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. Author manuscript; available in PMC: 2017 Mar 21.
Published in final edited form as: Dev Cell. 2016 Mar 21;36(6):624–638. doi: 10.1016/j.devcel.2016.02.023

Vascular Influence on Ventral Telencephalic Progenitors and Neocortical Interneuron Production

Xin Tan 1,2,4, Wenying Angela Liu 1,2,4, Xin-Jun Zhang 1, Wei Shi 1,3, Si-Qiang Ren 1, Zhizhong Li 1, Keith N Brown 1, Song-Hai Shi 1,2,3,*
PMCID: PMC4806403  NIHMSID: NIHMS764203  PMID: 27003936

SUMMARY

The neocortex contains glutamatergic excitatory neurons and GABAergic inhibitory interneurons. Extensive studies have revealed substantial insights into excitatory neuron production. However, our knowledge of the generation of GABAergic interneurons remains limited. Here we show that periventricular blood vessels selectively influence neocortical interneuron progenitor behavior and neurogenesis. Distinct from those in the dorsal telencephalon, radial glial progenitors (RGPs) in the ventral telencephalon responsible for producing neocortical interneurons progressively grow radial glial fibers anchored to periventricular vessels. This progenitor-vessel association is robust and actively maintained as RGPs undergo interkinetic nuclear migration and divide at the ventricular zone surface. Disruption of this association by selective removal of INTEGRIN β1 in RGPs leads to a decrease in progenitor division, a loss of PARVALBUMIN and SOMATOSTATIN-expressing interneurons, and defective synaptic inhibition in the neocortex. These results highlight a prominent interaction between RGPs and periventricular vessels important for proper production and function of neocortical interneurons.

Graphical Abstract

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INTRODUCTION

Neurons in the neocortex include excitatory glutamatergic neurons and inhibitory γ–aminobutyric acid (GABA)ergic interneurons, which are intricately interconnected to form functional circuits for neural representation and behavioral control. Proper function of the neocortex depends on the production of correct numbers and types of excitatory and inhibitory neurons that largely occurs during embryonic development (Batista-Brito and Fishell, 2009; Flames and Marin, 2005; Gotz and Huttner, 2005; Greig et al., 2013; Huang et al., 2007; Isaacson and Scanziani, 2011; Markram et al., 2004; Wonders and Anderson, 2006). Excitatory neurons are produced by radial glial progenitors (RGPs) in the ventricular zone (VZ) of the dorsal telencephalon (i.e. the developing neocortex) and migrate radially from their birthplace to constitute the future neocortex (Ayala et al., 2007; Florio and Huttner, 2014; Kriegstein and Alvarez-Buylla, 2009; Rakic, 1988). On the other hand, most if not all inhibitory interneurons are generated in the ventral telencephalon and migrate tangentially over long distances to reach the neocortex (Anderson et al., 1997; Marin and Rubenstein, 2001). In the mouse, genetic fate-mapping studies have shown that more than 70% of neocortical interneurons originate in the medial ganglionic eminence (MGE) and the preoptic area (PoA) (Butt et al., 2008; Fogarty et al., 2007; Gelman et al., 2011; Xu et al., 2008). Extensive studies over the past two decades have provided a comprehensive view on the production of glutamatergic excitatory neurons; however, the cellular mechanisms that regulate neocortical interneuron neurogenesis remain poorly understood.

RGPs in the dorsal telencephalon responsible for producing excitatory neurons in the neocortex have been well-characterized (Anthony et al., 2004; Malatesta et al., 2000; Tamamaki et al., 2001). With the cell bodies residing in the VZ, they possess a characteristic bipolar morphology consisting of a short ventricular endfoot reaching the VZ surface and a long radial glial fiber contacting the pial basement membrane. Guided by the ventricular endfoot, RGPs display interkinetic nuclear oscillation that is tightly coupled to cell cycle progression and divide exclusively at the VZ surface. During excitatory neuron genesis, RGPs predominantly divide asymmetrically to self-renew and, at the same time, to produce a neuron or an intermediate progenitor, which further divide to produce neurons in the subventricular zone (SVZ) (Englund et al., 2005; Haubensak et al., 2004; Noctor et al., 2004). Newborn neurons then migrate radially along the radial glial fibers of mother RGPs to reach their final destination in the future neocortex (Rakic, 1971, 1988). Similar to those in the VZ of the dorsal telencephalon, progenitors in the VZ of the MGE and PoA are RGPs in nature (Anthony et al., 2004; Brown et al., 2011). They divide asymmetrically at the VZ surface to produce neocortical interneurons either directly or indirectly through intermediate progenitors that divide symmetrically in the SVZ (Brown et al., 2011). Yet, little is known about the cellular organizations and behavior of these RGPs in the ventral telencephalon.

Besides the neural system, the other major cellular component of the developing telencephalon is the vascular network. Based on the anatomical location, growth pattern, and developmental regulation, the telencephalic vasculature has been suggested to comprise two general categories: pial vessels and periventricular vessels (Vasudevan and Bhide, 2008). The pial vessels surround the brain by embryonic day 9.5 (E9.5) and progressively perforate into the developing brain (Hogan et al., 2004). In comparison, the periventricular vessels, originating from a basal vessel on the telencephalic floor of the basal ganglia primordium, actively develop in the ventral telencephalon and form an elaborated network that progressively propagates into the dorsal telencephalon by E11.5 (Vasudevan et al., 2008). The early presence of the periventricular vessels in the ventral telencephalon raises the intriguing possibility that these vessels regulate progenitor behavior and neurogenesis. In this study, we uncover an interaction between RGPs and the periventricular vessels in the developing ventral but not dorsal telencephalon that influences neocortical interneuron production.

RESULTS

RGP Anchorage to Periventricular Vessels in the MGE and PoA

To explore the relationship between RGPs and the periventricular vessels, we performed in utero intraventricular injection of low-titer retroviruses expressing enhanced green fluorescent protein (EGFP) into mouse embryos at embryonic day 12.5 (E12.5). Brains were collected at E14.5, sectioned, and stained for EGFP (green), NKX2.1 (white), a transcription factor specifically expressed in the MGE/PoA progenitors of the developing telencephalon (Butt et al., 2008; Fogarty et al., 2007; Sussel et al., 1999; Xu et al., 2008), and ISOLECTIN B4 (red), a glycoprotein marker that reliably labels blood vessels including both endothelial cells and pericytes in the developing brain (Figures 1A, S1A, and S1B) (Vasudevan et al., 2008), as well as with DAPI (blue) (Figure 1A).

