina [6, 7]. Angpt1 loss results in severe defects in SC formation, recapitulating the same phenotype seen in Tie2-deficient mice [7], and within the retina causes reduced vascular growth and pericyte coverage [8].

The expression of Angpt1 in the central nervous system remains poorly characterized. Prior studies have reported conflicting results, with Angpt1 expression being proposed in pericytes [9, 10], endothelial cells [11, 12], neurons and astrocytes in the adult brain [13]. In the present study, we perform a systematic examination of Angpt1 expression in the developing and mature mouse brain. By utilizing Angpt1-GFP reporter mice and single-cell RNA sequencing (scRNA-seq) datasets, we demonstrate that Angpt1 shows temporally and regionally restricted expression patterns. Angpt1 expression is largely restricted to neurons in the cerebrum and hypothalamus, while it is expressed by interneuron progenitors and astrocytes in the hindbrain. Together, our findings document the precise expression of Angpt1 in the developing and adult brain and provide a resource for future studies on Angpt1 function in brain homeostasis and pathological states.

Mouse Colony and Genotyping Information

All mouse work was done according to institutional and IACUC review boards (University of Cincinnati). Angpt1-GFP knock-in mice (Angpt1tm1.1Sjm/J; Jax stock #028924) were obtained from the Jackson laboratory and were originally deposited by Dr. Sean Morrison [5]. The Angpt1-GFP colony was maintained by breeding heterozygotes to wild-type mice, and all offspring were genotyped by PCR as previously described [5]. The following primers were used for PCR genotyping: OLD308: 5′-gggaaagagtcaaacaagcag-3′ OLD309: 5′-aaccgaaagcgatcacttac-3′ and OLD292: 5′-cggacacgctgaacttgtgg-3.

Tissue Collection and Immunostaining

Brains were collected and processed as previously described [14, 15]. In brief, following euthanasia, brains were rapidly dissected in ice-cold phosphate buffered saline (PBS) and then fixed in 4% paraformaldehyde overnight. Adult mice were perfused with PBS, followed by 4% paraformaldehyde before brains were removed. Fixed brains were washed in PBS and then incubated at 4°C in a 30% sucrose/PBS solution overnight before embedding in tissue freezing media. Fifty-micrometer-thick free-floating sections were generated on a cryostat (Lecia). Free-floating sections were stained in blocking solution (PBS + 0.5% Triton X-100 + 10% normal donkey serum) containing combinations of primary antibodies and incubated at 4°C overnight. The next day, sections were washed in PBS and then transferred to blocking solution containing the appropriate secondary antibodies (1:500) and incubated at 4°C overnight. Finally, sections were washed in PBS, followed by a 10 min incubation in Hoechst (1:1,000 in PBS) before the final PBS washes and then mounting onto slides (Superfrost; Fisher Scientific) and coverslipping (Prolong Gold Antifade; ThermoFisher). Primary antibodies used include GFP-Alexa-488 (#A-21311; ThermoFisher), Hoechst (ThermoFisher), Cd31 (#550274; BD Biosciences), Pdgfr-beta (#14-1402-82; ebioscience), Gfap (#12389S; Cell signaling), Pax3 (MIgG2A; DSHB), NeuN (#MAB377; Millipore), and reelin (#MAB5366; Millipore). Corresponding secondary antibodies used were all purchased from Jackson ImmunoResearch. Images were acquired on a confocal microscope (Nikon A1), and image analysis was performed in ImageJ (National Institutes of Health).

RNA Preparation and Quantitative PCR

RNA was collected as previously described [15]. In brief, the dissected murine brain tissue was collected and stored in RNAlater (#AM7020; Thermo Fisher) before RNA isolation was performed using the NucleoSpin Plus RNA kit (Macherey-Nagel) according to the manufacturer’s instructions. The SuperScript VILO cDNA Synthesis Kit (#11754050; ThermoFisher) was used to synthesize cDNA from each sample using 1 μg of total RNA. Quantitative PCR (qPCR) was performed on a CFX96 Real-Time System (BioRad, USA). Relative Angpt1 mRNA expression was calculated by the 2−ΔΔCT method and normalized to the expression of Gapdh between samples. qPCR primer sequences used in the study: Angpt1: 5′-CAC ATA GGG TGC AGC AAC CA-3′ (forward) and 5′-CGT CGT GTT CTG GAA GAA TGA-3′ (reverse); Gapdh: 5′-AGG TCG GTG TGA ACG GAT TTG-3′ (forward); 5′- TGT AGA CCA TGT AGT TGA GGT CA -3′(reverse).

Whole-Transcriptome at the Tissue Level

Angpt1 expression dynamics across the human lifespan (8 postconception weeks to 40 years of age) in different brain regions was investigated using RPKM gene expression values obtained from the BrainSpan project [16]. Angpt1 RPKM levels were extracted for all samples (N = 524) and for each brain region (N = 16), and expression values were smoothed using LOESS regression. Analysis of Angpt1 expression within adult human organs was obtained from the genotype-tissue expression (GTEx) portal, GTEx Analysis Release V8, on January 10, 2020.

