RECK in Neural Precursor Cells Plays a Critical Role in Mouse Forebrain Angiogenesis

•Mice lacking RECK in Foxg1-positive neural precursor cells die shortly after birth•These mice show vascular defects similar to those in mice lacking endothelial RECK•The vascular phenotype can be suppressed by LiCl, an activator of WNT signaling•RECK in WNT7-producing cell enhances contact-dependent WNT signaling in adjacent cells RECK in neural precursor cells (NPCs) was previously found to support Notch-dependent neurogenesis in mice. On the other hand, recent studies implicate RECK in endothelial cells (ECs) in WNT7-triggered canonical WNT signaling essential for brain angiogenesis. Here we report that RECK in NPCs is also critical for brain angiogenesis. When Reck is inactivated in Foxg1-positive NPCs, mice die shortly after birth with hemorrhage in the forebrain, with angiogenic sprouts stalling at the periphery and forming abnormal aggregates reminiscent of those in EC-selective Reck knockout mice and Wnt7a/b-deficient mice. The hemorrhage can be pharmacologically suppressed by lithium chloride. An effect of RECK in WNT7-producing cells to enhance canonical WNT-signaling in reporter cells is detectable in mixed culture but not with conditioned medium. Our findings suggest that NPC-expressed RECK has a non-cell-autonomous function to promote forebrain angiogenesis through contact-dependent enhancement of WNT signaling in ECs, implying possible involvement of RECK in neurovascular coupling. RECK in neural precursor cells (NPCs) was previously found to support Notch-dependent neurogenesis in mice. On the other hand, recent studies implicate RECK in endothelial cells (ECs) in WNT7-triggered canonical WNT signaling essential for brain angiogenesis. Here we report that RECK in NPCs is also critical for brain angiogenesis. When Reck is inactivated in Foxg1-positive NPCs, mice die shortly after birth with hemorrhage in the forebrain, with angiogenic sprouts stalling at the periphery and forming abnormal aggregates reminiscent of those in EC-selective Reck knockout mice and Wnt7a/b-deficient mice. The hemorrhage can be pharmacologically suppressed by lithium chloride. An effect of RECK in WNT7-producing cells to enhance canonical WNT-signaling in reporter cells is detectable in mixed culture but not with conditioned medium. Our findings suggest that NPC-expressed RECK has a non-cell-autonomous function to promote forebrain angiogenesis through contact-dependent enhancement of WNT signaling in ECs, implying possible involvement of RECK in neurovascular coupling. Precise bidirectional communications between the nervous and vascular systems (i.e., neurovascular coupling) is essential for proper formation and functioning of the central nervous system (CNS) in vertebrates (Paredes et al., 2018Paredes I. Himmels P. Ruiz de Almodovar C. Neurovascular communication during CNS development.Dev. Cell. 2018; 45: 10-32Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Several neuroepithelium-derived molecules crucial for CNS-specific angiogenesis have been discovered, which include ID1/ID3 (Lyden et al., 1999Lyden D. Young A.Z. Zagzag D. Yan W. Gerald W. O'Reilly R. Bader B.L. Hynes R.O. Zhuang Y. Manova K. Benezra R. Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts.Nature. 1999; 401: 670-677Crossref PubMed Scopus (777) Google Scholar), integrin alpha-V (Bader et al., 1998Bader B.L. Rayburn H. Crowley D. Hynes R.O. Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all alpha v integrins.Cell. 1998; 95: 507-519Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, McCarty et al., 2002McCarty J.H. Monahan-Earley R.A. Brown L.F. Keller M. Gerhardt H. Rubin K. Shani M. Dvorak H.F. Wolburg H. Bader B.L. et al.Defective associations between blood vessels and brain parenchyma lead to cerebral hemorrhage in mice lacking alphav integrins.Mol. Cell. Biol. 2002; 22: 7667-7677Crossref PubMed Scopus (141) Google Scholar, McCarty et al., 2005McCarty J.H. Lacy-Hulbert A. Charest A. Bronson R.T. Crowley D. Housman D. Savill J. Roes J. Hynes R.O. Selective ablation of alphav integrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration and premature death.Development. 2005; 132: 165-176Crossref PubMed Scopus (186) Google Scholar), integrin beta-8 (Zhu et al., 2002Zhu J. Motejlek K. Wang D. Zang K. Schmidt A. Reichardt L.F. beta8 integrins are required for vascular morphogenesis in mouse embryos.Development. 2002; 129: 2891-2903Crossref PubMed Google Scholar, Proctor et al., 2005Proctor J.M. Zang K. Wang D. Wang R. Reichardt L.F. Vascular development of the brain requires beta8 integrin expression in the neuroepithelium.J. Neurosci. 2005; 25: 9940-9948Crossref PubMed Scopus (136) Google Scholar), WNT7A/B (Stenman et al., 2008Stenman J.M. Rajagopal J. Carroll T.J. Ishibashi M. McMahon J. McMahon A.P. Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature.Science. 2008; 322: 1247-1250Crossref PubMed Scopus (442) Google Scholar, Daneman et al., 2009Daneman R. Agalliu D. Zhou L. Kuhnert F. Kuo C.J. Barres B.A. Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis.Proc. Natl. Acad. Sci. U S A. 2009; 106: 641-646Crossref PubMed Scopus (497) Google Scholar), and TGFBR2 (Hellbach et al., 2014Hellbach N. Weise S.C. Vezzali R. Wahane S.D. Heidrich S. Roidl D. Pruszak J. Esser J.S. Vogel T. Neural deletion of Tgfbr2 impairs angiogenesis through an altered secretome.Hum. Mol. Genet. 2014; 23: 6177-6190Crossref PubMed Scopus (29) Google Scholar). However, it remains to be elucidated exactly how these molecules contribute to the neurovascular communication. RECK (reversion-inducing cysteine-rich protein with Kazal motifs) encodes a glycosylphosphatidylinositol-anchored glycoprotein capable of regulating several extracellular metalloproteinases (Takahashi et al., 1998Takahashi C. Sheng Z. Horan T.P. Kitayama H. Maki M. Hitomi K. Kitaura Y. Takai S. Sasahara R.M. Horimoto A. et al.Regulation of matrix metalloproteinase-9 and inhibition of tumor invasion by the membrane-anchored glycoprotein RECK.Proc. Natl. Acad. Sci. U S A. 1998; 95: 13221-13226Crossref PubMed Scopus (418) Google Scholar). Global Reck knockout mice die around embryonic day 10.5 (E10.5) with reduced tissue integrity, abdominal hemorrhage (Oh et al., 2001Oh J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. Alexander D.B. et al.The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis.Cell. 2001; 107: 789-800Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar), and precocious neuronal differentiation (Muraguchi et al., 2007Muraguchi T. Takegami Y. Ohtsuka T. Kitajima S. Chandana E.P. Omura A. Miki T. Takahashi R. Matsumoto N. Ludwig A. et al.RECK modulates Notch signaling during cortical neurogenesis by regulating ADAM10 activity.Nat. Neurosci. 2007; 10: 838-845Crossref PubMed Scopus (127) Google Scholar). Around E10.5, normal mice express RECK abundantly in blood vessels (both endothelial cells [ECs] and mural cells) as well as neural precursor cells (NPCs) (Oh et al., 2001Oh J. Takahashi R. Kondo S. Mizoguchi A. Adachi E. Sasahara R.M. Nishimura S. Imamura Y. Kitayama H. Alexander D.B. et al.The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis.Cell. 2001; 107: 789-800Abstract Full Text Full Text PDF PubMed Scopus (590) Google Scholar, Muraguchi et al., 2007Muraguchi T. Takegami Y. Ohtsuka T. Kitajima S. Chandana E.P. Omura A. Miki T. Takahashi R. Matsumoto N. Ludwig A. et al.RECK modulates Notch signaling during cortical neurogenesis by regulating ADAM10 activity.Nat. Neurosci. 