Figure 1. RGP anchorage to periventricular vessels in the MGE/PoA; See also Figures S1-S3 and Movie S1.

Figure 1

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(A) An E14.5 brain section with EGFP-expressing retrovirus injection at E12.5 stained for blood vessel marker ISOLECTIN B4 (red), and MGE/PoA-specific transcription factor NKX2.1 (white), and with DAPI (blue). High magnification images of an EGFP-expressing RGP in the MGE (white box) positive for NKX2.1 (area 2, arrowhead) with its radial glial fiber end wrapped around a periventricular vessel (area 1, arrows) are shown to the right. 3D reconstruction images of area 1 are shown at the bottom (A1’). Ncx, neocortex; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; PoA, preoptic area. Scale bars: 200 μm, 10 μm, 10 μm (from left to right); 10 μm (A1). (B) E13.5 and E16.5 MGE RGPs labeled by EGFP-expressing retrovirus (green) at E12.5 and stained for ISOLECTIN B4 (red) and with DAPI (blue). High magnification of the radial glial fiber end (areas 1 and 2) are shown to the right. 3D reconstruction images of area 2 are shown at the bottom (B2’). Scale bars: 10 μm. (C) The association between radial glial fiber end and periventricular vessels (arrows) in the MGE/PoA at different embryonic stages. Projection images are shown to the left and single-section images to the right. Scale bar: 10 μm. (D) Percentage of RGPs with a short radial glial fiber that were or were not anchored to the periventricular vessel.

As expected, RGPs labeled in the dorsal telencephalon exhibited a characteristic bipolar morphology with a short apical ventricular endfoot pointing towards the lateral ventricle and a long basal radial glial fiber reaching the pial surface with enlarged endings (Figure S2A). In contrast, we found that in the NKX2.1-labeled MGE/PoA, a majority of RGPs (Figure 1A, arrowheads) developed a radial glial fiber with a complex ending wrapped around the periventricular vessel labeled by ISOLECTIN B4 (Figure 1A, arrows) or Platelet endothelial cell adhesion molecule-1 (PECAM-1) (Figure S1C, arrows), a specific marker for endothelial cells (Williams et al., 1996), as revealed by three-dimensional (3D) reconstruction (Figure 1A, area 1 and A1’, and Movie S1) and cross-sectional imaging (Figure S1E) analyses. Note that the radial glial fiber endings did not associate with ISOLECTIN B4-positive but PECAM-1-negative pericytes (Figure S1C, arrowheads). At this developmental stage, a substantial fraction of endothelial cells and vessels in the MGE were not covered by NG2-positive pericytes (Figure S1D).

Intact RGPs were distinguished from RGPs with a truncated radial glial fiber by the existence of complex ending structures recovered by serial sectioning and 3D reconstruction analysis (Figures 1A). Similar tight association between RGPs and periventricular vessels were found in the MGE/PoA at different embryonic stages when neocortical interneurons were produced (Figures 1B-D and S1F). The ending structure associated with the vessel appeared diverse, ranging from simple club-shaped (Figure 1B, area 1) to complex claw-shaped with numerous branches (Figure 1B, area 2 and B2’, and Movie S1). Nearly all non-pia-reaching RGPs in the MGE/PoA were associated with the periventricular vessel (Figure 1D). Similar vessel-anchored RGPs were also readily found in the lateral ganglionic eminence (LGE) (Figure S3). In contrast, the radial glial fiber endings of RGPs in the dorsal telencephalon were rarely anchored to the vessel (Figure S2B); instead, they were anchored to the pial basement membrane labeled by LAMININ (Figure S2C). Together, these results suggest that periventricular vascular anchorage is a prominent and distinct property of the ventral telencephalic RGPs that produce neocortical interneurons, but not the dorsal telencephalic RGPs that produce neocortical excitatory neurons.

Progressive Generation of Vessel-anchored RGPs in MGE/PoA

To visualize the interaction between the radial glial fiber of RGPs and the vessel at the population level, we performed intraventricular injection of the lipophilic tracer 3H-Indolium, 5-[[[4-(chloromethyl)benzoyl]amino]methyl]-2-[3-(1,3-dihydro-3,3-dimethyl-1-octadecyl-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-1-octadecyl-, chloride (CM-DiI) into the lateral ventricles at E11.5 and E14.5 to label the VZ RGPs. Brains were later sectioned and stained with ISOLECTIN B4 (Figures 2A and S2D). At E11.5, the organization of RGPs in the MGE was similar to that in the neocortex; most if not all the DiI-labeled radial glial fibers projecting outside the VZ reached the pial surface in straight bundles (Figure 2A, left, area 3, red) and did not obviously associate with the vessels, despite their presence in the ventral telencephalon (Figure 2A, left, areas 1 and 2, green). In contrast, at E14.5, while the radial glial fibers in the neocortex remained extended to the pial surface (Figure S2D), only a few radial glial fibers in the MGE reached the pial surface (Figure 2A, right, area 6). Interestingly, they extensively covered the periventricular vessels (Figure 2A, right, areas 4 and 5, arrows), indicating a robust interaction between radial glial fibers and periventricular vessels. Moreover, these results suggest that the association between RGPs and periventricular vessels in the MGE occurs progressively as development proceeds.

Figure 2. Progressive generation of the periventricular vessel-anchored RGPs in the MGE/PoA; See also Figure S4 and Movie S2.