Analysis of scRNA-seq Datasets

The following scRNA-seq datasets and their associated web-based computational tools were utilized to investigate Angpt1 expression: (a) scRNA-seq of cells isolated from the adult mouse cortex and hippocampal formation was generated on the 10x Genomics Chromium platform by the Allen Brain Institute as described in their associated publication [17]. The expression of Angpt1 was analyzed using their online tool found at https://celltypes.brain-map.org/rnaseq/mouse_ctx-hip_10x. (b) scRNA-seq of cells isolated from the postnatal and adult mouse dentate gyrus (DG) was generated on the Fluidigm C1 and 10x Genomics Chromium platforms as described by Hochgerner et al. [18]. The expression of Angpt1 was analyzed using their online tool found at http://linnarssonlab.org/dentate/. (c) scRNA-seq of cells isolated from the adult mouse hypothalamic arcuate-median eminence (Arc-ME) complex was generated by Drop-seq as described by Campbell et al. [19]. The expression of Angpt1 was analyzed using the Broad Institute Single-Cell Portal found at https://singlecell.broadinstitute.org/single_cell/study/SCP97/a-molecular-census-of-arcuate-hypothalamus-and-median-eminence-cell-types. (d) Single-nuclei RNA-seq from the murine adult cerebellum was generated as described by Kozareva et al. [20]. The expression of Angpt1 was analyzed using the Broad Institute Single-Cell Portal found at https://singlecell.broadinstitute.org/single_cell/study/SCP795/a-transcriptomic-atlas-of-the-mouse-cerebellum. (e) scRNA-seq of cells isolated from 9 different regions of the adult mouse brain was generated by Drop-seq as described by Saunders et al. [21]. The expression of Angpt1 was analyzed using their online tool found at http://dropviz.org/. (f) scRNA-seq of the mouse visual cortex [22]. Single cells were isolated by FACS, poly(A)-RNA from each cell was reverse transcribed (SMARTer), cDNA was amplified and fragmented and then sequenced on a next-generation sequencing platform. (g) scRNA-seq of cells isolated from the developing mouse cerebellum from embryonic day 10 to postnatal day 14 (P14) was generated by 10X Genomics Chromium as described by Vladoiu et al. [23]. Clusters of cells belonging to the cerebellar interneuron lineage (interneuron precursors, differentiating interneurons, and interneurons) were subset using Seurat [24] (version 2.3.4) and dimensionality reduction was performed using UMAP [25]. Pseudotime was inferred using the R package monocle2 [26] (version 2.10.1). The mean expression of Angpt1 and selected canonical lineage-specific markers was computed for each single-cell cluster. (g) scRNA-seq of cells isolated from the developing pons and forebrain (E12-P6) was generated by 10X Genomics Chromium as described by Jessa et al. [27]. First, glial populations (progenitors, oligodendrocyte precursors, oligodendrocytes, astrocytes, and ependymal cells) were subset and re-embedded using UMAP as in (f). Second, astrocyte clusters from both brain regions were subset and re-embedded, and the differential expression of Angpt1 was performed using the Wilcoxon rank-sum test. Third, Cajal-Retzius neuron clusters from the forebrain were subset. For each analysis, the mean expression of Angpt1 and other selected genes was computed for each single-cell cluster.

Dynamic Expression of Angpt1 during Brain Development

To gain insights into Angpt1 expression, we first examined the BrainSpan transcriptional atlas [16], which contains data for multiple human brain regions spanning development to adulthood. Angpt1 expression displayed a unique peak in the developing cerebellum, which maintained the highest overall expression compared to other brain regions (shown in Fig. 1a). Relative to tissues previously identified to express Angpt1, such as the lung [4] and heart [3], adult human brain samples display low Angpt1 transcript levels, as detected in the GTEx whole-transcriptome dataset which contains samples from major human organs, including multiple brain regions (shown in Fig. 1b). To determine if this was consistent in the mouse brain, we performed qPCR for Angpt1 from regionally dissected mouse brain samples at postnatal (P0, P7, P14, and P21) and adult (6–7 weeks) ages. Across each brain regions (cortex, cerebellum, and brainstem), a general pattern emerged, with Angpt1 expression peaking by postnatal day 7, and gradually decreasing with age (shown in Fig. 1c). This pattern was most distinct in the cerebellum, which exhibited the greatest increase in Angpt1 expression at P7 relative to the cortex or brainstem regions (shown in online suppl. Fig. 1; for all online suppl. material, see www.karger.com/doi/10.1159/000518351). Our data show that the expression of Angpt1 in the brain varies by age and brain region, and that the mouse brain recapitulates the dynamic expression patterns found in the developing human brain.

Fig. 1.

Dynamic expression of Angpt1 during brain development. a Expression of Angpt1 across human brain development in the BrainSpan dataset, in cerebellar regions, and all other brain regions. The x-axis represents log (postconception weeks), and x-axis labels denote age in postconception weeks prenatally, and years postnatally. Vertical line represents birth. bAngpt1 expression across defined human organ samples in the GTEx dataset. c Expression of Angpt1 in the mouse cortex, brainstem, and cerebellum samples at noted postnatal time points. n = 3 samples/brain region, 3 independent experiments. Data are represented as mean with SD. *p < 0.05, **p < 0.001, ***p < 0.0001, the unpaired test with post hoc Tukey’s, comparing each time point to P0. Angpt1, angiopoietin1; CTX, cortex; BS, brainstem; CB, cerebellum, P0, postnatal day 0.

Fig. 1.

Dynamic expression of Angpt1 during brain development. a Expression of Angpt1 across human brain development in the BrainSpan dataset, in cerebellar regions, and all other brain regions. The x-axis represents log (postconception weeks), and x-axis labels denote age in postconception weeks prenatally, and years postnatally. Vertical line represents birth. bAngpt1 expression across defined human organ samples in the GTEx dataset. c Expression of Angpt1 in the mouse cortex, brainstem, and cerebellum samples at noted postnatal time points. n = 3 samples/brain region, 3 independent experiments. Data are represented as mean with SD. *p < 0.05, **p < 0.001, ***p < 0.0001, the unpaired test with post hoc Tukey’s, comparing each time point to P0. Angpt1, angiopoietin1; CTX, cortex; BS, brainstem; CB, cerebellum, P0, postnatal day 0.

Close modal

Angpt1 Is Transiently Expressed by GABA Interneuron Progenitors during Cerebellar Development

Our analysis of the human and mouse brains identified a peak of Angpt1 expression in the developing cerebellum. In order to define the location and identity of cell types that express Angpt1 in the cerebellum, we utilized previously generated Angpt1-GFP knock-in reporter mice, which express GFP from the endogenous Angpt1 locus [5] and an scRNA-seq dataset that spans cerebellar formation [23]. At P0, GFP-positive cells were present in the ventricular zone and adjacent dorsal regions of the developing cerebellum (shown in Fig. 2a). At P7, we found GFP-positive cells were restricted to the developing white matter tract of the cerebellum (shown in Fig. 2a). This population of GFP-positive cells decreased with age, with significantly fewer GFP-positive cells present in the white matter at P21 and adulthood (shown in Fig. 2a). The developing cerebellum harbors different proliferative progenitor populations, including granule neuron precursor cells in the external granule layer [28] and GABA interneuron progenitors derived from the embryonic ventricular zone [29, 30]. The location of Angpt1-GFP-positive cells in developing white matter tracts match the temporal and spatial pattern of GABA interneuron progenitor development, which migrate through the white matter before differentiating into Golgi and stellate/basket cells located in the internal granule layer and molecular layer (ML), respectively [31]. To reveal the identity of Angpt1-expressing cells, we utilized an scRNA-seq dataset that spans murine cerebellar development from embryonic day 10 to P14 [32]. We found that Angpt1 expression is enriched in cerebellar GABA interneuron lineages (shown in online suppl. Fig. 2a, b), consistent with our observation of GFP-positive cells in the cerebellar white matter tract during postnatal development. Pseudotime reconstruction and UMAP embedding of cells in the GABA interneuron lineage defined their developmental trajectory from proliferating Pax3-positive GABA interneuron progenitors to fully differentiated Pax2-positive GABA interneurons (shown in Fig. 2b, c). Angpt1 is expressed in Pax3-positive GABA interneuron progenitors and differentiating GABA interneurons but not in Pax2-positive differentiated GABA interneurons (shown in Fig. 2c). Co-labeling P7 Angpt1-GFP sections with a Pax3 antibody confirmed GFP-positive cells within this pool of GABA interneuron progenitor cells (shown in Fig. 2d).

