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• W2969245735 startingPage "2012" @default.
• W2969245735 abstract "•EGFR downregulation promotes postnatal radial glial differentiation into ependyma•Apical EGFR redistributes to ependymal basolateral domains, limiting its activation•Endocytic adaptor Numb traffics ependymal EGFR during adult neurogenic niche assembly•Progenitors differentially act on the same extracellular cues via receptor redistribution Specialized microenvironments, called niches, control adult stem cell proliferation and differentiation. The brain lateral ventricular (LV) neurogenic niche is generated from distinct postnatal radial glial progenitors (pRGPs), giving rise to adult neural stem cells (NSCs) and niche ependymal cells (ECs). Cellular-intrinsic programs govern stem versus supporting cell maturation during adult niche assembly, but how they are differentially initiated within a similar microenvironment remains unknown. Using chemical approaches, we discovered that EGFR signaling powerfully inhibits EC differentiation by suppressing multiciliogenesis. We found that EC pRGPs actively terminated EGF activation through receptor redistribution away from CSF-contacting apical domains and that randomized EGFR membrane targeting blocked EC differentiation. Mechanistically, we uncovered spatiotemporal interactions between EGFR and endocytic adaptor protein Numb. Ca2+-dependent basolateral targeting of Numb is necessary and sufficient for proper EGFR redistribution. These results reveal a previously unknown cellular mechanism for neighboring progenitors to differentially engage environmental signals, initiating adult stem cell niche assembly. Specialized microenvironments, called niches, control adult stem cell proliferation and differentiation. The brain lateral ventricular (LV) neurogenic niche is generated from distinct postnatal radial glial progenitors (pRGPs), giving rise to adult neural stem cells (NSCs) and niche ependymal cells (ECs). Cellular-intrinsic programs govern stem versus supporting cell maturation during adult niche assembly, but how they are differentially initiated within a similar microenvironment remains unknown. Using chemical approaches, we discovered that EGFR signaling powerfully inhibits EC differentiation by suppressing multiciliogenesis. We found that EC pRGPs actively terminated EGF activation through receptor redistribution away from CSF-contacting apical domains and that randomized EGFR membrane targeting blocked EC differentiation. Mechanistically, we uncovered spatiotemporal interactions between EGFR and endocytic adaptor protein Numb. Ca2+-dependent basolateral targeting of Numb is necessary and sufficient for proper EGFR redistribution. These results reveal a previously unknown cellular mechanism for neighboring progenitors to differentially engage environmental signals, initiating adult stem cell niche assembly. Adult stem cells are integral components of normal tissue homeostasis, and their dysfunctions can contribute to human disease (Gage and Temple, 2013Gage F.H. Temple S. Neural stem cells: generating and regenerating the brain.Neuron. 2013; 80: 588-601Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, Gonzales and Fuchs, 2017Gonzales K.A.U. Fuchs E. Skin and Its Regenerative Powers: An Alliance between Stem Cells and Their Niche.Dev. Cell. 2017; 43: 387-401Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, Tomasetti and Vogelstein, 2015Tomasetti C. Vogelstein B. Cancer etiology. Variation in cancer risk among tissues can be explained by the number of stem cell divisions.Science. 2015; 347: 78-81Crossref PubMed Scopus (1143) Google Scholar). To balance proliferation and differentiation, adult stem cells often reside in dedicated microenvironments called niches, which are cellular complexes composed of tissue-restricted stem cells interacting with neighboring cells (Gehart and Clevers, 2019Gehart H. Clevers H. Tales from the crypt: new insights into intestinal stem cells.Nat. Rev. Gastroenterol. Hepatol. 