Figure 2

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(A) E11.5 and E14.5 brain sections labeled by DiI (red) at the VZ of the MGE and stained for ISOLECTIN B4 (green). At E14.5 DiI-labeled radial glial fibers aggregate at the periventricular vessels (areas 4 and 5, arrows) with few reaching the pia (area 6), while those at E11.5 do not obviously associate with vessels (areas 1 and 2) and mostly reach the pia (area 3). Scale bars: 200 μm (left), 20 μm (right) and 20 μm (bottom). (B) E13.5 and E16.5 brain sections with retrovirus (green) injected two days earlier. High magnification images of a pia-anchored radial glial fiber (areas 1 and 3) and a periventricular vessel-anchored RGP (areas 2 and 4) are shown in the middle. 3D reconstruction images of the configuration of RGPs in the MGE/PoA are shown to the right. Blue and white lines indicate the contours of the pia and the VZ surface. Blue dots indicate RGP cell bodies, green lines indicate radial glial fibers, red dots indicate the radial glial fiber ends anchored to the vessel, and orange dots indicate the ends anchored to the pia. Scale bars: 200 μm, 20 μm, 20 μm, and 20 μm (from left to right). (C) Stereological quantification of the fraction of RGPs anchored to the vessel or the pia at different developmental stages. (D) An E13.5 brain section (left, retrovirus injected at E11.5) with a clone containing a pair of OLIG2-positive RGPs (right, yellow and white arrows, area 1). Note one RGP with a short radial glial fiber (yellow arrowheads) anchored to the periventricular vessel (yellow arrowheads, area 3), while the other with a long radial glial fiber reaching the pia (white arrowheads, area 4). Scale bars: 200 μm; 10 μm; 10 μm; 10 μm. (E) Schematic reconstructions of the RGP pair. The pia-reaching RGP is shown in blue and the vessel (v, red)-anchored RGP is shown in green. The highlighted areas (3 and 4) correspond to the same areas (3 and 4) in Figure 2D.

We next systematically analyzed the temporal development of RGP-vessel association in the MGE/PoA at the clonal level. We injected low-titer EGFP-expressing retrovirus into the lateral ventricle at E11.5, E12.5, E13.5, and E14.5 to label individual dividing RGPs in the MGE/PoA. Brains were harvested two days later and subjected to serial sectioning and 3D reconstruction analysis to recover all EGFP-labeled RGPs in the MGE/PoA and assess their radial glial fiber end location and configuration (Figures 2B-E and S4A-E). As expected, we observed simple club-shaped ends at the pial surface (Figures 2B, areas 1 and 3, arrowheads) that were not associated with the vessel, as well as branched, claw-shaped ends associated with the periventricular vessel in the mantle region (Figure 2B, areas 2 and 4, arrows). Notably, the pia-reaching radial glial fiber endings in the MGE/PoA shared a similar morphology with those in the dorsal telencephalon (Figure S2) and were also anchored to the pial basement membrane (Figure S4C). At E13.5 (with retroviruses injected at E11.5), the fraction of pia-anchored radial glial fiber endings was similar to that of vessel-anchored radial glial fiber endings (Figures 2B top and 2C, and Movie S2). However, as time proceeded, the fraction of pia-anchored endings progressively decreased, whereas the fraction of vessel-anchored endings concurrently increased. At E16.5 (with retroviruses injected at E14.5), the vast majority of RGPs in the MGE/PoA possessed a short radial glial fiber that was anchored to the periventricular vessel in the mantle region (Figures 2B bottom and 2C, and Movie S2).

The progressive increase in vessel-anchored RGPs raised the possibility that they were generated by pia-anchored RGPs through proliferative divisions. Indeed, in E11.5-E13.5 brains (i.e. retrovirus injection at E11.5 and analysis at E13.5) we observed pairs of RGPs in the same clonal cluster in the MGE/PoA labeled by OLIG2, a transcription factor abundantly expressed in the VZ of the ventral telencephalon (Petryniak et al., 2007), one containing a long radial glial fiber reached the pia (white arrow and arrowheads) and the other possessing a short radial glial fiber attached to the periventricular vessel (yellow arrow and arrowheads) (Figures 2D, 2E, S4A, and S4B). Notably, some RGP pairs remained connected with each other at the VZ surface (Figures S4D and S4E), likely representing the final stage of mitosis. In addition, we observed pairs of RGPs, both of which possessed a short radial glial fiber anchored to the periventricular vessel (Figure S4F), indicating a self-proliferation of vessel-anchored RGPs. Together, these results suggest that vessel-anchored RGPs in the MGE/PoA are gradually produced by proliferative divisions of pia-anchored or vessel-anchored RGPs.

Active Interaction between Radial Glial Fiber Endings and Vessels

The morphology of vessel-anchored radial glial fiber endings in the MGE/PoA was diverse and often complex, suggesting that this RGP-vessel interaction is dynamic. To test this, we performed live imaging experiments to monitor the interaction. We took advantage of the Tek-Cre transgenic mouse line, in which Cre recombinase is selectively expressed in endothelial cells under the Tek (i.e. Tie2) promoter (Cao et al., 2004). By crossing it with Ai14-tdTomato, a Cre-dependent fluorescent reporter mouse line (Madisen et al., 2010), we labeled the vessels in red fluorescence (Figure S5A). We then performed in utero intraventricular injection of low titer EGFP-expressing retrovirus into the Tek-Cre;Ai14-tdTomato mouse embryos at E12.5 to label individual dividing RGPs in the MGE/PoA. Organotypic brain slice cultures were prepared two days later and subjected to time-lapse imaging analysis as previously described (Brown et al., 2011).

We found that the radial glial fiber end continuously changed its morphology to probe the environment (Figures 3A and 3B). Upon contacting with the vessel, the end grew branches along the vessel and developed enlarged contact sites. Moreover, as the vessel remodeled its configuration, the radial glial fiber actively adjusted its length and orientation to maintain or resume its interaction with the vessel (Figures 3C and 3D, and Movie S3). Together, these results suggest that RGPs actively search for the vessel in the mantle region of the MGE/PoA and that the association between the radial glial fiber end and the vessel is actively maintained.

Figure 3. Dynamic and active interaction between the radial glial fiber and the periventricular vessel in the MGE/PoA; See also Figure S5 and Movie S3.

Figure 3

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(A) Time-lapse images of a radial glial fiber end (green) and the periventricular vessel (red) in the MGE of an organotypic slice culture prepared from an E14.5 Tek-Cre;Ai14-tdTomato mouse brain that received in utero intraventricular injection of EGFP-expressing retrovirus (green) at E12.5. Time is indicated on the top. Scale bar: 20 μm. (B) Schematic representation of the dynamic interaction between the radial glial fiber end and the vessel (v). (C) Time-lapse images of the interaction between two RGPs (green, R1 and R2) and a nearby periventricular vessel (red) in the MGE. Time is indicated on the top. Scale bar: 20 μm. (D) Schematic representation of the active interaction between the radial glial fiber and the vessel (v). Broken lines indicate the VZ surface.

