Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis
2016; Springer Nature; Volume: 35; Issue: 9 Linguagem: Inglês
10.15252/embj.201592372
ISSN1460-2075
AutoresChristian Lange, Miguel Turrero García, Ilaria Decimo, Francesco Bifari, Guy Eelen, Annelies Quaegebeur, Ruben Boon, Hui Zhao, Bram Boeckx, Junlei Chang, Christine Wu, Ferdinand le Noble, Diether Lambrechts, Mieke Dewerchin, Calvin J. Kuo, Wieland Β. Huttner, Peter Carmeliet,
Tópico(s)Epigenetics and DNA Methylation
ResumoArticle8 February 2016free access Source DataTransparent process Relief of hypoxia by angiogenesis promotes neural stem cell differentiation by targeting glycolysis Christian Lange Christian Lange Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Miguel Turrero Garcia Miguel Turrero Garcia Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Ilaria Decimo Ilaria Decimo Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Francesco Bifari Francesco Bifari Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Guy Eelen Guy Eelen Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Annelies Quaegebeur Annelies Quaegebeur Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Ruben Boon Ruben Boon Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Hui Zhao Hui Zhao Laboratory of Translational Genetics, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Bram Boeckx Bram Boeckx Laboratory of Translational Genetics, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Junlei Chang Junlei Chang Department of Medicine, Hematology Division, Stanford University, Stanford, CA, USA Search for more papers by this author Christine Wu Christine Wu Department of Medicine, Hematology Division, Stanford University, Stanford, CA, USA Search for more papers by this author Ferdinand Le Noble Ferdinand Le Noble Angiogenesis and Cardiovascular Pathology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Department of Cell and Developmental Biology, KIT, Karlsruhe, Germany Search for more papers by this author Diether Lambrechts Diether Lambrechts Laboratory of Translational Genetics, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Mieke Dewerchin Mieke Dewerchin Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Calvin J Kuo Calvin J Kuo Department of Medicine, Hematology Division, Stanford University, Stanford, CA, USA Search for more papers by this author Wieland B Huttner Wieland B Huttner Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Peter Carmeliet Corresponding Author Peter Carmeliet Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Christian Lange Christian Lange Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Miguel Turrero Garcia Miguel Turrero Garcia Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Ilaria Decimo Ilaria Decimo Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Francesco Bifari Francesco Bifari Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Guy Eelen Guy Eelen Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Annelies Quaegebeur Annelies Quaegebeur Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Ruben Boon Ruben Boon Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Hui Zhao Hui Zhao Laboratory of Translational Genetics, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Bram Boeckx Bram Boeckx Laboratory of Translational Genetics, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Junlei Chang Junlei Chang Department of Medicine, Hematology Division, Stanford University, Stanford, CA, USA Search for more papers by this author Christine Wu Christine Wu Department of Medicine, Hematology Division, Stanford University, Stanford, CA, USA Search for more papers by this author Ferdinand Le Noble Ferdinand Le Noble Angiogenesis and Cardiovascular Pathology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany Department of Cell and Developmental Biology, KIT, Karlsruhe, Germany Search for more papers by this author Diether Lambrechts Diether Lambrechts Laboratory of Translational Genetics, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Mieke Dewerchin Mieke Dewerchin Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Calvin J Kuo Calvin J Kuo Department of Medicine, Hematology Division, Stanford University, Stanford, CA, USA Search for more papers by this author Wieland B Huttner Wieland B Huttner Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany Search for more papers by this author Peter Carmeliet Corresponding Author Peter Carmeliet Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium Search for more papers by this author Author Information Christian Lange1,2, Miguel Turrero Garcia3, Ilaria Decimo1,2, Francesco Bifari1,2, Guy Eelen1,2, Annelies Quaegebeur1,2, Ruben Boon1,2, Hui Zhao4,5, Bram Boeckx4,5, Junlei Chang6, Christine Wu6, Ferdinand Le Noble7,8, Diether Lambrechts4,5, Mieke Dewerchin1,2, Calvin J Kuo6, Wieland B Huttner3 and Peter Carmeliet 1,2 1Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, VIB, Leuven, Belgium 2Laboratory of Angiogenesis and Neurovascular Link, Department of Oncology, KU Leuven, Leuven, Belgium 3Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 4Laboratory of Translational Genetics, Vesalius Research Center, VIB, Leuven, Belgium 5Laboratory of Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium 6Department of Medicine, Hematology Division, Stanford University, Stanford, CA, USA 7Angiogenesis and Cardiovascular Pathology, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany 8Department of Cell and Developmental Biology, KIT, Karlsruhe, Germany *Corresponding author. Tel: +32 16 37 32 02; Fax: +32 16 37 25 85; E-mail: [email protected] The EMBO Journal (2016)35:924-941https://doi.org/10.15252/embj.201592372 See also: JM Morante-Redolat & I Fariñas (May 2016) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Blood vessels are part of the stem cell niche in the developing cerebral cortex, but their in vivo role in controlling the expansion and differentiation of neural stem cells (NSCs) in development has not been studied. Here, we report that relief of hypoxia in the developing cerebral cortex by ingrowth of blood vessels temporo-spatially coincided with NSC differentiation. Selective perturbation of brain angiogenesis in vessel-specific Gpr124 null embryos, which prevented the relief from hypoxia, increased NSC expansion at the expense of differentiation. Conversely, exposure to increased oxygen levels rescued NSC differentiation in Gpr124 null embryos and increased it further in WT embryos, suggesting that niche blood vessels regulate NSC differentiation at least in part by providing oxygen. Consistent herewith, hypoxia-inducible factor (HIF)-1α levels controlled the switch of NSC expansion to differentiation. Finally, we provide evidence that high glycolytic activity of NSCs is required to prevent their precocious differentiation in vivo. Thus, blood vessel function is required for efficient NSC differentiation in the developing cerebral cortex by providing oxygen and possibly regulating NSC metabolism. Synopsis Blood vessel formation in mammalian brain development promotes neural stem cell differentiation by triggering a cascade of tissue oxygenation, reduced activity of HIF-1α and blunted glycolytic metabolism that favors the switch towards neurogenesis. An animated version of this synopsis is available online at: http://embopress.org/video_EMBOJ-2015-92372. Absence of blood vessels reduces neural stem cell (NSC) differentiation in development. Restoring oxygenation rescues NSC differentiation in the absence of normal vessels. HIF-1α levels regulate the switch of NSC expansion to differentiation in the cortex. The glycolytic regulator and HIF target gene Pfkfb3 is critically required for normal NSC expansion and upon HIF-1α stabilization. Introduction Radial glia (RGs) in the ventricular zone (VZ) of the developing cerebral cortex (simply referred to as “cortex”) are neural stem cells (NSCs) that generate neurons and glia in the cortex during development. RGs switch from expansion to generation of neurons by traversing from a symmetric division (producing two RGs) to an asymmetric division, mostly generating one RG plus one basal progenitor (BP; also termed intermediate progenitor cell, IPC), a committed neurogenic progenitor (Gotz & Huttner, 2005; Kriegstein & Alvarez-Buylla, 2009). The timing and extent of the switch from RG expansion to BP generation and, thus, neurogenesis determines the number of neurons in the cortex and their cellular identity in the cortical layers (Franco & Muller, 2013). Thus, proper regulation of RG expansion versus differentiation safeguards cortical development and prevents developmental disorders, associated with