2019; 16: 19-34Crossref PubMed Scopus (276) Google Scholar, Hogan et al., 2014Hogan B.L. Barkauskas C.E. Chapman H.A. Epstein J.A. Jain R. Hsia C.C. Niklason L. Calle E. Le A. Randell S.H. et al.Repair and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function.Cell Stem Cell. 2014; 15: 123-138Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, Ihrie and Alvarez-Buylla, 2011Ihrie R.A. Alvarez-Buylla A. Lake-front property: a unique germinal niche by the lateral ventricles of the adult brain.Neuron. 2011; 70: 674-686Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Although much is known about the cellular identities of stem cell niche components, the principles governing their differentiation from specified precursors into three-dimensional (3D) environments in adult tissues remain unclear. Understanding these principles will be important for tissue regeneration strategies that use in vivo and ex vivo platforms for engineering cellular transplants (Barrilleaux et al., 2006Barrilleaux B. Phinney D.G. Prockop D.J. O’Connor K.C. Review: ex vivo engineering of living tissues with adult stem cells.Tissue Eng. 2006; 12: 3007-3019Crossref PubMed Scopus (182) Google Scholar, Kim et al., 2012Kim H. Cooke M.J. Shoichet M.S. Creating permissive microenvironments for stem cell transplantation into the central nervous system.Trends Biotechnol. 2012; 30: 55-63Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, Pacelli et al., 2017Pacelli S. Basu S. Whitlow J. Chakravarti A. Acosta F. Varshney A. Modaresi S. Berkland C. Paul A. Strategies to develop endogenous stem cell-recruiting bioactive materials for tissue repair and regeneration.Adv. Drug Deliv. Rev. 2017; 120: 50-70Crossref PubMed Scopus (87) Google Scholar). In the adult rodent brain, the lateral ventricular (LV) neurogenic region supports continuous new neuron production throughout life (Bjornsson et al., 2015Bjornsson C.S. Apostolopoulou M. Tian Y. Temple S. It takes a village: constructing the neurogenic niche.Dev. Cell. 2015; 32: 435-446Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, Lim and Alvarez-Buylla, 2016Lim D.A. Alvarez-Buylla A. The Adult Ventricular-Subventricular Zone (V-SVZ) and Olfactory Bulb (OB) Neurogenesis.Cold Spring Harb. Perspect. Biol. 2016; 8: a018820Crossref PubMed Scopus (269) Google Scholar). This subependymal zone (SEZ) and/or subventricular zone (SVZ) niche containing adult neural stem cells (NSCs) is constructed within the first 2 weeks after birth from embryonically specified radial glial progenitors (Paez-Gonzalez et al., 2011Paez-Gonzalez P. Abdi K. Luciano D. Liu Y. Soriano-Navarro M. Rawlins E. Bennett V. Garcia-Verdugo J.M. Kuo C.T. Ank3-dependent SVZ niche assembly is required for the continued production of new neurons.Neuron. 2011; 71: 61-75Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and serves as an excellent model system to study mechanisms regulating adult stem cell niche assembly. For the SEZ niche, differentiation of at least 2 types of postnatal radial glial progenitors (pRGPs) is required for its functional assembly at the LV surface: (1) pRGPs that retain proliferative capacity to become future adult NSCs (Fuentealba et al., 2015Fuentealba L.C. Rompani S.B. Parraguez J.I. Obernier K. Romero R. Cepko C.L. Alvarez-Buylla A. Embryonic Origin of Postnatal Neural Stem Cells.Cell. 2015; 161: 1644-1655Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) and (2) pRGPs that differentiate into neighboring niche ependymal cells (ECs) (Paez-Gonzalez et al., 2011Paez-Gonzalez P. Abdi K. Luciano D. Liu Y. Soriano-Navarro M. Rawlins E. Bennett V. Garcia-Verdugo J.M. Kuo C.T. Ank3-dependent SVZ niche assembly is required for the continued production of new neurons.Neuron. 