Dividing RGPs Maintain Vessel Anchorage

A defining feature of RGPs is their mitotic capability. As we previously showed, RGPs in the MGE/PoA displayed interkinetic nuclear migration and divided at the VZ surface to produce neocortical interneurons (Brown et al., 2011). We found that throughout the process, RGPs maintained their association with the vessel, even though the morphology of the radial glial fiber endings changed dynamically (Figure 4 and Movie S4). Notably, a vast majority of dividing RGPs (25 out of 29) observed in our live imaging experiments had their radial glial fiber associated with the vessel, suggesting that the vascular anchorage may facilitate RGP division and neocortical interneuron neurogenesis. This maintenance of the radial glial fiber during mitosis also indicates that these vessel-anchored RGPs are likely different from the short neural precursors observed in the developing dorsal telencephalon (Gal et al., 2006).

Figure 4. RGPs divide while maintaining anchorage to the periventricular vessel in the MGE/PoA; See also Movie S4.

Figure 4

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Time-lapse images of an RGP in the MGE that undergoes mitosis in an organotypic culture slice prepared from an E14.5 Tek-Cre;Ai14-tdTomato (red) mouse brain with EGFP-expressing retrovirus (green) injection at E12.5. Time is indicated on the top. Note that the RGP undergoes interkinetic nuclear migration (white arrows) and divides at the VZ surface (broken line), and throughout the process, the radial glial fiber end remains attached to the periventricular vessel (asterisks). The double-arrowed broken line indicates the cleavage plane. White arrowheads indicate the new born daughter cell and yellow arrowheads indicate a previously born daughter cell of the RGP that progressively migrates away. Scale bar: 30 μm.

Removal of ITGβ1 in RGPs Disrupts Vessel Anchorage

To explore the molecular basis of radial glial fiber ending-vessel interaction, we examined the adhesion molecules that mediate cell-cell or cell-extracellular matrix (ECM) contacts (Buck and Horwitz, 1987; Troyanovsky, 1999). It is known that the vessel is covered by the basement membrane, a sheet-like extracellular matrix mainly composed of LAMININ (Quinones-Hinojosa et al., 2006). Indeed, we found a LAMININ-enriched sheet outside of the ISOLECTIN B4-labeled vessel that was in direct contact with the radial glial fiber ending of RGPs in the MGE/PoA (Figure 5A). Notably, a majority of the vascular basement membrane at this stage was devoid of pericyptes labeled by NG2 staining (Figure S5B). A well-known family of receptors for LAMININ is heterodimer INTEGRIN α6β1 (Belkin and Stepp, 2000; Miranti and Brugge, 2002). Both INTEGRIN β1 and α6 were abundantly expressed in the MGE/PoA RGPs, especially in the radial glial fibers labeled by NESTIN and BLBP (Figure 5B, arrows). Notably, they were also expressed in the vessels (Figure 5B, arrowheads).

Figure 5. ITGβ1 mediates vessel anchorage of the radial glial fiber end in the MGE/PoA; See also Figure S6 and Movies S5.

Figure 5

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(A) A radial glial fiber end (arrows) labeled by EGFP-expressing retrovirus (green) and anchored to the periventricular vessel labeled by ISOLECTIN B4 (red) and LAMININ (blue) staining. Cross-sectional images are shown to the right. Scale bars: 10 μm. (B) An E16.5 MGE stained for ITGβ1 (green, top) or ITGα6 (green, bottom) and NESTIN (top, red) or BLBP (bottom, red), two RGP-specific markers. Note the co-expression of ITGβ1 and ITGα6 in radial glial fibers labeled by NESTIN and BLBP (arrows), and the vessels (arrowheads). Scale bars: 100 μm. (C) E14.5 MGE of the wild type (WT, left) and Itgβ1 conditional knockout (Itgβ1 cKO, right) mice with retrovirus (green) injected at E12.5 and stained for ISOLECTIN B4 (red) and with DAPI (blue). Scale bars: 10 μm. (D) Radial glial fiber ends in E14.5 (left) and E16.5 (right) MGE/PoA of Itgβ1 cKO mice, labeled by EGFP-expressing retrovirus (green, arrows) injection at E12.5. Note the clear separation between radial glial fiber ends and the vessels (red, open arrowheads). Scale bar: 10 μm. (E) Percentage of pia-anchored RGPs in the MGE/PoA of the WT (n=3) and Itgβ1 cKO (n=4) mice. (F) Percentage of non-pia reaching RGPs anchored to the periventricular vessel in the MGE/PoA of the WT (n=6) and Itgβ1 cKO (n=7) mice. (G) Number of branches of individual non-pia-reaching radial glial fiber ends in the MGE/PoA. Data are presented as mean ± s.e.m. Student's two-tailed t-test was used to perform statistical significance. N.S., not significant.

The expression patterns of LAMININ on the surface of the vessels and INTEGRIN in the radial glial fiber of MGE/PoA RGPs suggest that they likely mediate the association between RGPs and vessels. To test this, we selectively removed INTEGRIN β1 (ITGβ1) in the MGE/PoA RGPs by crossing mice carrying the conditional allele of Itgβ1 (Itgβ1fl/fl) (Raghavan et al., 2000) with the Nkx2.1-Cre mice (Xu et al., 2008), in which Cre recombinase is selectively expressed in MGE/PoA progenitors under the Nkx2.1 promoter. As confirmed by immunohistochemistry, the expression of ITGβ1 was drastically reduced in RGPs labeled by NESTIN (arrows), but not in nearby vessels (arrowheads), in the MGE/PoA of Itgβ1 conditional knockout (Itgβ1 cKO) mice (Figure S6A). Consistent with this, the vessel morphology and density was not affected in the Itgβ1 cKO mice (Figure S6B, green). In addition, there was no obvious defect in the integrity of the junction formed between the apical ventricular endfeet of neighboring RGPs at the VZ surface, revealed by the ZO-1 staining (Figure S6C).

Interestingly, while radial glial fibers of MGE/PoA RGPs labeled by EGFP-expressing retrovirus were anchored to the periventricular vessel in the littermate wild type (WT) control mice (Figures 5C left, 5F, S6D left and Movie S5), more than half failed to anchor to the vessel in the Itgβ1 cKO mice (Figures 5C right, 5D, 5F, S6D right and Movie S5), suggesting that ITGβ1 is indeed crucial for the association between the radial glial fiber ending and vessel in the MGE/PoA. Consistent with this, we found that DiI-labeled radial glial fibers did not obviously converge to the periventricular vessels in the MGE of Itgβ1 cKO mice (Figure S6B). In addition, we observed that the radial glial fiber ending of MGE/PoA RGPs grew more branches in the Itgβ1 cKO mice than those in the wild type control mice (Figure 5G). Importantly, there was no obvious difference in pia-anchored RGPs between the wild type and Itgβ1 cKO brains (Figures 5E and S6D), suggesting that ITGβ1 removal does not impair the radial glial fiber anchorage to the pial basement membrane.