Fig. 2.

Angpt1 is transiently expressed by GABA interneuron progenitors during cerebellar development. a Representative 10× (top) and corresponding 40× (bottom) magnification images of Angpt1-GFP brain sections stained with anti-GFP and Hoechst. b UMAP embedding of GABA interneuron progenitors and interneurons from the murine cerebellum (E10-P14), cells colored by cell type (top) and by pseudotime inferred using monocle (bottom). c Heat map of the mean expression of Angpt1, Top2a, Pax3, and Pax2 in each cluster. The expression is z-scored across columns. d Immunostaining for Pax3 and Angpt1-GFP in the P7 cerebellum. Scale bars, 20 µm (top panels), 10 µm (bottom panels). Angpt1, angiopoietin1; E10, embryonic day 10; P14, postnatal day 14.

Fig. 2.

Angpt1 is transiently expressed by GABA interneuron progenitors during cerebellar development. a Representative 10× (top) and corresponding 40× (bottom) magnification images of Angpt1-GFP brain sections stained with anti-GFP and Hoechst. b UMAP embedding of GABA interneuron progenitors and interneurons from the murine cerebellum (E10-P14), cells colored by cell type (top) and by pseudotime inferred using monocle (bottom). c Heat map of the mean expression of Angpt1, Top2a, Pax3, and Pax2 in each cluster. The expression is z-scored across columns. d Immunostaining for Pax3 and Angpt1-GFP in the P7 cerebellum. Scale bars, 20 µm (top panels), 10 µm (bottom panels). Angpt1, angiopoietin1; E10, embryonic day 10; P14, postnatal day 14.

Close modal

Angpt1 Is Expressed by Glial Progenitors and Subsets of Astrocytes

In the developing cerebellum, we also noted that Angpt1 expression is enriched in astrocyte/Bergmann glia precursors and differentiating astrocytes (shown in online suppl. Fig. 2a, b). We found similar enrichment of Angpt1 in glial progenitors and astrocytes in a developing pons/hindbrain scRNA-seq dataset [27]. UMAP embedding of glial populations from the E12-P6 murine pons/hindbrain defined cell type clusters progressing along developmental lineages related to astrocyte, oligodendrocyte, and ependymal cellular programs (shown in Fig. 3a). Angpt1 is expressed by glial progenitor (glial progenitors 2), astrocyte (astrocytes 1, 2 and 3), and ependymal (ependymal 1) cell type clusters but absent in oligodendrocyte (newly forming oligo and myelinating oligo) related lineages (shown in Fig. 3b). While Angpt1 is also detected in glial progenitors and astrocytes of the developing forebrain (shown in online suppl. Fig. 3a, b), further analysis in the adult brain suggests there may be regional differences in expression. Following the differentiation of Pax3-positive GABA interneuron progenitors by P14, we noted that a population of Angpt1-GFP-positive cells persisted in the cerebellar white matter into adulthood (shown in online suppl. Fig. 3c). GFP-positive cells displayed a fibrous astrocyte-like morphology, which was verified by co-labeling with the astrocyte marker Gfap (shown in Fig. 3c). Analysis of an adult murine cerebellum scRNA-seq dataset [20] confirmed cerebellar astrocytes are the major source of Angpt1 expression in the mature cerebellum (shown in Fig. 3d). Unlike the cerebellum, scRNA-seq datasets from other adult brain regions, including the visual cortex [22] and hypothalamus [19], revealed that astrocytes in these regions were not major contributors of Angpt1 expression (shown in online suppl. Fig. 3d, e). Astrocytes encompass a heterogenous population of cells, displaying local and regional heterogeneity [33, 34]. In a previous study [34] on purified astrocytes from adult olfactory bulb, cortex, hippocampus, and brainstem, Angpt1 was among the significantly differentially expressed genes when comparing the cortex to all other regions (−1.68 log2FC; adj. p value = 0.0065). Furthermore, cerebellar astrocytes (non-Bergmann glia) ranked as the highest Angpt1-expressing astrocytes when examining astrocyte cell type clusters from an scRNA-seq dataset that sampled across 9 different adult mouse brain regions [21] (shown in online suppl. Fig. 3f).

Fig. 3.

Angpt1 is expressed by glial progenitors and subsets of astrocytes. a UMAP embedding of glial populations from the murine pons (E12-P6), cells colored by cell type. b Heat map of the mean expression of Angpt1, Top2a, and different glial markers in each cluster. The expression is z-scored across columns. c Representative immunofluorescent images of GFP and Gfap staining in the Angpt1-GFP mouse cerebellum at P7, P14, P21, and 6–7 weeks. Scale bars, 20 µm (top panels), 10 µm (bottom panels). d Dot plot of Angpt1 and Gfap expression in scRNA-seq data of the adult cerebellum [20]. Dot size encodes the proportion of cells in each cluster where the gene is detected, and dot color encodes the scaled expression of the gene. Scale bars, 20 µm. Angpt1, angiopoietin1; scRNA-seq, single-cell RNA sequencing; P14, postnatal day 14.

Fig. 3.