2011; 71: 61-75Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, Spassky et al., 2005Spassky N. Merkle F.T. Flames N. Tramontin A.D. García-Verdugo J.M. Alvarez-Buylla A. Adult ependymal cells are postmitotic and are derived from radial glial cells during embryogenesis.J. Neurosci. 2005; 25: 10-18Crossref PubMed Scopus (487) Google Scholar). Although little is known about the steps governing transition of pRGPs into adult GFAP+ SEZ NSCs, differentiation of niche ECs from pRGPs requires timely and coordinated activation of numerous transcription factors, including Mcidas, Myb, and Foxj1 (Spassky and Meunier, 2017Spassky N. Meunier A. The development and functions of multiciliated epithelia.Nat. Rev. Mol. Cell Biol. 2017; 18: 423-436Crossref PubMed Scopus (161) Google Scholar). This transcriptional cascade results in EC morphological specialization, including basal body duplication and multiciliogenesis (Stubbs et al., 2012Stubbs J.L. Vladar E.K. Axelrod J.D. Kintner C. Multicilin promotes centriole assembly and ciliogenesis during multiciliate cell differentiation.Nat. Cell Biol. 2012; 14: 140-147Crossref PubMed Scopus (160) Google Scholar). Proper EC differentiation is critical assembling SEZ niches and sustaining adult neurogenesis (Paez-Gonzalez et al., 2011Paez-Gonzalez P. Abdi K. Luciano D. Liu Y. Soriano-Navarro M. Rawlins E. Bennett V. Garcia-Verdugo J.M. Kuo C.T. Ank3-dependent SVZ niche assembly is required for the continued production of new neurons.Neuron. 2011; 71: 61-75Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), as well as preventing hydrocephalus (Abdi et al., 2018Abdi K. Lai C.H. Paez-Gonzalez P. Lay M. Pyun J. Kuo C.T. Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus.Nat. Commun. 2018; 9: 1655Crossref PubMed Scopus (28) Google Scholar, Del Bigio, 2010Del Bigio M.R. Ependymal cells: biology and pathology.Acta Neuropathol. 2010; 119: 55-73Crossref PubMed Scopus (211) Google Scholar, Tissir et al., 2010Tissir F. Qu Y. Montcouquiol M. Zhou L. Komatsu K. Shi D. Fujimori T. Labeau J. Tyteca D. Courtoy P. et al.Lack of cadherins Celsr2 and Celsr3 impairs ependymal ciliogenesis, leading to fatal hydrocephalus.Nat. Neurosci. 2010; 13: 700-707Crossref PubMed Scopus (238) Google Scholar). The cerebrospinal fluid (CSF), containing various signaling molecules and growth factors during development (Dani and Lehtinen, 2016Dani N. Lehtinen M.K. CSF Makes Waves in the Neural Stem Cell Niche.Cell Stem Cell. 2016; 19: 565-566Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar), contacts the apical membranes of both pRGP populations during postnatal SEZ niche development. Because extracellular environments are temporally and contextually similar for pRGPs destined to becoming adult NSCs and niche ECs, it has generally been assumed that cell-intrinsic programs differentially drive the differentiation of pRGP subpopulations during SEZ niche development. Given the comparable local microenvironments along the LV wall, it remains unclear how specified niche EC progenitors initiate their differentiation while acting on similar extracellular cues as adult NSC progenitors. Although embryonically specified progenitors mature postnatally into adult SEZ niche ECs and NSCs (Ortiz-Alvarez et al., 2019Ortiz-Alvarez G. Daclin M. Shihavuddin A. Lansade P. Fortoul A. Faucourt M. Clavreul S. Lalioti M.E. Taraviras S. Hippenmeyer S. et al.Adult Neural Stem Cells and Multiciliated Ependymal Cells Share a Common Lineage Regulated by the Geminin Family Members.Neuron. 2019; 102: 159-172Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, Redmond et al., 2019Redmond S.A. Figueres-Onate M. Obernier K. Nascimento M.A. Parraguez J.I. López-Mascaraque L. Fuentealba L.C. Alvarez-Buylla A. Development of Ependymal and Postnatal Neural Stem Cells and Their Origin from a Common Embryonic Progenitor.Cell Rep. 2019; 27: 429-441Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), EC progenitor differentiation can be specifically tracked and readily studied via Foxj1 expression (Paez-Gonzalez et al., 2011Paez-Gonzalez P. Abdi K. Luciano D. Liu Y. Soriano-Navarro M. Rawlins E. Bennett V. Garcia-Verdugo J.M. Kuo C.T. Ank3-dependent SVZ niche assembly is required for the continued production of new neurons.Neuron. 