epilepsy, autism, and schizophrenia (Sun & Hevner, 2014), but the underlying signals and mechanisms are incompletely understood. Radial glia differentiation is regulated by signals from the cortical stem cell niche (Johansson et al, 2010; Bjornsson et al, 2015). In various developing organs, blood vessels are an essential component of stem cell niches that regulate the balance between precursor expansion and differentiation (Cleaver & Dor, 2012; Ramasamy et al, 2015). For the brain, co-culturing embryonic NSCs with immortalized endothelial cells in vitro increases NSC expansion and directs their fate toward neurons (Shen et al, 2004). In vivo, the pattern of angiogenesis in the cortex seems to correlate with the initiation of neurogenesis (Miyama et al, 1997; Shen et al, 2004), but conclusive evidence for an interrelation is lacking. Further, vessels attract newborn BPs in the VZ and induce their division in the vicinity of vessels (Javaherian & Kriegstein, 2009; Stubbs et al, 2009) and impaired vessel formation by deletion of VEGF reduces neural precursor cell (NPC) proliferation and survival, but direct effects of VEGF on NPCs were not excluded (Haigh et al, 2003; Raab et al, 2004). Importantly, for all observations, the molecular mechanisms are elusive. Signals from niche vessels have been implicated in the regulation of NSC quiescence in the adult brain (Goldman & Chen, 2011; Delgado et al, 2014; Ottone et al, 2014), and hypoxia and the hypoxia-inducible transcription factor HIF-1α have been involved in NSC proliferation and differentiation, and neuronal maturation in the adult brain (Mazumdar et al, 2010; Li et al, 2014). In contrast, the functional role of vessels and a possible function of HIF-1α in developmental neurogenesis in the embryonic brain are unclear. As the molecular mechanisms governing embryonic versus adult NSC expansion and differentiation differ (Urban & Guillemot, 2014), and embryonic NSCs rely on rapid proliferation for expansion, while adult NSCs rely on long periods of quiescence for self-renewal (Kippin et al, 2005; Lange et al, 2009), it is unknown whether vessels and HIF-1α in the embryonic NSC niche exert a similar or distinct function as in the adult niches. Mediators of HIF-1α in NSC regulation also remain to be identified. HIF-1α regulates glycolytic metabolism (Iyer et al, 1998), but only circumstantial evidence suggests a role of metabolism in NSC fate regulation. Drosophila neuroblasts switch from anaerobic metabolism to oxidative phosphorylation during development, and induction of oxidative phosphorylation is required for cell cycle exit and differentiation of neuroblasts (Homem et al, 2014). Similarly, adult mammalian NSCs in vitro increase oxygen consumption upon differentiation and inhibition of the electron transport chain increases proliferation (Wang et al, 2010; Bartesaghi et al, 2015). However, it is unknown whether NPC metabolism is altered in the in vivo niche during mammalian brain development, and whether alteration of metabolism alone functionally regulates NSC differentiation. Thus, it remains unclear whether and how niche vessels influence NPC proliferation and cell fate during prenatal brain development in vivo, and whether they regulate this process by supplying oxygen. We therefore characterized the role of blood vessels in regulating neurogenesis in the developing cerebral cortex. Results Angiogenesis is linked to neurogenesis during cortical development Previous studies documented the onset of angiogenesis and neurogenesis during cortical development (Miyama et al, 1997; Shen et al, 2004), but their temporo-spatial relationship has never been established side-by-side in the same study. Thus, we stained the developing mouse cerebral cortex for the endothelial marker isolectin B4 (IB4), the neurogenic marker Ngn1 (Kim et al, 2011), specific for both differentiating RGs and newborn BPs, and the BP marker Tbr2 (Englund et al, 2005) during cortical development (see Appendix Fig S1 for scheme). We found a striking temporo-spatial congruence of intraparenchymal vessel formation and induction of RG differentiation. At E10.5, vessels were absent and only single, scattered Ngn1+ and Tbr2+ NPCs were present in the cortex (Fig EV1A and B). In contrast, by E11.5, vessels had formed in the lateral but not the dorsal