Angpt1 is expressed by glial progenitors and subsets of astrocytes. a UMAP embedding of glial populations from the murine pons (E12-P6), cells colored by cell type. b Heat map of the mean expression of Angpt1, Top2a, and different glial markers in each cluster. The expression is z-scored across columns. c Representative immunofluorescent images of GFP and Gfap staining in the Angpt1-GFP mouse cerebellum at P7, P14, P21, and 6–7 weeks. Scale bars, 20 µm (top panels), 10 µm (bottom panels). d Dot plot of Angpt1 and Gfap expression in scRNA-seq data of the adult cerebellum [20]. Dot size encodes the proportion of cells in each cluster where the gene is detected, and dot color encodes the scaled expression of the gene. Scale bars, 20 µm. Angpt1, angiopoietin1; scRNA-seq, single-cell RNA sequencing; P14, postnatal day 14.

Close modal

Neurons Are the Major Source of Angpt1 Expression in the Cerebrum

In the cerebrum, Angpt1-GFP-positive cells were found within specific regions, with some areas showing changes during maturation. The entorhinal cortex (ENT), which is a major input and output of the hippocampal formation [35], displayed strong GFP expression in clusters of cells (shown in Fig. 4a). In layer VI of the dorsal cortex, GFP-positive cells with neuronal morphologies sat adjacent to the corpus callosum and were visible from P7 to adulthood (shown in Fig. 4b). Within the hippocampus, the GFP expression was arranged in two main areas of the DG. First, GFP-positive cells were distributed around the outer ML of the DG in all ages examined (shown in Fig. 4c). Second, the subgranular (SG) layer displayed a diffuse GFP pattern at early postnatal time points, which became more restricted to areas of the SG zone (SGZ) at later postnatal and adult time points (shown in Fig. 4c). We also identified a transient population of GFP-positive cells at the pial surface of the cortex that were present at P0 but not found at subsequent ages (shown in Fig. 4d). Of note, we did not find Angpt1-GFP-positive endothelial cells or vascular associated pericytes (shown in Fig. 4e), suggesting that these cell types are not a significant source of Angpt1 in the brain. No GFP was detected in wild-type Angpt1-GFP reporter littermates which were used as controls.

Fig. 4.

Angpt1 expression maps to distinct regions of the cerebrum. Representative immunostaining images of GFP and Hoechst staining in the Angpt1-GFP mouse brain sections at P0, P7, P14, P21, and 6–7 weeks. a Images of GFP-positive cells in layer II/III of the ENT region. b Images of the dorsal cortex showing GFP-positive cells in layer VI. Dashed lines demarcate the CC. c Immunofluorescent images showing GFP-positive cells in the hippocampal DG. d Images showing GFP-positive cells at the cortical-pial interface at P0 but absent at P14. Arrowheads denote Angpt1-GFP-positive cells in each panel. e Immunostaining for CD31, Pdgfrb, and Angpt1-GFP in the cerebrum, hippocampus, and hypothalamus at P14. Scale bars, 20 µm. CC, corpus callosum; LV, lateral ventricle; Angpt1, angiopoietin1; ML, molecular layer; DG, dentate gyrus; SG, subgranular; P14, postnatal day 14; ENT, entorhinal cortex.

Fig. 4.

Angpt1 expression maps to distinct regions of the cerebrum. Representative immunostaining images of GFP and Hoechst staining in the Angpt1-GFP mouse brain sections at P0, P7, P14, P21, and 6–7 weeks. a Images of GFP-positive cells in layer II/III of the ENT region. b Images of the dorsal cortex showing GFP-positive cells in layer VI. Dashed lines demarcate the CC. c Immunofluorescent images showing GFP-positive cells in the hippocampal DG. d Images showing GFP-positive cells at the cortical-pial interface at P0 but absent at P14. Arrowheads denote Angpt1-GFP-positive cells in each panel. e Immunostaining for CD31, Pdgfrb, and Angpt1-GFP in the cerebrum, hippocampus, and hypothalamus at P14. Scale bars, 20 µm. CC, corpus callosum; LV, lateral ventricle; Angpt1, angiopoietin1; ML, molecular layer; DG, dentate gyrus; SG, subgranular; P14, postnatal day 14; ENT, entorhinal cortex.

Close modal

Next, we utilized available scRNA-seq datasets that include cells isolated from the adult mouse isocortex and hippocampal formation [17], the developing postnatal and adult hippocampus DG [18], and developing forebrain [27], to validate and further define the cellular identities of Angpt1-GFP-positive cells. In the adult isocortex and hippocampal formation dataset [17], cell clusters associated with layer II/III neurons in the medial and lateral ENT displayed the highest levels of Angpt1 expression (shown in Fig. 5a). These data independently mirrored our finding of Angpt1-GFP-positive cells in this region, which we verified as neurons by co-immunolabeling with the neuronal marker NeuN (shown in Fig. 5d). Within this dataset, additional cell type clusters expressing Angpt1 included Car3 Layer VI neurons (L6 Car3 and L6 IT/CT CTX clusters), subclusters of GABAergic vasoactive intestinal peptide-expressing interneurons and vascular smooth muscle cells (SMCs) (shown in Fig. 5a). A separate scRNA-seq dataset that spans the postnatal and adult hippocampal DG [18] revealed strong expression of Angpt1, specifically in Cajal-Retzius neurons (shown in Fig. 5b). Since Cajal-Retzius neurons in the cortex/forebrain are a transient cell population born during embryonic development and mostly eliminated shortly after birth [36], we analyzed Angpt1 expression within Cajal-Retzius neuron clusters in a forebrain scRNA-seq dataset spanning E12.5 to P6 [27]. Interestingly, strong expression of Angpt1 was detected in P0 Cajal-Retzius cells but not in Cajal-Retzius cells at either E12.5 or E15.5 (shown in Fig. 5c). Together, these data aligned with our observation of a transient GFP-positive cell present at P0 near the cortical pial surface, in addition to the sustained presence of Angpt1-GFP-positive cells in the DG ML, where Cajal-Retzius cells continually reside. To validate the identity of these cells, we immunolabeled P0 and P14 Angpt1-GFP sections with reelin, a marker of Cajal-Retzius cells. GFP-positive cells at the cortical pial surface and DG ML were reelin-positive, cross-validating the cellular identity of these cells (shown in Fig. 5d, f). Taken together, the identity of Angpt1-expressing cells detected in scRNA-seq datasets was concordant with expression patterns detected in the brains of Angpt1-GFP reporter mice, revealing that distinct subsets of neurons are the main source of Angpt1 expression in the cerebrum.

Fig. 5.