2011; 71: 61-75Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), unlike postnatal NSC progenitors, which at present cannot be easily identified. Using a chemical screen to uncover signaling pathways controlling pRGP differentiation into SEZ niche ECs, we uncovered a previously unknown cellular mechanism for neighboring radial glial progenitors to differentially act on the same extracellular cues, enabling distinct downstream programs in EC progenitors for assembling the adult LV neurogenic niche. To interrogate molecular signals controlling the initiation of SEZ niche EC differentiation, we first grew postnatal day 0 (P0) LV progenitors using our established EC culture assay. We showed previously that this culture can efficiently differentiate into ECs following a reduction of media serum concentration from 10% to 2% (Paez-Gonzalez et al., 2011Paez-Gonzalez P. Abdi K. Luciano D. Liu Y. Soriano-Navarro M. Rawlins E. Bennett V. Garcia-Verdugo J.M. Kuo C.T. Ank3-dependent SVZ niche assembly is required for the continued production of new neurons.Neuron. 2011; 71: 61-75Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). We wondered whether reducing certain factors within the serum is necessary to initiate niche EC differentiation from specified pRGPs. To test this, we used chemical inhibitors of fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or transforming growth factor β (TGF-β) pathways in EC cultures incubated in 10% serum and screened for their potential to initiate EC differentiation. Although FGF, PDGF, and TGF-β pathway inhibitors showed no significant effects, we found that epidermal growth factor receptor (EGFR)-specific inhibitor (Erlotinib) enabled Foxj1+ EC differentiation from pRGPs while incubated in 10% serum (Figures 1A–1D and S1A). To confirm that EGFR activity can directly block the initiation of niche EC differentiation, upon initiating EC differentiation in 2% serum, we added EGF to the culturing media. This effectively prevented EC Foxj1 expression and multiciliogenesis (Figures 1B–1E). Chemical inhibition of MEK1/2 (signaling effector kinases downstream of activated EGFR) was also sufficient to initiate Foxj1 expression and EC differentiation under 10% serum condition (Figures S1A and S1B). Immunohistochemical (IHC) staining for phospho-EGFR (pEGFR, the Y1068-activated form of the receptor) on differentiating EC cultures with the addition of EGF showed strong cellular enrichment compared with controls (Figure S1C). Cultures harvested from P0 FOXJ1-GFP reporter mice (Ostrowski et al., 2003Ostrowski L.E. Hutchins J.R. Zakel K. O’Neal W.K. Targeting expression of a transgene to the airway surface epithelium using a ciliated cell-specific promoter.Mol. Ther. 2003; 8: 637-645Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) showed a lack of GFP expression following EGF addition during EC differentiation (compared with robust GFP upregulation in control condition), suggesting lack of foxj1 transcriptional activation instead of protein stability (Abdi et al., 2018Abdi K. Lai C.H. Paez-Gonzalez P. Lay M. Pyun J. Kuo C.T. Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus.Nat. Commun. 