Loss of Vessel Anchorage Impairs Progenitor Division

Active anchorage of dividing RGPs to the periventricular vessel in the MGE/PoA suggests that this progenitor-vessel interaction affects RGP behavior and neocortical interneuron production. To test this, we examined progenitor proliferation in the MGE/PoA of the wild type control and Itgβ1 cKO mice at E12.5, E14.5, and E16.5 (Figures 6A, 6B, S7A, and S7B). Similar to those in the dorsal telencephalon, RGPs in the MGE/PoA divide at the VZ surface to give rise to postmitotic interneurons or intermediate progenitors that divide away from the VZ surface to produce neocortical interneurons (Brown et al., 2011; Harwell et al., 2015). To identify the dividing progenitors, we stained the brain sections for phosphorylated histone H3 (PHH3), a specific marker for cells undergoing mitosis.

Figure 6. Loss of vessel anchorage leads to a decrease in progenitor division; See also Figures S7.

Figure 6

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(A) E14.5 (top) and E16.5 (bottom) WT (left) or Itgβ1 cKO (right) MGE/PoA stained for ITGβ1 (red) and PHH3 (green), and with DAPI (blue). Higher magnification images (white boxes) are shown to the bottom of E16.5 MGE/PoA images. Yellow dash lines indicate the VZ surface of the targeted MGE/PoA. Note a selective loss of ITGβ1 in the MGE/PoA of Itgβ1 cKO mice (asterisks). Scale bar: 100 μm. (B) Quantification of the number of PHH3+ cells at the VZ surface and away from the VZ surface (Extra-VZ surface) at E14.5 and E16.5 (n=4 mice for each genotype). Data are presented as mean ± s.e.m. Student's two-tailed t-test was used to perform statistical significance. N.S., not significant.

As expected, we observed PHH3+ cells at the VZ surface as well as away from the VZ surface (Extra-VZ surface) (Figures 6A and S7A). Interestingly, while there was no significant difference in the number of PHH3+ cells at the VZ or Extra-VZ surface in the MGE/PoA between the wild type control and Itgβ1 cKO mice at E12.5 (Figures S7A and S7B), there was a significant decrease in the number of PHH3+ cells at the VZ surface in the MGE/PoA of the Itgβ1 cKO mice compared to that of the wild type control mice at E14.5 (Figures 6A and 6B, top). As development proceeded, this decrease at the VZ surface became more pronounced at E16.5 (Figures 6A and 6B, bottom). Moreover, there was a significant decrease in the number of PHH3+ cells at the Extra-VZ surface (Figures 6A and 6B, bottom). Notably, the number of PHH3+ cells in the LGE did not obviously change (Figures S7C and S7D), suggesting that the selective reduction in the MGE/PoA progenitor division is due to ITGβ1 removal and a loss of vessel anchorage. In addition, we observed a significant reduction in the short-term BrdU labeling index (i.e. the S-phase index) in the VZ and SVZ at E16.5 (Figures S7E and S7F). Notably, there was no obvious spatial organization between Ki67-expressing (Figure S7G) or PHH3+ dividing (Figure S7H) intermediate progenitors and the periventricular vessels in the SVZ of the MGE/PoA. We did not observe any substantial increase in apoptosis in the MGE/PoA of Itgβ1 cKO mice compared to the control (Figure S7I). Together, these results suggest that disruption of radial glial fiber anchorage to the periventricular vessels leads to a decrease in RGP division at the VZ surface and a subsequent decrease in intermediate progenitor division away from the VZ surface in the MGE/PoA. Consistent with this, we found that in vivo injection of vascular endothelial growth factor receptor 2 (VEGFR2) blocking antibody (Prewett et al., 1999) caused a retraction of the periventricular vessels without affecting the pial basement membrane in the developing brain (Figure 7A). This resulted in a significant decrease in progenitor cell division in the MGE (Figures 7B and 7C) where vessel-anchored RGPs were abundant, but not in the neocortex (Figures 7D and 7E) where vessel-anchored RGPs were not abundant. A previous study suggested that VEGFR2 promotes the survival of neural stem cells in culture (Wada et al., 2006). On the other hand, VEGFR2 has been shown to be expressed in vessels (Breier et al., 1995) but not neocortical progenitor cells in vivo (Javaherian and Kriegstein, 2009). Notably, VEGFR2 blocking antibody treatment did not cause any significant neural progenitor cell apoptosis in the MGE/PoA (Figures 7F and 7G).

Figure 7. In vivo injection of VEGFR2 blocking antibodies causes periventricular vessel retraction and a decrease in progenitor cell division in the MGE/PoA.

Figure 7

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(A) Representative images of E13.5 brains that received in vivo injection of control IgG (left) or VEGFR2 blocking antibody (right) at E12.5 and stained for ISOLECTIN B4 (red) and LAMININ (green), and with DAPI (blue). Note that VEGFR2 antibody injection leads to a retraction of periventricular vessels in both the MGE (arrows) and Ncx with no obvious effect on the pial basement membrane (arrowheads). Scale bars: 120 μm. (B) Representative images of the MGE stained for PHH3 (green) and ISOLECTIN B4 (red), and with DAPI (blue). The arrow indicates the retraction of the periventricular vessels in the MGE. Scale bar: 100 μm. (C) Quantification of the density of PHH3+ cells at the VZ surface (left) and away from the VZ surface (right) in the MGE (n=4 brains for each condition). (D) Representative images of the Ncx stained for PHH3 (green) and ISOLECTIN B4 (red), and with DAPI (blue). Scale bar: 80 μm. (E) Quantification of the density of PHH3+ cells at the VZ surface (left) and away from the VZ surface (right) in the Ncx (n=4 brains for each condition). (F) Representative images of the MGE stained for CLEAVED CASPASE-3 (green) and ISOLECTINB4 (red), and with DAPI (blue). Arrowheads indicate ISOLECTIN B4–/CASPASE-3+ cells. Scale bar: 80 μm. (G) Quantification of the number of ISOLECTIN B4–/CASPASE-3+ cells in the MGE (n=4 brains for each condition). Data are presented as mean ± s.e.m. Student's two-tailed t-test was used to perform statistical significance. N.S, not significant.