cortex, accompanied by an increase in Ngn1+ and Tbr2+ NPCs in the lateral but not the avascular dorsal cortex (Fig EV1C and D). One day later at E12.5, vessels had now also formed in the dorsal cortex, together with a higher number of Ngn1+ and Tbr2+ NPCs at this location as compared to E11.5 (Fig EV1F and G). Similar results were obtained when using the Tis21-GFP mouse model, which labels all neurogenic NPCs (Haubensak et al, 2004) (Fig EV1E and H). The pattern of angiogenesis was confirmed using CD31 as endothelial marker (Fig EV1I–K). These data reveal a close temporo-spatial correlation between blood vessel formation and the switch to neurogenesis in vivo. Click here to expand this figure. Figure EV1. Congruence of angiogenesis and NSC differentiation in the developing mouse cortex A–H. Staining for isolectin B4 (IB4)+ blood vessels and Ngn1+ neurogenic NPCs (A, A', C, F), Tbr2+ BPs (B, B', D, G) and Tis21-GFP+ (E, H) neurogenic NPCs of the mouse cortex at E10.5 (A-B'), E11.5 (C-E), and E12.5 (F–H). Panels (A') and (B') show larger magnifications of the E10.5 cortex region above the vascular front. Arrows mark the border between lateral and dorsal cortex. Arrowheads in (A–E) denote the position of the vascular front. I–K. Immunostaining for CD31 in the cortex at E10.0 (I), E11.5 (J), and E12.5 (K). Arrowheads in (I) and (J) denote the position of the vascular front. L–O. Staining for Ki67 (green), EdU (red), and IB4 (white) in the lateral (L, N) and the dorsal (M, O) cortex at E11.5 (L, M) and E 12.5 (N, O), showing a temporo-spatial relationship between the appearance of isolectin B4+ blood vessels in the parenchyma of lateral cortex from E11.5 (M) to E 12.5 (O) and the generation of Ki67− EdU+ neurons. Asterisks indicate non-parenchymal, meningeal blood vessels. P, Q. Quantifications of the generation of Ki67− EdU+ neurons (P) and the presence of IB4+ Ki67+ endothelial cells (Q) in the lateral and dorsal cortex shown in (L–M) (mean ± SEM; N = 4; ***P < 0.001). Data information: Full and dotted lines indicate basal and apical boundaries of the cortex, respectively. N.S., not significant. Scale bars: 100 μm. Download figure Download PowerPoint We also studied neuron formation from proliferating precursors in the context of vessel ingrowth in the cortex. EdU was injected in pregnant mice at E10.5 or E11.5 and 24 h later, the EdU+ progeny of the proliferating precursors that initially incorporated the label was identified as proliferating precursors by the presence of the proliferation marker Ki67, while postmitotic neurons were Ki67-negative. When EdU was injected at E10.5 and the EdU+ progeny was analyzed at E11.5 (over a time window when vessels form in the lateral cortex), formation of EdU+/Ki67− neurons was lower in the avascular dorsal than vascularized lateral cortex (Fig EV1L–Q). When EdU was injected at E11.5 and the analysis was performed at E12.5, the rate of neurogenesis was not changed in the lateral cortex but threefold more EdU+/Ki67− neurons were detected in the newly vascularized dorsal cortex, when compared to E11.5 (Fig EV1N–Q). The neocortex has undergone substantial evolutionary expansion in mammals, implicating changes in the regulation of neural precursor expansion and differentiation between lissencephalic (mouse) and gyrencephalic (ferret) species. We thus investigated whether vessel formation and RG differentiation were also congruent in the ferret cortex at developmental stages corresponding to E10.5, E11.5, and E12.5 of mouse development, that is, E20, E24, and E28 in the ferret (Noctor et al, 1997). Staining of vessels with IB4 and BPs with Tbr2 revealed a similar pattern of angiogenesis and BP generation as seen in the mouse cortex, that is, the congruence of increased generation of BPs in the VZ from RGs and cortex vascularization at E20 (Fig EV2A–C,), E24 (Fig EV2D–F), and E28 (Fig EV2H–I). These results identified a conserved correlation between angiogenesis and the induction of RG differentiation in the cortex in lissencephalic and gyrencephalic brains. Together, these data revealed a tight temporo-spatial link between blood vessel formation and the switch from NPC expansion to neurogenesis in the developing cortex. Click here to expand this figure. Figure EV2. Congruence of angiogenesis and NSC differentiation in the developing gyrencephalic