2007; 10: 838-845Crossref PubMed Scopus (127) Google Scholar, Chandana et al., 2010Chandana E.P. Maeda Y. Ueda A. Kiyonari H. Oshima N. Yamamoto M. Kondo S. Oh J. Takahashi R. Yoshida Y. et al.Involvement of the Reck tumor suppressor protein in maternal and embryonic vascular remodeling in mice.BMC Dev. Biol. 2010; 10: 84Crossref PubMed Scopus (41) Google Scholar). To explore the functions of RECK in mice beyond E10.5, we generated Reck-flox mice (Chandana et al., 2010Chandana E.P. Maeda Y. Ueda A. Kiyonari H. Oshima N. Yamamoto M. Kondo S. Oh J. Takahashi R. Yoshida Y. et al.Involvement of the Reck tumor suppressor protein in maternal and embryonic vascular remodeling in mice.BMC Dev. Biol. 2010; 10: 84Crossref PubMed Scopus (41) Google Scholar, Yamamoto et al., 2012Yamamoto M. Matsuzaki T. Takahashi R. Adachi E. Maeda Y. Yamaguchi S. Kitayama H. Echizenya M. Morioka Y. Alexander D.B. et al.The transformation suppressor gene Reck is required for postaxial patterning in mouse forelimbs.Biol. Open. 2012; 1: 458-466Crossref PubMed Scopus (15) Google Scholar). Our earlier study using temporally inducible Reck knockout mice revealed that inactivation of Reck at around E11 results in vascular defects including forebrain hemorrhage and vascular malformation by E15.5 and embryonic death before birth (Chandana et al., 2010Chandana E.P. Maeda Y. Ueda A. Kiyonari H. Oshima N. Yamamoto M. Kondo S. Oh J. Takahashi R. Yoshida Y. et al.Involvement of the Reck tumor suppressor protein in maternal and embryonic vascular remodeling in mice.BMC Dev. Biol. 2010; 10: 84Crossref PubMed Scopus (41) Google Scholar). The roles of RECK in different cell types, however, could not be discriminated in such system. A more recent study using cell type-selective Reck knockout mice revealed that Reck inactivation in mural cells recapitulates the E10.5 lethality of global knockout mice, whereas Reck inactivation in ECs results in perinatal death with brain hemorrhage (Almeida et al., 2015Almeida G.M. Yamamoto M. Morioka Y. Ogawa S. Matsuzaki T. Noda M. Critical roles for murine Reck in the regulation of vascular patterning and stabilization.Sci. Rep. 2015; 5: 17860Crossref PubMed Scopus (21) Google Scholar), further highlighting the importance of RECK in vascular development. Recent studies also indicate that RECK binds and cooperates with GPR124, an orphan G-protein-coupled receptor, to facilitate the canonical WNT signaling in ECs triggered by WNT7A/B that is required for proper tip cell function, CNS angiogenesis, and blood-brain barrier maturation (Vanhollebeke et al., 2015Vanhollebeke B. Stone O.A. Bostaille N. Cho C. Zhou Y. Maquet E. Gauquier A. Cabochette P. Fukuhara S. Mochizuki N. et al.Tip cell-specific requirement for an atypical Gpr124- and Reck-dependent Wnt/beta-catenin pathway during brain angiogenesis.Elife. 2015; 4 (e06489)Crossref PubMed Scopus (146) Google Scholar; Ulrich et al., 2016Ulrich F. Carretero-Ortega J. Menendez J. Narvaez C. Sun B. Lancaster E. Pershad V. Trzaska S. Veliz E. Kamei M. et al.Reck enables cerebrovascular development by promoting canonical Wnt signaling.Development. 2016; 143: 147-159Crossref PubMed Scopus (41) Google Scholar; Cho et al., 2017Cho C. Smallwood P.M. Nathans J. Reck and Gpr124 are essential receptor cofactors for Wnt7a/Wnt7b-specific signaling in mammalian CNS angiogenesis and blood-brain barrier regulation.Neuron. 