Loss of Vessel Anchorage Leads to interneuron loss and Reduced Synaptic Inhibition

MGE/PoA progenitors are responsible for producing a majority of neocortical interneurons, mostly PARVALBUMIN (PV)- and SOMATOSTATIN (SST)-expressing interneurons (Butt et al., 2008; Fogarty et al., 2007; Gelman et al., 2011; Xu et al., 2008). To test if the decrease in MGE/PoA progenitor division in the Itgβ1 cKO mice indeed leads to a loss of neocortical interneurons, we crossed Nkx2.1-Cre;Itgβ1fl/+ or Nkx2.1-Cre;Itgβ1fl/fl mice with Ai14-tdTomato reporter mice, which allowed selective labeling of all progeny arising from the NKX2.1-expressing MGE/PoA progenitors in the presence or absence of ITGβ1 (Figure 8A). We stereologically analyzed the density of tdTomato-expressing cells in the somatosensory neocortex at P21-30 that were positive for PV or SST. Interestingly, we found that compared to the control, the density of PV-expressing interneurons was significantly decreased in both the superficial and deep layers in the Itgβ1 cKO brain (Figures 8A and 8B, left). In addition, the density of SST-expressing interneurons was significantly decreased in the deep but not superficial layers (Figures 8A and 8B, right). Notably, we did not observe any obvious defect in the tangential migration of MGE/PoA-derived interneurons at the embryonic stage (Figure S8A) or any change in the density of MGE/PoA-derived interneurons in the striatum at P21-30 (Figures S8B and S8C), suggesting that the loss of neocortical interneurons is unlikely due to a migration defect. Together, these results suggested that vascular anchorage of RGPs in the MGE/PoA is crucial for proper production of neocortical interneurons.

Figure 8. Loss of vessel anchorage results in a loss of interneurons and an impaired synaptic inhibition in the somatosensory cortex; See also Figures S8.

Figure 8

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(A) Representative images of the somatosensory neocortex of P30 Nkx2.1-Cre;Itgβ1fl/+;Ai14-tdTomato (control, left) and Nkx2.1-Cre;Itgβ1fl/fl;Ai14-tdTomato (Itgβ1 cKO, right) mice stained for PV (blue) and SST (green). High magnification images of the superficial (Layer 1-4; areas 1 and 3) and deep (Layer 5-6; areas 2 and 4) layers are shown to the right. The arrow indicates an example PV+/tdTomato+ interneuron and the arrowhead indicates an example SST+/tdTomato+ interneuron. Scale bars: 100 μm. (B) Stereological quantification of the density of interneurons positive for PV+/tdTomato+ or SST+/tdTomato+ in the somatosensory neocortex of the WT (n=6) and Itgβ1 cKO (n=6) mice. (C, E) Representative sample traces of mIPSCs recorded from superficial (C) and deep (E) layer excitatory neurons in the somatosensory neocortex of P21 WT or Itgβ1 cKOmice. Scale bars: 25 pA and 200 msec. (D, F) Quantification of the frequency and amplitude of mIPSCs. Data are presented as mean ± s.e.m. Student's two-tailed t-test was used to perform statistical significance. N.S., not significant.

PV- and SST-expressing interneurons provide essential synaptic inhibition to excitatory neurons in the neocortex (Isaacson and Scanziani, 2011; Markram et al., 2004; Pfeffer et al., 2013). To test whether the loss of MGE/PoA-derived interneurons causes any functional deficit in the neocortex, we performed whole-cell patch clamp recording on both the superficial (2-4) and deep (5-6) excitatory neurons in the somatosensory neocortex and examined the miniature inhibitory postsynaptic currents (mIPSCs) (Figures 8C-F). We found that, while the amplitude of mIPSCs did not significantly change between the wild type control and Itgβ1 cKO neurons, the frequency of mIPSCs was substantially reduced in the Itgβ1 cKO neurons. These results demonstrated that loss of RGP anchorage to the periventricular vessel in the MGE/PoA leads to a reduction in synaptic inhibition onto excitatory neurons in the neocortex.

DISCUSSION

While it is well established that neocortical interneurons are produced in the ventral telencephalon (Batista-Brito and Fishell, 2009; Flames and Marin, 2005; Wonders and Anderson, 2006), the cellular properties of the progenitors remain largely unknown. Here, we report a distinct organization of RGPs in the developing ventral telencephalon including those in the MGE/PoA that are responsible for producing a majority of neocortical interneurons. At the early embryonic stage (before E11.5-12.5), the majority of RGPs are anchored to the pial basement membrane, similar to those in the dorsal telencephalon responsible for producing neocortical excitatory neurons. However, as time proceeds, mitotic RGPs in the MGE/PoA become progressively anchored to the periventricular vessels. This shift in the anchorage of radial glial fiber endings coincides with the rapid expansion of the ventral telencephalon and the emergence of the striatum between the VZ and the pia, which would demand the development of exceedingly long radial glial fibers for RGPs to maintain the characteristic bipolar morphology, should they be anchored to the pial surface. Thus, the shift from the pial basement membrane anchorage to the periventricular vessel anchorage can be an effective way for RGPs to cope with the distinct developmental change in structural organization of the ventral telencephalon. A recent study showed that many progenitors in the human ganglionic eminence lack a prominent long radial glial fiber (Hansen et al., 2013), raising the possibility of a conserved progenitor-vessel interaction for interneuron progenitors in humans.

Besides serving as the progenitors, RGPs also provide the scaffold for the migration of neuronal progeny. In the dorsal telencephalon, the radial glial fibers of RGPs are essential for radial migration of excitatory neurons (Noctor et al., 2001; Rakic, 1971). Generated by RGPs in the ventral telencephalon, interneurons undergo an initial radial migration along mother radial glial fiber (Brown et al., 2011) and then a lengthy tangential migration to reach the neocortex (Marin and Rubenstein, 2001; Yokota et al., 2007). There are two general tangential migratory routes for MGE/PoA-derived interneurons, including a superficial route along the marginal zone beneath the pial surface and a deep route in the SVZ and the IZ. Interestingly, the deep interneuron migratory stream appears around E12.5 and becomes the main route after E14.5 (Marin and Rubenstein, 2001). Temporally, this change from the superficial to deep migration route correlates with the appearance of the vessel-anchored RGPs that possess relatively short radial glial fibers, suggesting that the increasing population of vessel-anchored RGPs in the MGE/PoA likely influence the developmental switch of the migratory routes of neocortical interneurons.