Neurons are the major source of Angpt1 expression in the cerebrum. a Heat map of Angpt1 expression in cell clusters from scRNA-seq of the adult cortex and hippocampus, ordered from high to low expression [17]. bAngpt1 expression within cell types identified in scRNA-seq data from the developing and adult DG [18]. c Mean expression of Angpt1 in Cajal-Retzius neurons detected at E12, E15, and P0 in the mouse forebrain [27]. d Co-labeling of Angpt1-GFP-positive cells with NeuN in layer 2/3 of the ENT. e Co-labeling of Angpt1-GFP-positive cells with reelin in the DG at P14. f Co-labeling of Angpt1-GFP-positive cells with reelin in the dorsal cortex at P0. Scale bars, 20 µm. CTX, cortex; Astro, astrocyte; CR, Cajal-Retzius; IT, intratelencephalic; Angpt1, angiopoietin1; scRNA-seq, single-cell RNA sequencing; ML, molecular layer; ENT, entorhinal cortex; DG, dentate gyrus; SG, subgranular; SMC, smooth muscle cell; P14, postnatal day 14.

Fig. 5.

Neurons are the major source of Angpt1 expression in the cerebrum. a Heat map of Angpt1 expression in cell clusters from scRNA-seq of the adult cortex and hippocampus, ordered from high to low expression [17]. bAngpt1 expression within cell types identified in scRNA-seq data from the developing and adult DG [18]. c Mean expression of Angpt1 in Cajal-Retzius neurons detected at E12, E15, and P0 in the mouse forebrain [27]. d Co-labeling of Angpt1-GFP-positive cells with NeuN in layer 2/3 of the ENT. e Co-labeling of Angpt1-GFP-positive cells with reelin in the DG at P14. f Co-labeling of Angpt1-GFP-positive cells with reelin in the dorsal cortex at P0. Scale bars, 20 µm. CTX, cortex; Astro, astrocyte; CR, Cajal-Retzius; IT, intratelencephalic; Angpt1, angiopoietin1; scRNA-seq, single-cell RNA sequencing; ML, molecular layer; ENT, entorhinal cortex; DG, dentate gyrus; SG, subgranular; SMC, smooth muscle cell; P14, postnatal day 14.

Close modal

Angpt1 Is Expressed by Populations of Hypothalamic Neurons in the Arc-ME Complex

We identified Angpt1-GFP-positive cells in the hypothalamus, including in the Arc-ME complex, starting at early postnatal ages and persisting into adulthood. In the anterior hypothalamus, GFP-positive cells with neuronal morphologies were observed in dorsal areas adjacent to the third ventricle (shown in Fig. 6a) and in ventral regions neighboring the pial surface (shown in Fig. 6b). In more posterior sections, GFP-positive cells aligned along each side of the ME (shown in Fig. 6c), where different populations of Arc neurons reside. Leveraging a scRNA-seq dataset generated from the Arc-ME tissue [19], we found that a specific subset of pro-opiomelanocortin neurons defined by their expression of Glipr1 displayed the highest level of Angpt1 (shown in Fig. 6d, e). Pro-opiomelanocortin neurons are critical regulators of metabolism and reproduction, and project to multiple brain regions, including the ME [37]. Angpt1 expression was also detected at lower levels in a small proportion of Arc neurons defined by the expression of Htr3b, as well as in non-neural cells from tissues adjacent to the Arc-ME, namely pituitary cells from pars tuberalis (shown in Fig. 6d, e). Whether Angpt1 expressed by these hypothalamic Arc neurons participate in regulating metabolic or hormone responses, either directly or indirectly through modulating vascular dynamics, will be of interest in future studies.

Fig. 6.

Hypothalamic neurons in the Arc-ME complex express Angpt1. a, b Images of Angpt1-GFP-positive cells in the anterior hypothalamus at P0 and 6–7 weeks. c Images of GFP-positive cells in the Arc-ME area at P7 and 6–7 weeks. d tSNE plot of scRNA-seq data from the adult hypothalamic Arc-ME region [19], colored by Angpt1 expression. e Top: insets depict regions of the tSNE plot indicated (d). Bottom: violin plot of Angpt1 expression across cell type clusters. Scale bars, 20 µm. Angpt1, angiopoietin1; scRNA-seq, single-cell RNA sequencing; Arc-ME, arcuate-median eminence.

Fig. 6.

Hypothalamic neurons in the Arc-ME complex express Angpt1. a, b Images of Angpt1-GFP-positive cells in the anterior hypothalamus at P0 and 6–7 weeks. c Images of GFP-positive cells in the Arc-ME area at P7 and 6–7 weeks. d tSNE plot of scRNA-seq data from the adult hypothalamic Arc-ME region [19], colored by Angpt1 expression. e Top: insets depict regions of the tSNE plot indicated (d). Bottom: violin plot of Angpt1 expression across cell type clusters. Scale bars, 20 µm. Angpt1, angiopoietin1; scRNA-seq, single-cell RNA sequencing; Arc-ME, arcuate-median eminence.

Close modal

Our findings define Angpt1 expression patterns within the brain and demonstrate regional and temporal regulation across different cell populations. We show that in the cerebellum, Angpt1 displayed a distinct expression pattern, with high expression in dividing GABA interneuron progenitors during development, and astrocytic expression at later postnatal and adult ages. In the cortex, hippocampus and hypothalamus neurons are the major source of Angpt1 expression. By using available scRNA-seq datasets of cells isolated from developing and adult brain regions, we were able to confirm our results and further define the specific cell types that express Angpt1.

Angpt1 promotes vascular stability through its ability to activate the endothelial Tie2 receptor [38]. Previous studies have characterized Angpt1 expression outside of the central nervous system, showing that fibroblasts and pericytes are frequent sources of expression [4, 5]. While our data demonstrate brain pericytes do not express Angpt1, expression by other cell types, including populations of astrocytes, neurons, and vascular SMCs we identified could still participate in regulating aspects of vascularization and homeostasis.