2017; 95: 1221-1225Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar; Vallon et al., 2018Vallon M. Yuki K. Nguyen T.D. Chang J. Yuan J. Siepe D. Miao Y. Essler M. Noda M. Garcia K.C. Kuo C.J. A RECK-WNT7 receptor-ligand interaction enables isoform-specific regulation of Wnt bioavailability.Cell Rep. 2018; 25: 339-349.e9Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Interestingly, RECK was found to directly bind WNT7A/B and confer ligand specificity to the FZD4-LRP5/6 receptor complex (Eubelen et al., 2018Eubelen M. Bostaille N. Cabochette P. Gauquier A. Tebabi P. Dumitru A.C. Koehler M. Gut P. Alsteens D. Stainier D.Y.R. et al.A molecular mechanism for Wnt ligand-specific signaling.Science. 2018; 361 (eaat1178)Crossref PubMed Scopus (116) Google Scholar, Vallon et al., 2018Vallon M. Yuki K. Nguyen T.D. Chang J. Yuan J. Siepe D. Miao Y. Essler M. Noda M. Garcia K.C. Kuo C.J. A RECK-WNT7 receptor-ligand interaction enables isoform-specific regulation of Wnt bioavailability.Cell Rep. 2018; 25: 339-349.e9Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). As our earlier study using global Reck knockout mice implicated RECK in CNS development (Muraguchi et al., 2007Muraguchi T. Takegami Y. Ohtsuka T. Kitajima S. Chandana E.P. Omura A. Miki T. Takahashi R. Matsumoto N. Ludwig A. et al.RECK modulates Notch signaling during cortical neurogenesis by regulating ADAM10 activity.Nat. Neurosci. 2007; 10: 838-845Crossref PubMed Scopus (127) Google Scholar), we attempted to confirm and extend that finding by inactivating Reck selectively in the Foxg1-positive NPCs in mice, expecting to find some neural deficiency. Characterization of these mice, however, revealed an unexpected role for NPC-expressed RECK in CNS angiogenesis. To selectively inactivate Reck in NPCs, we chose to use a Foxg1-Cre transgenic line (Hebert and McConnell, 2000Hebert J.M. McConnell S.K. Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures.Dev. Biol. 2000; 222: 296-306Crossref PubMed Scopus (432) Google Scholar). When visualized with the mTmG reporter system (Muzumdar et al., 2007Muzumdar M.D. Tasic B. Miyamichi K. Li L. Luo L. A global double-fluorescent Cre reporter mouse.Genesis. 2007; 45: 593-605Crossref PubMed Scopus (2258) Google Scholar), Foxg1-Cre-expressed cells (i.e., green cells in mTmG;Foxg1-Cre mice) are abundant in telencephalon at E8.5 and persist in a large area of the forebrain from E9.5 onward (Figure 1A; green signals). We generated mice carrying this Foxg1-Cre allele and one or two Reck-flox allele(s). The Reck-flox heterozygotes (Reckflex1/+;Foxg1-Cre) are normal in gross morphology, fertile, and hence used as a control in this study. On the other hand, the Reck-flox homozygotes (Reckflex1/flex1;Foxg1-Cre), which we call Reck-cKO (Foxg1), are not found among the adult littermates obtained from the mating supposed to yield such offspring at the 25% frequency (i.e., Reckflex1/+;Foxg1-Cre x Reckflex1/flex1). Careful examination of newborn pups revealed that Reck-cKO (Foxg1) mice are viable up to the day of birth (P0) but die shortly after birth exhibiting forebrain hemorrhage (Figure 1B). When traced back, visible forebrain hemorrhage occurred in 74% (29/39) of Reck-cKO (Foxg1) mice at E12.5 (Figure 1C, red diamond) and in all Reck-cKO (Foxg1) mice at E13.5 (Figure 1D, arrow) as well as at later embryonic time points (Figure 1C, E13.5–E18.5). Histological examinations indicate that hemorrhage mainly occurs in ganglionic eminence (GE) at early stages (Figure 1E-1, arrows) but becomes prominent in cerebral cortex (Cx) at later stages (Figure 1E-3, arrows). Immunofluorescence staining of brain sections