Our live imaging experiments in organotypic brain slice cultures showed that the interaction between the radial glial fiber of RGPs and the periventricular vessels is active and robust, indicating an attractive communication between the vessels and the radial glial fibers. Notably, the vessels tend to degenerate as the culture time proceeds to the late time point. Therefore, the organotypic brain slice culture preparation may not perfectly represent the in vivo condition. Nonetheless, these experiments provide important insights into the progenitor-vessel interaction as well as progenitor cell behavior in situ. We identified cell adhesion molecule ITGβ1 in RGPs as a key component in maintaining this progenitor-vessel interaction. ITGβ1 acts as a receptor for LAMININ that is abundantly present at the vessel surface. Selective deletion of ITGβ1 in the MGE/PoA RGPs results in a dissociation between the radial glial fiber and the periventricular vessel, and a progressive decrease in progenitor division. A previous study showed that intraventricular injection of ITGβ1 blocking antibody caused a detachment of the apical ventricular endfeet of RGPs in the developing neocortex, likely due to a compromised junction integrity and inter-RGP interaction at the VZ surface (Loulier et al., 2009). In consequence, interkinetic nuclear migration and division of RGPs were affected. In contrast, genetic deletion of Itgβ1 in the developing neocortex did not cause any obvious defect in the VZ organization and RGP proliferation (Graus-Porta et al., 2001; Haubst et al., 2006). Consistent with the previous genetic studies, we found that selective deletion of Itgβ1 in the MGE/PoA did not cause any obvious junction defect and/or detachment of the RGP apical ventricular endfeet. Instead, it disrupted the unique association between RGPs and periventricular vessels in the MGE/PoA. The increase in the branch number of radial glial fiber ending after ITGβ1 removal indicates that vessel anchorage likely stabilizes the radial glial fiber ending structure. Without the vessel association, the radial glial fiber ends appear to grow additional branches to probe the environment.

Notably, RGPs in the MGE/PoA divide while maintaining the association with the periventricular vessels, indicating that the vascular anchorage may regulate RGP maintenance and/or division. The decrease in progenitor division initially at the VZ surface then in the SVZ in the Itgβ1 cKO mice likely reflects that a decrease in RGP division at the VZ surface progressively leads to a reduction in intermediate progenitors that divide in the SVZ. ITGβ1 has previously been shown to be expressed in RGPs of the dorsal telencephalon and mediate the attachment of radial glial fibers to the pial basement membrane (Graus-Porta et al., 2001). However, its removal or disruption of the pial basement membrane attachment does not affect RGP proliferation in the dorsal telencephalon (Graus-Porta et al., 2001; Haubst et al., 2006). Interestingly, removal of ITGβ1 in the MGE/PoA did not affect the anchorage of radial glial fibers to the pial basement membrane. This difference is likely related to the distinct cellular organization of RGPs in the ventral versus dorsal telencephalon, and also suggests that certain secreted or membrane-anchored signaling factors sourced from the periventricular vessels may regulate RGP division in the MGE/PoA, as suggested for stem cells in the adult SVZ (Crouch et al., 2015; Kokovay et al., 2010). Future identification of these signaling factors will facilitate our understanding of the regulation of interneuron production and the vascular niche of neural progenitor/stem cells in general.

The decrease in progenitor division in the MGE/PoA in the Itgβ1 cKO mice led to a substantial loss of PV-expressing and, to a lesser degree, SST-expressing interneurons in the neocortex, and a clear reduction of synaptic inhibition in neocortical excitatory neurons at both the superficial and deep layers. Previous fate mapping study suggests that SST-expressing interneurons are largely generated at a relative early embryonic time window, whereas PV-expressing interneurons are produced persistently during embryonic development (Miyoshi et al., 2007). We found that at the early embryonic stage pia-anchored RGPs account for the major population of RGPs in the MGE/PoA. As time proceeds, vessel-associated RGPs become the dominant population of RGPs in the MGE/PoA. The subtle difference in the loss of PV- versus SST-expressing interneurons in the Itgβ1 cKO brain suggests that pia-anchored and vessel-associated RGPs may produce different subtypes of interneurons.

Endothelial cells have been shown to stimulate self-renewal and promote neurogenesis of neural stem cells in culture (Shen et al., 2004). However, the context of this vascular regulation in the developing brain in vivo remains largely unexplored. A previous study suggested that the vessels are a niche for TBR2-positive intermediate progenitors in the developing neocortex (Javaherian and Kriegstein, 2009). Currently, there is no well-established marker that selectively labels intermediate progenitors in the MGE/PoA; however, we found that there was no obvious spatial organization between proliferative or dividing progenitors in the SVZ and the periventricular vessels in the MGE/PoA. Interestingly, our study demonstrates the cellular organizational heterogeneity of RGPs and intermediate progenitors with regard to the periventricular vessel association in the developing dorsal versus ventral telencephalon and links it to neocortical interneuron production. The embryonic vascular network has also been suggested to support interneuron tangential migration (Won et al., 2013). In addition, it has been shown that RGPs in the developing dorsal telencephalon signal to influence vessel development and stabilization (Ma et al., 2013). In comparison, disruption of the association between RGPs and periventricular vessels by ITGβ1 removal in RGPs did not obviously affect vessel density and organization in the MGE. This difference may be due to the timing difference in periventricular vessel development between the ventral and dorsal telencephalon. Previous study showed that periventricular vessels develop initially in the ventral telencephalon and then propagate towards the dorsal telencephalon (Vasudevan et al., 2008). In the MGE/PoA, the vascular network forms largely before the appearance of vessel-associated RGPs, whereas in the developing neocortex, the vascular network forms while RGPs actively proliferate and expand. The concurrent development of RGPs and vessels in the developing neocortex may facilitate the regulation between RGPs and vessels.