2018; 9: 1655Crossref PubMed Scopus (28) Google Scholar) (Figure S1C). To evaluate EGF’s strong effects on inhibiting niche EC differentiation from pRGPs, we performed transcriptome analyses on differentiating EC cultures in low serum with or without EGF addition. Microarray analyses showed that expression levels of ∼3,200 genes were altered when comparing EGF-treated with untreated conditions (Figure 1F). Gene Ontology (GO) analysis identified the main biological process difference as being related to motile cilium assembly, with the top five terms including cilium organization, assembly, movement, and multicilia formation (Figures 1G–1I, S1D, and S1E). Key factors known to be required for multiciliated cell differentiation (Brooks and Wallingford, 2014Brooks E.R. Wallingford J.B. Multiciliated cells.Curr. Biol. 2014; 24: R973-R982Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, Marshall and Kintner, 2008Marshall W.F. Kintner C. Cilia orientation and the fluid mechanics of development.Curr. Opin. Cell Biol. 2008; 20: 48-52Crossref PubMed Scopus (93) Google Scholar), including Mcidas, Gemc1, Myb, and Foxj1, were repressed by the addition of EGF to the culturing media (Figure S1F). Additional downstream genes, such as DNAH6, DNAH9, Kif9, and Kif27 (Choksi et al., 2014Choksi S.P. Lauter G. Swoboda P. Roy S. Switching on cilia: transcriptional networks regulating ciliogenesis.Development. 2014; 141: 1427-1441Crossref PubMed Scopus (179) Google Scholar), were uniformly downregulated by at least 5-fold (Figure S1G). STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) protein-network analyses diagramming gene associations within our dataset specifically indicated EGF’s strong effects on attenuating the initiation of EC differentiation from pRGPs (Figure S1H). Although the upstream control of multiciliogenesis transcriptional regulator Foxj1 remains poorly understood, these results suggest that reduced MEK activation in EC progenitors may induce Foxj1 expression, resulting in multiciliogenesis initiation. However, it remains possible that downregulation of MEK activity directly control aspects of multiciliogenesis gene induction. Our ependymal culture results suggested that terminating developmental EGF signaling after birth is required for initiating niche EC differentiation from pRGPs. Because EGF is known to be present in the CSF contacting the apical surfaces of differentiating ECs (Cieślak et al., 1986Cieślak D. Szulc-Kuberska J. Stepień H. Klimek A. Epidermal growth factor in human cerebrospinal fluid: reduced levels in amyotrophic lateral sclerosis.J. Neurol. 1986; 233: 376-377Crossref PubMed Scopus (11) Google Scholar, Doetsch et al., 2002Doetsch F. Verdugo J.M. Caille I. Alvarez-Buylla A. Chao M.V. Casaccia-Bonnefil P. Lack of the cell-cycle inhibitor p27Kip1 results in selective increase of transit-amplifying cells for adult neurogenesis.J. Neurosci. 2002; 22: 2255-2264Crossref PubMed Google Scholar), we next wondered whether EGFR expression may be dynamically regulated during this important developmental time window. To track pRGPs fated to become niche ECs, we imaged FOXJ1-GFP-expressing cells along the LV neurogenic niche in P1, P3, P7, and P10 animals. Although FOXJ1-GFP can mislabel some neural progenitors postnatally (Abdi et al., 2018Abdi K. Lai C.H. Paez-Gonzalez P. Lay M. Pyun J. Kuo C.T. Uncovering inherent cellular plasticity of multiciliated ependyma leading to ventricular wall transformation and hydrocephalus.Nat. Commun. 2018; 9: 1655Crossref PubMed Scopus (28) Google Scholar, Ostrowski et al., 2003Ostrowski L.E. Hutchins J.R. Zakel K. O’Neal W.K. Targeting expression of a transgene to the airway surface epithelium using a ciliated cell-specific promoter.Mol. Ther. 2003; 8: 637-645Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), it remains a highly efficient reporter for ECs during their differentiation. At P1, LV germinal matrix GFP+ cells retained a radial glial-like morphology with smaller apical surface areas and single basal processes (Figure S2A). By P3, these GFP+ cells showed apical surface expansion while retaining their basal processes (Figure S2A). This apical surface expansion continued during EC differentiation at P7 and P10, with concurrent disappearance of basal processes from