of mTmG;Foxg1-Cre reporter mice, as shown in Figure 1A, indicates that Foxg1-Cre-expressed cells are neither CD31 positive (vascular ECs) nor NG2 positive (vascular mural cells) (Figure 1F). A previous study by Hellbach et al. also indicated that Foxg1-Cre is expressed in neuronal cells, but not in vascular cells, in the forebrain in this transgenic line (Hellbach et al., 2014Hellbach N. Weise S.C. Vezzali R. Wahane S.D. Heidrich S. Roidl D. Pruszak J. Esser J.S. Vogel T. Neural deletion of Tgfbr2 impairs angiogenesis through an altered secretome.Hum. Mol. Genet. 2014; 23: 6177-6190Crossref PubMed Scopus (29) Google Scholar). These data support the idea that the phenotype of Reck-cKO (Foxg1) mice results from the lack of RECK in NPCs rather than vascular cells. CD31 is known to be expressed in ECs and some blood cells (Privratsky et al., 2010Privratsky J.R. Newman D.K. Newman P.J. PECAM-1: conflicts of interest in inflammation.Life Sci. 2010; 87: 69-82Crossref PubMed Scopus (128) Google Scholar). When forebrain sections from E12.5 embryos were stained with anti-CD31, a line of regularly spaced small loops (representing cross sections of blood vessels) was found near the ventricular edge of both GE and Cx in control mice (Figures 2B and 2C , arrows). In Reck-cKO (Foxg1) mice, however, abnormal aggregates of CD31-positive cells or loops are found in GE near the perineural vascular plexus or midway toward the ventricle (Figure 2E, arrowheads); these abnormal vessels are proliferative (Figure S1A) and reminiscent of the glomeruloid malformations found in Wnt7A/Wnt7b double-knockout mice (Stenman et al., 2008Stenman J.M. Rajagopal J. Carroll T.J. Ishibashi M. McMahon J. McMahon A.P. Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature.Science. 2008; 322: 1247-1250Crossref PubMed Scopus (442) Google Scholar, Daneman et al., 2009Daneman R. Agalliu D. Zhou L. Kuhnert F. Kuo C.J. Barres B.A. Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis.Proc. Natl. Acad. Sci. U S A. 2009; 106: 641-646Crossref PubMed Scopus (497) Google Scholar) and Gpr124 knockout mice (Kuhnert et al., 2010Kuhnert F. Mancuso M.R. Shamloo A. Wang H.T. Choksi V. Florek M. Su H. Fruttiger M. Young W.L. Heilshorn S.C. Kuo C.J. Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124.Science. 2010; 330: 985-989Crossref PubMed Scopus (214) Google Scholar). On the other hand, very few vessels were found in the cortex of Reck-cKO (Foxg1) mice (Figure 2F). Less severe but similar vascular abnormalities were found in cKO (Foxg1) mice at E11.5 (Figures 2L–2P). In GE, CD31-positive (red) cells form multilayered tubes with wide opening that are associated with NG2-positive (green) cells (Figure 2O, arrowheads). At E13.5, Reck-cKO (Foxg1) mice show more advanced vascular abnormalities, including the lack of a line of regularly spaced periventricular vessels (Figures 3G and 3H ; see arrows in Figures 3C and 3D); large aggregates of CD31-positive, proliferative cells midway toward the ventricle (Figure 3G, arrowheads; Figures S1B–S1D); and a central region of apparent tissue damage (Figure 3G, asterisk) in GE. In the cortex, some vessels consisting of multilayered CD31-positive cells with round luminal space are found in the cortex (Figure 3H, arrowheads). Taken together, these findings indicate that Reck expressed in NPCs is critical for proper angiogenesis in the forebrain, especially in GE and Cx. The vascular abnormalities in the forebrains of Reck-cKO (Foxg1) mice are accompanied by the increased number and widened zone of TUJ1-positive differentiated neurons (Figure 2Q, compare green signals in panels 1 and 2) and the reduced number and narrower zone of Ki67-positive proliferative cells in both Cx and GE (Figure S2). Hence, precocious neuronal differentiation, a phenotype previously found in global Reck knockout mice (Muraguchi et al., 2007Muraguchi T. Takegami Y. Ohtsuka T. Kitajima S. Chandana E.P. Omura A. Miki T. Takahashi R. Matsumoto N. Ludwig A. et al.RECK modulates Notch signaling during cortical neurogenesis by regulating ADAM10 activity.Nat. Neurosci. 