Surveying astrocyte populations across the brain, we find the highest Angpt1 expression in a subset of cerebellar astrocytes corresponding to clusters that are in part transcriptionally defined by their expression of Gfap and myocilin [21, 39]. Interestingly, myocilin and Angpt1 are also co-expressed by mesenchymal cells that make up the TM [7], a specialized eye tissue derived from the periocular mesenchyme that is essential for the regulation of intraocular pressure [40, 41]. Loss of Angpt1 results in a specific defect in the development of SC [7], a specialized hybrid vessel with lymphatic and venous features that develops next to the TM in the iridocorneal angle [41, 42]. While there are no lymphatics inside the cerebellum, Angpt1 expression by these astrocytes could be involved in promoting vascular sprouting or remodeling, or in specifying venous identity within the cerebellar white matter where they reside. Indeed, prior work in the heart has suggested Angpt1 promotes coronary vein formation [43], and Tie2 deletion in embryos disrupts specification and maintenance of venous identity [44].

Our findings indicate that within the cerebrum, subsets of neurons are the main source of Angpt1 expression. Specific populations of neurons expressed high levels of Angpt1, including Cajal-Retzius cells in the hippocampus and developing cortex, and subsets of specialized neurons in the ENT and hypothalamus/Arc-ME. Neuronal Angpt1 expression could participate in conventional neurovascular coupling, or in the case of Arc-ME neurons, Angpt1 may help establish a boundary between permeable fenestrated blood vessels in the median eminence and neighboring non-permeable vasculature in the hypothalamus. Angpt1 may also participate in non-traditional mechanisms of neurovascular coupling. Relative to non-permeable brain capillaries, arteriolar endothelial cells contain abundant caveolae, which they use to relay signals to adjacent SMCs to regulate vasoconstriction and dilation [45]. Angpt1 expression by SMCs in the brain could participate in crosstalk between these cell types, providing stabilizing signals during mechanical stress associated with changes in the vessel diameter and blood flow.

While examining the granular zone/sub-granular zone (GZ/SGZ) of the hippocampus, we also noted diffuse Angpt1-GFP expression. Besides Cajal-Retzius cells, no other cell types display substantial Angpt1 expression within hippocampal scRNA-seq datasets. Moreover, Angpt1-GFP expression did not appear to be within GZ/SGZ cell bodies, suggesting the expression could be originating from another source. The DG does receive inputs from the ENT [46], whose neurons we identify as a major source of Angpt1 expression. Thus, Angpt1-GFP signal in the GZ/SGZ may originate from innervating ENT neurons. Neurogenesis in the GZ/SGZ can be influenced by factors that induce angiogenesis or by changes in the blood flow that are linked to neurovascular coupling [47]. We hypothesize that Angpt1 secreted from ENT neurons could participate in this relationship by acting as a vascular stabilizing factor to balance angiogenic or hemodynamic changes induced by neuronal activity.

We find the strongest expression of Angpt1 in cerebellar GABA interneuron progenitor cells during early postnatal development. This apex of Angpt1 expression coincides with rapid postnatal growth of the cerebellum. While little is known about cerebellar vascular development, one recent study showed low vascularization and elevated levels of hypoxia at early postnatal ages [48]. Hypoxia peaked within the cerebellum at postnatal day 9, and its relief corresponds with increased vascularization, which more than doubled between postnatal day 5 and 15 [48]. High levels of Angpt1 may be important to coordinate this rapid vascular growth, as it could help promote vascular sprouting and stabilization during cerebellar growth and maturation.

Beyond a role in cerebellar vascularization, Angpt1 could also participate in the developmental trajectory of interneuron progenitors, which proliferate and migrate tangentially throughout the presumptive cerebellar white matter before exiting to differentiate in the internal granule layer and ML [31]. Independent of Tie2, Angpt1 can directly interact with integrin receptors to promote cellular migration [8]. Determining if Angpt1 plays a role in regulating the proliferation, migration, or differentiation of these cell types through a Tie2-independent mechanism, and/or coordinates cerebellar vascular development by activating Tie2 in neighboring endothelial cells, will be of future interest.

Our data define the pattern and cellular identity of Angpt1-expressing cells in the developing and mature brain by pairing analysis of Angpt1-GFP reporter brains with scRNA sequencing datasets. This includes expression in populations of neurons in the cerebrum and hypothalamus, glial progenitors and astrocytes, and GABA interneuron progenitors in the cerebellum. These findings provide a detailed map of Angpt1 expression in the developing and adult brain and will prove useful for future studies examining the function of Angpt1 in brain development, homeostasis and pathological conditions.

All mouse work was done according to institutional and IACUC review boards (University of Cincinnati).

The authors have no competing interests to declare.

This work was supported by the Pediatric Brain Tumor Foundation Early Career Development Award and start-up funds provided by the University of Cincinnati and Cincinnati Children’s Hospital Medical Center (T.N.P.).

X.W. conducted experiments and contributed to data analysis and writing of the manuscript. S.J. performed data analyses for scRNA-seq datasets and contributed to writing and editing of the manuscript. C.L.K. supervised S.J. and contributed to writing and editing of the manuscript. T.N.P. conceived the project and supervised X.W., assisted with planning of experiments and cowriting of the manuscript.

Publicly available datasets were used in this study. Each dataset cited is deposited in GEO, and can be found at the following accession numbers: [17] GSE115746; [18] GSE95752 and GSE95315; [19] GSE90806; [20] GSE165371; [21] GSE116470; [22] GSE71585; [23] GSE118068; [24] GSE110513; [27] GSE133531.