GFP+ cells (Figure S2A). Using orthogonal imaging of GFP+ cells on the x-z plane, at P1 we found EGFR expression in GFP+ pRGPs at their apical domain, as well as intracellularly (Figure 2A). By P3, EGFR was mostly intracellular, with decreased apical surface expression, while some cells began to show EGFR enrichment at their basolateral membranes (Figure 2A). At P7, we observed EGFR concentrating at the lateral domains of differentiating GFP+ ECs, which became mainly basolateral by P10 (Figure 2A). Quantifications of the apically to laterally localized EGFR ratio confirmed redistribution during postnatal EC maturation (Figure 2B). To quantify EGFR redistribution in ependymal pRGPs, we turned to super-resolution stimulated emission depletion (STED) microscopy (Klar and Hell, 1999Klar T.A. Hell S.W. Subdiffraction resolution in far-field fluorescence microscopy.Opt. Lett. 1999; 24: 954-956Crossref PubMed Scopus (590) Google Scholar). We detected distinct EGFR+ vesicular particles on the apical and lateral membrane domains of developing P1 FOXJ1-GFP+ progenitors (Figure 2C). Using Imaris 3D reconstructing of STED microscopic images and volumetric analysis of EGFR apical density, we were able to calculate the average numbers of EGFR particles per GFP+ cell at the apical domain in P1 and P7 animals, showing a significant reduction in the total numbers of apical EGFR particles from P1 to P7 (Figures 2D and 2E). To determine whether this postnatal downregulation of EGFR from EC progenitors corresponded to receptor signaling activity, we performed IHC staining for activated pEGFR on LV whole mounts from P3 and P7 FOXJ1-GFP animals. Consistently, although we detected pEGFR signals on the apical domains of GFP+ pRGPs at P3, they were absent by P7 (Figure S2B). The presence of nearby pEGFR+ (but GFP-negative) cells within the developing neurogenic niche at P7 suggested that differentiating ECs were able to terminate their EGFR activity within a microenvironment that is activating EGFR in other cell types (Figure S2B). In the P35 mature SEZ niche, tamoxifen induction of nestion-CreERtm4; R26R-tdTomato; FOXJ1-GFP animals at P14 labels both niche ECs and NSCs (Figure S2C). Although as expected we readily detected EGFR IHC staining in tdTomato+ SEZ niche astrocytes at P35, we did not find EGFR expression in neighboring GFP+ niche ECs (Figure S2C). Because the dynamic redistribution of EGFR from apical to basolateral cellular domains during EC development had not been described previously, we next wanted to examine whether this is functionally required for EC differentiation. We used a well-characterized EGFR mutation, located within the juxtamembrane domain (EGFR-P667A), resulting in receptor misaccumulation at the cellular apical domains, in addition to basolateral targeting (He et al., 2002He C. Hobert M. Friend L. Carlin C. The epidermal growth factor receptor juxtamembrane domain has multiple basolateral plasma membrane localization determinants, including a dominant signal with a polyproline core.J. Biol. Chem. 2002; 277: 38284-38293Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We generated hemagglutinin (HA)-tagged wild type (WT)-EGFR and P667A-EGFR lentiviral constructs, and injected them into P1 animal LVs. To minimize overexpression of EGFR, we limited in vivo viral infection to 1%–5% of differentiating pRGPs. IHC staining of LV whole mounts 3 days after injection confirmed that pRGPs can efficiently express WT-EGFR-HA and P667A-EGFR-HA constructs (Figures S2D and S2E). While WT-EGFR-HA was redistributed temporally from apical to lateral domains similar to endogenously expressed EGFR (Figures S2D and S2F), P667A-EGFR-HA was retained apically concurrent with a block in pRGP transition to EC morphology (Figures S2E and S2F). We next performed LV lentiviral injections in P1 FOXJ1-GFP+ mice. IHC staining of P14 LV whole mounts readily showed control