2007; 10: 838-845Crossref PubMed Scopus (127) Google Scholar), is recapitulated in Reck-cKO (Foxg1) mice, suggesting that RECK produced by NPCs affects both neurogenesis and angiogenesis. For comparison, we generated EC-selective conditional knockout mice using a Tie2-Cre transgenic line (Kisanuki et al., 2001Kisanuki Y.Y. Hammer R.E. Miyazaki J. Williams S.C. Richardson J.A. Yanagisawa M. Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo.Dev. Biol. 2001; 230: 230-242Crossref PubMed Scopus (932) Google Scholar). The Reckflex1/flex1;Tie2-Cre mice (Reck-cKO [Tie2] mice in short) show phenotypes reminiscent of those of Reck-cKO (Foxg1) mice. For instance, at E13.5, the lack of a line of regularly spaced periventricular vessels in both the GE and cortex (Figures 3O and 3P; see arrows in Figures 3K and 3L), large aggregates of CD31-positive cells (Figure 3O, arrowheads), and a central tissue damage (Figure 3O, asterisk) in GE are evident. On the other hand, misexpression of TUJ1 is not observed in Reck-cKO (Tie2) mice (Figure S3), suggesting distinct effects of Reck in ECs and NPCs (see Figures 2Q and S2) on neuronal differentiation. Nevertheless, our data indicate that Reck deficiency in ECs and Reck deficiency in NPCs give rise to a vascular phenotype very similar both in timing and locations in the forebrain. Importance of the WNT7a/WNT7b-dependent canonical WNT signaling in brain angiogenesis (Stenman et al., 2008Stenman J.M. Rajagopal J. Carroll T.J. Ishibashi M. McMahon J. McMahon A.P. Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature.Science. 2008; 322: 1247-1250Crossref PubMed Scopus (442) Google Scholar, Daneman et al., 2009Daneman R. Agalliu D. Zhou L. Kuhnert F. Kuo C.J. Barres B.A. Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis.Proc. Natl. Acad. Sci. U S A. 2009; 106: 641-646Crossref PubMed Scopus (497) Google Scholar) and the EC-autonomous function of RECK to specifically enhance such signaling (Vanhollebeke et al., 2015Vanhollebeke B. Stone O.A. Bostaille N. Cho C. Zhou Y. Maquet E. Gauquier A. Cabochette P. Fukuhara S. Mochizuki N. et al.Tip cell-specific requirement for an atypical Gpr124- and Reck-dependent Wnt/beta-catenin pathway during brain angiogenesis.Elife. 2015; 4 (e06489)Crossref PubMed Scopus (146) Google Scholar; Ulrich et al., 2016Ulrich F. Carretero-Ortega J. Menendez J. Narvaez C. Sun B. Lancaster E. Pershad V. Trzaska S. Veliz E. Kamei M. et al.Reck enables cerebrovascular development by promoting canonical Wnt signaling.Development. 2016; 143: 147-159Crossref PubMed Scopus (41) Google Scholar; Cho et al., 2017Cho C. Smallwood P.M. Nathans J. Reck and Gpr124 are essential receptor cofactors for Wnt7a/Wnt7b-specific signaling in mammalian CNS angiogenesis and blood-brain barrier regulation.Neuron. 2017; 95: 1221-1225Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar; Eubelen et al., 2018Eubelen M. Bostaille N. Cabochette P. Gauquier A. Tebabi P. Dumitru A.C. Koehler M. Gut P. Alsteens D. Stainier D.Y.R. et al.A molecular mechanism for Wnt ligand-specific signaling.Science. 