ferret cortex A–I. Stainings for Tbr2 (A, C, D, F, G, I) and isolectin B4 (IB4; B, C, E, F, H, I) in the ferret cortex at E20 (A–C), E24 (D–F) and E28 (G–I), showing induction of Tbr2+ BPs specifically in the vascularized areas of the cortex. Panels (C, F, I) are merged magnifications of the boxed areas in (A) and (B), (D) and (E), or (G) and (H), respectively. Arrowheads in (A, B, D, E) denote the position of the vascular front. Full and dotted lines indicate basal and apical boundaries of the cortex, respectively. Scale bars: 200 μm. Download figure Download PowerPoint Perturbing periventricular vessel ingrowth causes cortical hypoxia and impairs neurogenesis To investigate a possible requirement of angiogenesis for the switch of NPCs from expansion to neurogenesis, we studied cortical development in homozygous Gpr124 LacZ knock-in null (Gpr124KO) embryos, which show central nervous system (CNS)-restricted impairment and perturbation of angiogenesis in the forebrain and ventral spinal cord, but not in peripheral organs (Kuhnert et al, 2010; Anderson et al, 2011; Cullen et al, 2011). Gpr124 is an Wnt7a/b-specific co-activator of Wnt signaling in CNS endothelial cells (Zhou & Nathans, 2014). Since Gpr124 expression in the developing CNS is restricted to endothelial cells and pericytes (Kuhnert et al, 2010; Anderson et al, 2011; Cullen et al, 2011), the effect of global Gpr124 deficiency is blood vessel specific in the brain. Since Gpr124KO embryos are embryonic lethal from E15.5 on (Kuhnert et al, 2010), we used these embryos until E13.5 to exclude general effects of embryonic morbidity. Consistent with previous reports (Kuhnert et al, 2010; Anderson et al, 2011; Cullen et al, 2011), the normal periventricular vascular plexus was nearly completely absent, and only disorganized vascular tufts were formed in the ventral forebrain and neocortex, but not in the cortical hem (Fig 1A and B, asterisk) of Gpr124KO embryos at E13.5. Wild-type (WT) and heterozygous-deficient littermates were indistinguishable (together referred to as controls). Staining for the hypoxia marker pimonidazole revealed increased levels of hypoxia, and neuroepithelial expression of the hypoxia-inducible gene Glut1 was elevated in the cortex of E13.5 Gpr124KO embryos (Appendix Fig S2A–D). Figure 1. Suppression of brain angiogenesis expands radial glia cells A, B. Immunostaining for CD31+ blood vessels in control (ctrl, A) and Gpr124KO (B) forebrains. Note the normal vasculature in the cortical hem (asterisk) of Gpr124KO mice. C, D. Immunostaining for Pax6 in control (C) and Gpr124KO (D) forebrains to reveal the lateral extension of the neocortex (dashed line) between the cortical hem (arrowhead) and the lateral ganglionic eminence (arrow). E. Quantification of the lateral extension of the cortex in control and Gpr124KO brains (mean ± SEM; N = 10; ***P < 0.001). F, G. Staining for EdU (red) and Ki67 (green) in control (F) and Gpr124KO (G) cortices at E13.5, 24 h after EdU injection. H. Quantification of Ki67+ NPCs in the VZ (blue), SVZ/CP (gray) and of Ki67− neurons (cyan) generated from EdU-labeled NPCs in control and Gpr124KO embryos (mean ± SEM; N = 4 (control) and N = 3 (Gpr124KO); *P < 0.05, **P < 0.01). I, J. Immunostaining for Tbr2 (red) and Pax6 (green) in control (I) and Gpr124KO (J) cortices. K. Quantification of newborn BPs in the VZ of control and Gpr124KO cortices (mean ± SEM; N = 4; *P < 0.05). L, M. Immunostaining for Tbr2 (red) and Ngn1 (green) in control (L) and Gpr124KO (M) cortices. N, O. Quantification of neurogenic (Ngn1+) RGs and BPs (N) or expanding (Tbr2− Ngn1−) RGs (O) in control and Gpr124KO cortices (mean ± SEM; N = 4; *P < 0.05, ***P < 0.001). Note that the fraction of total BPs is significantly reduced (see K), while the fraction of Ngn1+ BPs is insignificantly reduced (Fig 1N), because the proportion of Ngn1+ Tbr2+ cells within all BPs is slightly, though statistically insignificantly higher in the Gpr124KO brains (see Appendix Fig S2M). P, Q. Stainings for Tbr2 (red) and for Tis21-GFP expression (green) in control (P) and Gpr124KO (Q) cortices. R, S. Quantification of neurogenic (Tis21-GFP+) RGs and BPs (R) or expanding (Tbr2− Tis21-GFP−) RGs (S) in control and Gpr124KO cortices (mean ± SEM; N = 4; ***P < 0.001). Data information: Full, dotted, and dashed lines