A vascular niche of adult neurogenesis has also been extensively characterized (Leventhal et al., 1999; Palmer et al., 2000; Shen et al., 2008; Tavazoie et al., 2008). In the adult subependymal zone (SEZ), neural stem cells extend a radial/basal process which contacts nearby blood vessels. This similar organization between stem/progenitor cells and vessels in the adult and embryonic stages suggests that a substantial fraction of adult SEZ stem cells may originate from the vessel-anchored RGPs in the ventral telencephalon. A recent study suggests that a large fraction of adult SEZ neural stem cells is derived from a slowly dividing subpopulation of embryonic neural progenitors that are produced around E13.5-15.5 (Furutachi et al., 2015), which coincides with the generation of vessel-anchored RGPs in the ventral telencephalon. Future effort to link the vessel-anchored RGPs in the ventral telencephalon and the slowly dividing embryonic progenitors may provide important insights into the embryonic origin and distinct properties of adult neural stem cells (Fuentealba et al., 2015).

EXPERIMENTAL PROCEDURES

Animals, Retrovirus Production, and In Utero Intraventricular Injection

All mice were maintained at Memorial Sloan Kettering Cancer Center (MSKCC) and handled according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC). Replication-incompetent EGFP-expressing retroviruses were produced and injected into the lateral ventricle of mouse embryos in utero as previously described (Yu et al., 2009).

Tissue Preparation, Immunohistochemistry, and Imaging

Mice were transcardially perfused with ice-cold PBS (pH 7.4) followed by 4% paraformaldehyde (PFA) in PBS (pH 7.4). Brains were dissected out and post-fixed in the same fixation solution at 4°C. Coronal sections (~60 μm) were prepared using a vibratome (Leica Microsystems). For cryosections, fixed brains were placed in 30% sucrose in PBS overnight at 4°C until submerged. After embedding in O.C.T. tissue-freezing medium, coronal cryosections (16-20 m for embryonic brains; 40 μm for postnatal brains) were prepared and mounted onto gelatin-coated slides. Immunohistochemistry was performed as previously described (Brown et al., 2011). Images were acquired with a laser scanning confocal microscope (FV1000, Olympus), and analyzed with FluoView (Olympus) and Photoshop (Adobe). Three-dimensional reconstruction was performed using Imaris (Bitplane) or Neurolucida (MBF Bioscience).

For lipophilic tracer 3H-Indolium, 5-[[[4-(chloromethyl)benzoyl]amino]methyl]-2-[3-(1,3-dihydro-3,3-dimethyl-1-octadecyl-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-1-octadecyl-, chloride (CM-DiI) labeling, mouse embryonic brains were dissected and fixed with 4% PFA in PBS for 4-6 hours. CM-DiI (Invitrogen, 1 mg/mL dissolved in DMSO) was intraventricularly injected into the lateral ventricles. Brains were incubated in PBS at 37°C for 2 days and sectioned using a vibratome.

For BrdU pulse-chase labeling, a single dose of BrdU at 25 mg per kg body weight was administrated via intraperitoneal injection 30 minutes prior to sacrifice. For in vivo blocking antibody injection, Rat monoclonal anti-VEGFR2 antibody (DC101, Bioxcell) (Prewett et al., 1999) or non-specific IgG as a control was injected intraperitoneally into the mouse embryos in utero at E12.5 at a concentration of ~80 μg per mg body weight. One to two days later, brains were recovered and serially sectioned using a cryostat (16 μm). Consecutive sections covering the MGE/PoA were collected, immunostained, and mounted on gelatin-coated slides. Proliferative and apoptotic cells were stereologically quantified using Neurolucida and Stereo Investigator (MBF Bioscience).

Organotypic brain slice culture and live imaging analysis were performed as previously described (Brown et al., 2011).

Three-Dimensional Reconstruction and Stereologic Analysis of RGP Morphology and Neuronal Density

EGFP-expressing retroviruses were injected into the lateral ventricle at E11.5, E12.5, E13.5 and E14.5, respectively. Two days later, brains were processed and serially sectioned using a vibratome. Consecutive sections covering the entire MGE/PoA were collected, immunostained and mounted on gelatin-coated slides. All EGFP-labeled RGPs in the MGE/PoA of each section were traced using Neurolucida (MBF Bioscience) on an upright microscope equipped with epifluoresence illumination and cooled CCD camera (Zeiss). All traced sections were then aligned to recover the entire RGPs. At least six brains were analyzed for each time point. For the quantitative analysis, at least three animals for each condition were examined.

For each postnatal brain, the number of interneurons in the somatosensory cortex was stereologically analyzed on both hemispheres from four consecutive coronal sections using Neurolucida and Stereo Investigator (MBF Bioscience). Cortical laminar position was determined by cell packing density revealed by DAPI staining. Data were presented as mean ± s.e.m. and statistical significance was determined using Student's two-tailed t-test.

Electrophysiology

Acute brain slices were prepared at P21 and whole-cell patch clamping recordings were performed as previously described (Yu et al., 2009). Tetrodotoxin (1 μM), D-AP5 (50 μM) and NBQX (10 μM) (Tocris Biosciences, MI) were included to isolated miniature inhibitory synaptic currents (mIPSCs). Recordings were collected and analyzed using Axopatch 700B amplifier and pCLAMP10 software (Molecular Devices). Miniature IPSCs were analyzed using mini Analysis Program (Synaptosoft Inc). Data were presented as mean ± s.e.m. and statistical significance was determined using Student's two-tailed t-test.

Supplementary Material

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HIGHLIGHTS.

  • Selective association of ventral telencephalic RGPs with periventricular vessels.

  • Progressive generation of vessel-anchored RGPs as development proceeds.

  • Progenitor-vessel association is robust and actively maintained as RGPs divide.

  • Loss of vessel association affects cortical interneuron production and function.

ACKNOWLEDGEMENTS

We thank Dr. Stewart A. Anderson (University of Pennsylvania) for the Nkx2.1-Cre mouse line, and Drs. Margaret E. Ross, Stewart A. Anderson, Costantino Iadecola, Holly Moore, Julia A. Kaltschmidt, and Shi Laboratory members for inputs and comments. This work was supported by NIH grants (R01DA024681 and P01NS048120 to S.-H.S.) and a NYSTEM fellowship (C026879 to X.-J.Z).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AUTHOR CONTRIBUTIONS

X.T. and S.-H.S. designed the study; X.T. performed the majority of the experiments and analyses together with W.A.L.; X.-J.Z. and S.-Q.R. performed the electrophysiology experiments; W.S. carried out the pilot DiI labeling experiment; Z.L. generated the retrovirus; K.N.B. identified the progenitor-vessel association initially; X.T. and S.-H.S. wrote the manuscript with inputs from all other authors.

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