2018; 361 (eaat1178)Crossref PubMed Scopus (116) Google Scholar; Vallon et al., 2018Vallon M. Yuki K. Nguyen T.D. Chang J. Yuan J. Siepe D. Miao Y. Essler M. Noda M. Garcia K.C. Kuo C.J. A RECK-WNT7 receptor-ligand interaction enables isoform-specific regulation of Wnt bioavailability.Cell Rep. 2018; 25: 339-349.e9Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) has been demonstrated. However, because RECK is a membrane-anchored protein, it is not obvious how the RECK expressed in NPCs can also affect angiogenesis. To address this question, we first tested whether canonical WNT signaling is affected in Reck-cKO (Foxg1) mice by daily administration of lithium chloride (LiCl) into the pregnant mice followed by morphological examination of Reck-cKO (Foxg1) embryos. LiCl is known to inhibit glycogen synthase kinase-3β thereby bypassing the ligand-receptor interactions and directly activating downstream cascade of canonical WNT signaling (Klein and Melton, 1996Klein P.S. Melton D.A. A molecular mechanism for the effect of lithium on development.Proc. Natl. Acad. Sci. U S A. 1996; 93: 8455-8459Crossref PubMed Scopus (2079) Google Scholar); the agent has been used to demonstrate the involvement of canonical WNT signaling during various developmental processes in mouse embryos (Cohen et al., 2007Cohen E.D. Wang Z. Lepore J.J. Lu M.M. Taketo M.M. Epstein D.J. Morrisey E.E. Wnt/beta-catenin signaling promotes expansion of Isl-1-positive cardiac progenitor cells through regulation of FGF signaling.J. Clin. Invest. 2007; 117: 1794-1804Crossref PubMed Scopus (213) Google Scholar, Cohen et al., 2009Cohen E.D. Ihida-Stansbury K. Lu M.M. Panettieri R.A. Jones P.L. Morrisey E.E. Wnt signaling regulates smooth muscle precursor development in the mouse lung via a tenascin C/PDGFR pathway.J. Clin. Invest. 2009; 119: 2538-2549Crossref PubMed Scopus (151) Google Scholar, Kugimiya et al., 2007Kugimiya F. Kawaguchi H. Ohba S. Kawamura N. Hirata M. Chikuda H. Azuma Y. Woodgett J.R. Nakamura K. Chung U.I. GSK-3beta controls osteogenesis through regulating Runx2 activity.PLoS One. 2007; 2: e837Crossref PubMed Scopus (120) Google Scholar, Tian et al., 2010Tian Y. Yuan L. Goss A.M. Wang T. Yang J. Lepore J.J. Zhou D. Schwartz R.J. Patel V. Cohen E.D. Morrisey E.E. Characterization and in vivo pharmacological rescue of a Wnt2-Gata6 pathway required for cardiac inflow tract development.Dev. Cell. 2010; 18: 275-287Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, Griffin et al., 2011Griffin C.T. Curtis C.D. Davis R.B. Muthukumar V. Magnuson T. The chromatin-remodeling enzyme BRG1 modulates vascular Wnt signaling at two levels.Proc. Natl. Acad. Sci. U S A. 2011; 108: 2282-2287Crossref PubMed Scopus (86) Google Scholar, Curtis and Griffin, 2012Curtis C.D. Griffin C.T. The chromatin-remodeling enzymes BRG1 and CHD4 antagonistically regulate vascular Wnt signaling.Mol. Cell. Biol. 2012; 32: 1312-1320Crossref PubMed Scopus (31) Google Scholar, Cornett et al., 2013Cornett B. Snowball J. Varisco B.M. Lang R. Whitsett J. Sinner D. Wntless is required for peripheral lung differentiation and pulmonary vascular development.Dev. Biol. 2013; 379: 38-52Crossref PubMed Scopus (45) Google Scholar, Cambier et al., 2014Cambier L. Plate M. Sucov H.M. Pashmforoush M. Nkx2-5 regulates cardiac growth through modulation of Wnt signaling by R-spondin3.Development. 