1.
Suri
C
,
Jones
PF
,
Patan
S
,
Bartunkova
S
,
Maisonpierre
PC
,
Davis
S
,
.
Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis
.
Cell
.
1996 Dec 27
;
87
(
7
):
1171
80
.
2.
Jeansson
M
,
Gawlik
A
,
Anderson
G
,
Li
C
,
Kerjaschki
D
,
Henkelman
M
,
.
Angiopoietin-1 is essential in mouse vasculature during development and in response to injury
.
J Clin Invest
.
2011 Jun
;
121
(
6
):
2278
89
.
3.
Kim
KH
,
Nakaoka
Y
,
Augustin
HG
,
Koh
GY
.
Myocardial angiopoietin-1 controls atrial chamber morphogenesis by spatiotemporal degradation of cardiac jelly
.
Cell Rep
.
2018 May 22
;
23
(
8
):
2455
66
.
4.
Kato
K
,
Diéguez-Hurtado
R
,
Park
DY
,
Hong
SP
,
Kato-Azuma
S
,
Adams
S
,
.
Pulmonary pericytes regulate lung morphogenesis
.
Nat Commun
.
2018 Jun 22
;
9
(
1
):
2448
.
5.
Zhou
BO
,
Ding
L
,
Morrison
SJ
.
Hematopoietic stem and progenitor cells regulate the regeneration of their niche by secreting angiopoietin-1
.
Elife
.
2015 Mar 30
;
4
:
e05521
.
6.
Park
DY
,
Lee
J
,
Kim
J
,
Kim
K
,
Hong
S
,
Han
S
,
.
Plastic roles of pericytes in the blood-retinal barrier
.
Nat Commun
.
2017 May 16
;
8
:
15296
.
7.
Thomson
BR
,
Souma
T
,
Tompson
SW
,
Onay
T
,
Kizhatil
K
,
Siggs
OM
,
.
Angiopoietin-1 is required for Schlemm’s canal development in mice and humans
.
J Clin Invest
.
2017 Dec 1
;
127
(
12
):
4421
36
.
8.
Lee
J
,
Kim
KE
,
Choi
DK
,
Jang
JY
,
Jung
JJ
,
Kiyonari
H
,
.
Angiopoietin-1 guides directional angiogenesis through integrin αvβ5 signaling for recovery of ischemic retinopathy
.
Sci Transl Med
.
2013 Sep 18
;
5
(
203
):
203ra127
.
9.
Hori
S
,
Ohtsuki
S
,
Hosoya
K
,
Nakashima
E
,
Terasaki
T
.
A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro
.
J Neurochem
.
2004 Apr
;
89
(
2
):
503
13
.
10.
Teichert
M
,
Milde
L
,
Holm
A
,
Stanicek
L
,
Gengenbacher
N
,
Savant
S
,
.
Pericyte-expressed Tie2 controls angiogenesis and vessel maturation
.
Nat Commun
.
2017 Jul 18
;
8
:
16106
.
11.
Stratmann
A
,
Risau
W
,
Plate
KH
.
Cell type-specific expression of angiopoietin-1 and angiopoietin-2 suggests a role in glioblastoma angiogenesis
.
Am J Pathol
.
1998 Nov
;
153
(
5
):
1459
66
.
12.
Sun
J
,
Yu
L
,
Huang
S
,
Lai
X
,
Milner
R
,
Li
L
.
Vascular expression of angiopoietin1, α5β1 integrin and tight junction proteins is tightly regulated during vascular remodeling in the post-ischemic brain
.
Neuroscience
.
2017 Oct 24
;
362
:
248
56
.
13.
Beck
H
,
Acker
T
,
Wiessner
C
,
Allegrini
PR
,
Plate
KH
.
Expression of angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery occlusion in the rat
.
Am J Pathol
.
2000 Nov
;
157
(
5
):
1473
83
.
14.
Phoenix
TN
,
Patmore
DM
,
Boop
S
,
Boulos
N
,
Jacus
MO
,
Patel
YT
,
.
Medulloblastoma genotype dictates blood brain barrier phenotype
.
Cancer Cell
.
2016 Apr 11
;
29
(
4
):
508
22
.
15.
Patel
SK
,
Hartley
RM
,
Wei
X
,
Furnish
R
,
Escobar-Riquelme
F
,
Bear
H
,
.
Generation of diffuse intrinsic pontine glioma mouse models by brainstem-targeted in utero electroporation
.
Neuro Oncol
.
2020 Mar 5
;
22
(
3
):
381
92
.
16.
Miller
JA
,
Ding
SL
,
Sunkin
SM
,
Smith
KA
,
Ng
L
,
Szafer
A
,
.
Transcriptional landscape of the prenatal human brain
.
Nature
.
2014 Apr 10
;
508
(
7495
):
199
206
.
17.
Tasic
B
,
Yao
Z
,
Graybuck
LT
,
Smith
KA
,
Nguyen
TN
,
Bertagnolli
D
,
.
Shared and distinct transcriptomic cell types across neocortical areas
.
Nature
.
2018 Nov
;
563
(
7729
):
72
8
.
18.
Hochgerner
H
,
Zeisel
A
,
Lönnerberg
P
,
Linnarsson
S
.
Conserved properties of dentate gyrus neurogenesis across postnatal development revealed by single-cell RNA sequencing
.
Nat Neurosci
.
2018 Feb
;
21
(
2
):
290
9
.
19.
Campbell
JN
,
Macosko
EZ
,
Fenselau
H
,
Pers
TH
,
Lyubetskaya
A
,
Tenen
D
,
.
A molecular census of arcuate hypothalamus and median eminence cell types
.
Nat Neurosci
.
2017 Mar
;
20
(
3
):
484
96
.
20.
Kozareva
V
,
Martin
C
,
Osorno
T
,
Rudolph
S
,
Guo
C
,
Vanderburg
C
,
.
A transcriptomic atlas of the mouse cerebellum reveals regional specializations and novel cell types
.
bioRxiv
.
2020 Mar 5
.
21.
Saunders
A
,
Macosko
EZ
,
Wysoker
A
,
Goldman
M
,
Krienen
FM
,
de Rivera
H
,
.
Molecular diversity and specializations among the cells of the adult mouse brain
.
Cell
.
2018 Aug 9
;
174
(
4
):
1015
16
.
22.
Tasic
B
,
Menon
V
,
Nguyen
TN
,
Kim
TK
,
Jarsky
T
,
Yao
Z
,
.
Adult mouse cortical cell taxonomy revealed by single cell transcriptomics
.
Nat Neurosci
.
2016 Feb
;
19
(
2
):
335
46
.
23.
Vladoiu
MC
,
El-Hamamy
I
,
Donovan
LK
,
Farooq
H
,
Holgado
BL
,
Sundaravadanam
Y
,
.
Childhood cerebellar tumours mirror conserved fetal transcriptional programs
.
Nature
.
2019 Aug
;
572
(
7767
):
67
73
.
24.
Butler
A
,
Hoffman