WT-EGFR-HA construct expression in GFP+ ECs (Figure S3A). Foxj1 and acetylated tubulin antibody IHC co-staining confirmed that WT-EGFR-HA-expressing cells were able to properly differentiate into ECs (Figures 2F–2H and S3B). Although most control WT-EGFR-HA expression co-localized with ECs, we also detected as expected its expression in neighboring SEZ astrocytes from targeting P1 pRGPs (Figures S3A). In contrast to controls, most mutant P667A-EGFR-HA protein expression co-localized with GFAP+ SEZ astrocytes (Figures 2F and S3C). It was difficult to detect P667A-EGFR-HA expression in GFP+ Foxj1+-multiciliated cells (Figures 2F–2H, S3A, and S3B). Quantifications of control and mutant EGFR construct expressions in Foxj1+/multiciliated cells over multiple experiments revealed significant reductions of their cellular co-localization with the mutant receptor (Figures 2G and 2H), consistent with our earlier in vitro data showing that persistent EGFR activation can effectively block pRGP differentiation into niche ECs. Furthermore, our results showed that instead of simply regulating receptor expression, selective protein redistribution is a key step in terminating EGFR activation and allowing postnatal niche EC differentiation to proceed. We next wondered which molecular mechanisms control EGFR redistribution in pRGP to downregulate extracellular growth factor signaling. The endocytic adaptor protein Numb was first identified as a key mediator of cellular asymmetric division (Rhyu et al., 1994Rhyu M.S. Jan L.Y. Jan Y.N. Asymmetric distribution of numb protein during division of the sensory organ precursor cell confers distinct fates to daughter cells.Cell. 1994; 76: 477-491Abstract Full Text PDF PubMed Scopus (625) Google Scholar, Roegiers and Jan, 2004Roegiers F. Jan Y.N. Asymmetric cell division.Curr. Opin. Cell Biol. 2004; 16: 195-205Crossref PubMed Scopus (195) Google Scholar, Uemura et al., 1989Uemura T. Shepherd S. Ackerman L. Jan L.Y. Jan Y.N. numb, a gene required in determination of cell fate during sensory organ formation in Drosophila embryos.Cell. 1989; 58: 349-360Abstract Full Text PDF PubMed Scopus (403) Google Scholar). Subsequent experiments have shown that Numb can interact with plasma membrane receptors to control their recycling and degradation (Gulino et al., 2010Gulino A. Di Marcotullio L. Screpanti I. The multiple functions of Numb.Exp. Cell Res. 2010; 316: 900-906Crossref PubMed Scopus (152) Google Scholar). At P1, Numb IHC staining on LV whole mounts from FOXJ1-GFP reporter animals showed strong intracellular distribution in GFP+ ependymal pRGPs (Figure 3A). Between P3 to P7, Numb became localized to lateral cellular domains in GFP+ cells during niche EC differentiation (Figure 3A). STED super-resolution imaging of differentiating EC pRGPs showed EGFR and Numb co-localizing to vesicular structures (Figures 3B, 3C, and S4A). At P1, there was robust EGFR and Numb co-localization at apical and basolateral domains, which became more basolateral by P7 (Figures 3B, 3C, and S4A). Upregulation of Numb protein in FOXJ1-GFP+ pRGPs consistently coincided with EGFR expression (Figure S4B). To determine whether this Numb protein redistribution was responsible for EGFR trafficking, we deleted Numb and its closely related homolog Numblike (Nbl) in EC progenitors by inter-crossing FOXJ1-Cre; NumbF/+; NblKO/+ and NumbF/+; NblKO/+ animals to generate FOXJ1-Cre; NumbF/+; NblKO/KO (control) and FOXJ1-Cre; NumbF/F; NblKO/KO (conditional double knockout [cDKO]) mice. The CAG-GFP Cre-dependent reporter line was also crossed into the genetic background to label FOXJ1-Cre-targeted cells. Numb cDKO mice developed significant ventriculomegaly, glial scaring, and reduced multicilia coverage on the LV wall postnatally (Figures S4B–S4G) and did not survive past 5 weeks of age. IHC staining of P14 control versus cDKO ventricular sections showed that