indicate basal and apical boundaries of the cortex or the basal boundary of the VZ, respectively. CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; and VZ, ventricular zone. Scale bars: 250 μm (B, D) or 50 μm (G, J, M, Q), respectively. Download figure Download PowerPoint Suppression of periventricular vessel ingrowth inhibits the switch from RG expansion to neurogenesis Gpr124KO brains showed notably wider and thinner cortices, a hallmark of increased RG expansion (Farkas et al, 2008; Siegenthaler et al, 2009). We thus measured the lateral extension of the neocortex marked by Pax6 expression, between its border with the cortical hem (Fig 1C and D, arrowhead) and the border between the neocortex and lateral ganglionic eminence (Fig 1C and D, arrow). Notably, lateral extension of the cortex was increased in Gpr124KO embryos, indicative of increased RG expansion (Fig 1E). To assess whether inhibition of brain angiogenesis in Gpr124KO embryos affected neurogenesis, we injected EdU at E12.5 and analyzed the cellular identity of EdU+ cells at E13.5. The proportion of EdU+/Ki67− newborn neurons was lower in Gpr124KO than WT embryos, while correspondingly the proportion of EdU+/Ki67+ precursors in the VZ was increased (Fig 1F–H). Thus, suppression of vessel formation inhibits neurogenesis and increases NSC expansion. To explore whether changes in the RG or BP population were underlying the reduced neurogenesis in Gpr124KO as compared to WT embryos, we stained for the cortical RG marker Pax6 and the BP marker Tbr2. The total proportion of newborn RG-derived BPs, migrating through the VZ at E13.5, was decreased in Gpr124KO embryos (Fig 1I–K). Importantly, all Tbr2− cells in the VZ expressed Pax6, identifying them as RGs (Fig 1I and J). In addition, we performed birthdating of newborn BPs by EdU injection at E13.5, and after an 8-h chase to allow division of the labeled cells, we counted only newly generated EdU+ BPs in the VZ (Appendix Fig S2E–J). EdU+ BPs in the VZ were approaching but had not reached the subventricular zone (SVZ) in WT and Gpr124KO embryos, enabling us to distinguish them from the EdU+ progeny of preexisting BPs in the SVZ. Strikingly, the fraction of EdU+ VZ cells that were newborn RG-derived BPs was reduced in Gpr124KO brains (Appendix Fig S2K), confirming that RGs generate fewer BPs upon perturbation of brain angiogenesis. To further investigate whether the switch of RGs, BPs, or both NPCs from expansion to neurogenesis was impaired in Gpr124KO embryos, we quantified the proportion of neurogenic RGs (Ngn1+/Tbr2−) and newborn neurogenic BPs (Ngn1+/Tbr2+) in the cortical VZ at E13.5. While the proportion of neurogenic Ngn1+/Tbr2+ BPs was only insignificantly reduced, the neurogenic Ngn1+/Tbr2− RG population was strikingly reduced in Gpr124KO embryos (Fig 1L–N). Consequently, the proportion of expanding RGs that are Ngn1−/Tbr2− was increased (Fig 1L–O). Since Ngn1 is only present during the G1 phase of the cell cycle (Britz et al, 2006), we confirmed our findings in Gpr124KO and control embryos expressing Tis21-GFP, which is present in all neurogenic NPCs. The proportions of both neurogenic Tis21-GFP+ BPs and RGs were reduced in Gpr124KO embryos compared to controls, while that of expanding RGs (Tbr2−Tis21-GFP−) was enlarged (Fig 1P–S). In addition, we confirmed that apoptosis in the cortex was low and not altered by Gpr124 deletion (Appendix Fig S2L). Together, suppression of vessel formation in Gpr124KO embryos reduced neurogenesis and RG differentiation. Inhibition of brain angiogenesis does not change BP fate We then investigated whether the fate of BPs was changed upon perturbation of brain angiogenesis by quantifying the expression of the neurogenic markers Ngn1 and Tis21-GFP in the BP population. The proportion of Ngn1+/Tbr2+ as well as Tis21-GFP+/Tbr2+ BPs in the VZ was similar in control and Gpr124KO BPs (Appendix Fig S2M and N), suggesting that cell fate of the BPs was not altered after inhibition of brain angiogenesis and that the regulation of the switch to neurogenesis by angiogenesis occurred most likely at the level of RGs rather than BPs. Vascular deletion of Gpr124 is sufficient for increased RG expansion To exclude the possibility that non-vascular changes were responsible for the
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