P
,
Smibert
P
,
Papalexi
E
,
Satija
R
.
Integrating single-cell transcriptomic data across different conditions, technologies, and species
.
Nat Biotechnol
.
2018 Jun
;
36
(
5
):
411
20
.
25.
McInnes
L
,
Healy
J
,
Melville
J
.
UMAP: uniform manifold approximation and projection for dimension reduction
.
2018
. Available from: https://arxivorg/abs/180203426.
26.
Qiu
X
,
Mao
Q
,
Tang
Y
,
Wang
L
,
Chawla
R
,
Pliner
HA
,
.
Reversed graph embedding resolves complex single-cell trajectories
.
Nat Methods
.
2017 Oct
;
14
(
10
):
979
82
.
27.
Jessa
S
,
Blanchet-Cohen
A
,
Krug
B
,
Vladoiu
M
,
Coutelier
M
,
Faury
D
,
.
Stalled developmental programs at the root of pediatric brain tumors
.
Nat Genet
.
2019 Dec
;
51
(
12
):
1702
13
.
28.
Wallace
VA
.
Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum
.
Curr Biol
.
1999 Apr 22
;
9
(
8
):
445
8
.
29.
Hoshino
M
,
Nakamura
S
,
Mori
K
,
Kawauchi
T
,
Terao
M
,
Nishimura
YV
,
.
Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum
.
Neuron
.
2005 Jul 21
;
47
(
2
):
201
13
.
30.
Leto
K
,
Rolando
C
,
Rossi
F
.
The genesis of cerebellar GABAergic neurons: fate potential and specification mechanisms
.
Front Neuroanat
.
2012
;
6
:
6
.
31.
Leto
K
,
Carletti
B
,
Williams
IM
,
Magrassi
L
,
Rossi
F
.
Different types of cerebellar GABAergic interneurons originate from a common pool of multipotent progenitor cells
.
J Neurosci
.
2006 Nov 8
;
26
(
45
):
11682
94
.
32.
Vladoiu
MC
,
El-Hamamy
I
,
Donovan
LK
,
Farooq
H
,
Holgado
BL
,
Sundaravadanam
Y
,
.
Childhood cerebellar tumours mirror conserved fetal transcriptional programs
.
Nature
.
2019 Aug
;
572
(
7767
):
67
73
.
33.
Khakh
BS
,
Deneen
B
.
The emerging nature of astrocyte diversity
.
Annu Rev Neurosci
.
2019 Jul 8
;
42
:
187
207
.
34.
Lozzi
B
,
Huang
TW
,
Sardar
D
,
Huang
AY
,
Deneen
B
.
Regionally distinct astrocytes display unique transcription factor profiles in the adult brain
.
Front Neurosci
.
2020
;
14
:
61
.
35.
Witter
MP
,
Doan
TP
,
Jacobsen
B
,
Nilssen
ES
,
Ohara
S
.
Architecture of the entorhinal cortex a review of entorhinal anatomy in rodents with some comparative notes
.
Front Syst Neurosci
.
2017
;
11
:
46
.
36.
Soriano
E
,
Del Río
JA
.
The cells of cajal-retzius: still a mystery one century after
.
Neuron
.
2005 May 5
;
46
(
3
):
389
94
.
37.
Toda
C
,
Santoro
A
,
Kim
JD
,
Diano
S
.
POMC neurons: from birth to death
.
Annu Rev Physiol
.
2017 Feb 10
;
79
:
209
36
.
38.
Thurston
G
,
Suri
C
,
Smith
K
,
McClain
J
,
Sato
TN
,
Yancopoulos
GD
,
.
Leakage-resistant blood vessels in mice transgenically overexpressing angiopoietin-1
.
Science
.
1999 Dec 24
;
286
(
5449
):
2511
4
.
39.
Zeisel
A
,
Hochgerner
H
,
Lönnerberg
P
,
Johnsson
A
,
Memic
F
,
van der Zwan
J
,
.
Molecular architecture of the mouse nervous system
.
Cell
.
2018 Aug 9
;
174
(
4
):
999
e22
.
40.
Abu-Hassan
DW
,
Acott
TS
,
Kelley
MJ
.
The trabecular meshwork: a basic review of form and function
.
J Ocul Biol
.
2014 May
;
2
(
1
). http://dx.doi.org/10.13188/2334-2838.1000017.
41.
Park
DY
,
Lee
J
,
Park
I
,
Choi
D
,
Lee
S
,
Song
S
,
.
Lymphatic regulator PROX1 determines Schlemm’s canal integrity and identity
.
J Clin Invest
.
2014 Sep
;
124
(
9
):
3960
74
.
42.
Kizhatil
K
,
Ryan
M
,
Marchant
JK
,
Henrich
S
,
John
SW
.
Schlemm’s canal is a unique vessel with a combination of blood vascular and lymphatic phenotypes that forms by a novel developmental process
.
PLoS Biol
.
2014 Jul
;
12
(
7
):
e1001912
.
43.
Arita
Y
,
Nakaoka
Y
,
Matsunaga
T
,
Kidoya
H
,
Yamamizu
K
,
Arima
Y
,
.
Myocardium-derived angiopoietin-1 is essential for coronary vein formation in the developing heart
.
Nat Commun
.
2014 Jul 29
;
5
:
4552
.
44.
Chu
M
,
Li
T
,
Shen
B
,
Cao
X
,
Zhong
H
,
Zhang
L
,
.
Angiopoietin receptor Tie2 is required for vein specification and maintenance via regulating COUP-TFII
.
Elife
.
2016 Dec 22
;
5
:
5
.
45.
Chow
BW
,
Nuñez
V
,
Kaplan
L
,
Granger
AJ
,
Bistrong
K
,
Zucker
HL
,
.
Caveolae in CNS arterioles mediate neurovascular coupling
.
Nature
.
2020 Mar
;
579
(
7797
):
106
10
.
46.
Amaral
DG
,
Scharfman
HE
,
Lavenex
P
.
The dentate gyrus: fundamental neuroanatomical organization (dentate gyrus for dummies)
.
Prog Brain Res
.
2007
;
163
:
3
22
.
47.
Shen
J
,
Wang
D
,
Wang
X
,
Gupta
S
,
Ayloo
B
,
Wu
S
,
.
Neurovascular coupling in the dentate gyrus regulates adult hippocampal neurogenesis
.
Neuron
.
2019 Sep 4
;
103
(
5
):
878
e3
.
48.
Kullmann
JA
,
Trivedi
N
,
Howell
D
,
Laumonnerie
C
,
Nguyen
V
,
Banerjee
SS
,
.
Oxygen tension and the VHL-Hif1α pathway determine onset of neuronal polarization and cerebellar germinal zone exit
.
Neuron
.
2020 May 20
;
106
(
4
):
607
e5
.