2014; 141: 2959-2971Crossref PubMed Scopus (39) Google Scholar, Briggs et al., 2015Briggs L.E. Burns T.A. Lockhart M.M. Phelps A.L. Van den Hoff M.J. Wessels A. Wnt/beta-catenin and sonic hedgehog pathways interact in the regulation of the development of the dorsal mesenchymal protrusion.Dev. Dyn. 2015; 245: 103-113Crossref PubMed Scopus (23) Google Scholar, Da Silva et al., 2017Da Silva F. Rocha A.S. Motamedi F.J. Massa F. Basboga C. Morrison H. Wagner K.D. Schedl A. Coronary artery formation is driven by localized expression of R-spondin3.Cell Rep. 2017; 20: 1745-1754Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). First, untreated Reck-cKO (Foxg1) mice at E15.5 show hemorrhage in both GE and the cortex (Cx) at 100% penetrance (Figure 4A; Table 1, a; Figure S4). In contrast, daily administration of LiCl from E8.5 to E14.5 suppressed macroscopic hemorrhage completely in the cortex and partially in GE (Table 1, b). We therefore compared the effects of LiCl injected at different timings. The results indicate that the treatments from E10.5 to E14.5 (Table 1, d) are essential for this suppression. Histological examinations of the LiCl-treated mice indicated that small hemorrhagic legions were found in GE in some of the animals but not in their cortices (Figures 4B and 4C); this was in sharp contrast to the numerous large hemorrhagic legions found in both the GE and the cortex of untreated animals that resulted in the leakage of blood into the ventricles (Figure 4A). These findings support the idea that RECK in NPCs influences canonical WNT signaling.Table 1Suppression of Forebrain Hemorrhage by LiCl-AdministrationSchedule for Daily Administration of LiCl (200 mg/kg/day, i.p.)Number of Embryos[(Hemorrhage at E15.5)/(cKO Embryos Examined)]Stage (E)8.59.510.511.512.513.514.5Ganglionic EminenceCerebral Cortexa45/4545/45b×××××××3/40/4c×××××2/22/2d×××××9/110/11e××××5/51/5f××3/33/3i.p., intraperitoneal injection; cKO, conditional knockout (Reckflex1/flex1;Foxg1-Cre); x, LiCl injected. See also Figure S4. Open table in a new tab i.p., intraperitoneal injection; cKO, conditional knockout (Reckflex1/flex1;Foxg1-Cre); x, LiCl injected. See also Figure S4. To gain some insights into the possible mechanism by which RECK in NPCs may affect canonical WNT signaling and vascular development in the forebrain, we determined the levels of mRNAs encoding some molecules relevant to brain development, angiogenesis, and WNT signaling using total RNA extracted from forebrain of embryos at three time points (E11.5, E12.5, and E13.5) by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) (Figure 5A). At the first time point (E11.5; blue symbols), significant downregulation of an NPC marker, Nes, was detected (Figure 5A, group 23). At the second time point (E12.5; red symbols), upregulation of Mmp2 (protease), Id3 (transcription factor), and downregulation of Wnt7a, Itg8b (cell adhesion receptor), and two endogenous targets of canonical WNT signaling, Apcdd1 and Sox17, became significant (Figure 5A, groups 3, 6, 12, 38, and 41). At the third time point (E13.5; green symbols), significant upregulation of Mmp2, Vegfa (vascular endothelial growth factor A), downregulation of Wnt7a, Wnt7b, Apcdd1, and Sox17 were noted (Figure 5A, groups 4, 7, 10, 22, 39, and 42). Downregulation of Apcdd1 and Sox17 provides additional evidence suggesting that canonical WNT signaling is affected by the Reck deficiency. Some of the other alterations in gene expression may directly or indirectly contribute to the mutant phenotype (see Discussion). Neuroepithelium-derived WNT7A/B (leading to canonical Wnt signaling in CNS ECs) is known to be specifically required for CNS vascularization (Stenman et al., 2008Stenman J.M. Rajagopal J. Carroll T.J. Ishibashi M. McMahon J. McMahon A.P. Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature.Science. 2008; 322: 1247-1250Crossref PubMed Scopus (442) Google Scholar, Daneman et al., 2009Daneman R. Agalliu D. Zhou L. Kuhnert F. Kuo C.J. Barres B.A. Wnt/beta-catenin signaling is require