although, as expected, we did not detect EGFR expression in GFP+ ECs in control samples, there was strong EGFR expression in GFP+ cells in cDKO samples (Figure 3D). Furthermore, IHC staining of P14 LV whole mounts detected aberrant pEGFR on the apical membrane of cDKO mutant cells (Figure 3E). LV whole-mount western blot analyses comparing P14 control and cDKO animals showed that although total AKT and ERK1/2 protein levels were equivalent, pAKT, pERK1/2, and pEGFR levels (activated forms) were all significantly increased in cDKO samples (Figure 3F), confirming abnormal EGFR activation. Quantifications of GFP+ cells comparing P10 cDKO with control LV whole mounts showed cDKO cells had significantly increased GLAST expression and reduced apical surface diameter, indicative of pRGP to EC maturation defects (Figures S4F and S4G). There was phenotypic variability in cDKO GFP+ cells postnatally, and we believe this is caused by our timing of conditional Numb deletion and Numb protein perdurance. Using onset of foxj1 gene transcription to express Cre for Numb deletion also initiates the Foxj1-driven multiciliogenesis program in Cre-targeted cells. Ideally, one would need an EC progenitor-specific Cre driver that turns on postnatally before Foxj1-initiated multiciliogenesis, but to our knowledge such a driver line has not been identified. To examine potential biochemical interactions between Numb and EGFR, we first preformed immunoprecipitation (IP) experiments using tagged protein expression constructs. Co-transfections of HA-EGFR and Numb-GFP into HEK293 cells, followed by protein lysate IP with anti-GFP antibody, resulted in a robust pull-down of HA-EGFR (Figure 4A). As a control, co-transfections of HA-EGFR and GFP followed by anti-GFP IP did not pull down HA-EGFR (Figure 4A). IP experiments on acutely isolated P3 LV whole mounts showed efficient co-precipitation of EGFR and Numb proteins (Figure 4B). EGFR is internalized and recycled from the plasma membrane in a clathrin-dependent manner through activity of AP-2 protein containing complexes (Tomas et al., 2014Tomas A. Futter C.E. Eden E.R. EGF receptor trafficking: consequences for signaling and cancer.Tre" @default.
• W2969245735 created "2019-08-29" @default.
• W2969245735 creator A5020254351 @default.
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• W2969245735 date "2019-08-01" @default.
• W2969245735 modified "2023-10-18" @default.
• W2969245735 title "EGFR Signaling Termination via Numb Trafficking in Ependymal Progenitors Controls Postnatal Neurogenic Niche Differentiation" @default.
• W2969245735 cites W1548839433 @default.
• W2969245735 cites W1617074755 @default.
• W2969245735 cites W1965238080 @default.
• W2969245735 cites W1968013691 @default.
• W2969245735 cites W1978478424 @default.
• W2969245735 cites W1984243695 @default.
• W2969245735 cites W1988896202 @default.
• W2969245735 cites W2003717699 @default.
• W2969245735 cites W2007086703 @default.
• W2969245735 cites W2008009288 @default.
• W2969245735 cites W2010054551 @default.
• W2969245735 cites W2016043593 @default.
• W2969245735 cites W2019078072 @default.
• W2969245735 cites W2022311164 @default.
• W2969245735 cites W2024035512 @default.
• W2969245735 cites W2024490989 @default.
• W2969245735 cites W2024502310 @default.
• W2969245735 cites W2028480293 @default.
• W2969245735 cites W2033965339 @default.
• W2969245735 cites W2034947180 @default.
• W2969245735 cites W2034975029 @default.
• W2969245735 cites W2046457668 @default.
• W2969245735 cites W2048430961 @default.
• W2969245735 cites W2053216321 @default.
• W2969245735 cites W2061944004 @default.
• W2969245735 cites W2069644127 @default.
• W2969245735 cites W2071397266 @default.
• W2969245735 cites W2072417992 @default.
• W2969245735 cites W2076195597 @default.
• W2969245735 cites W2084652685 @default.
• W2969245735 cites W2097492158 @default.
• W2969245735 cites W2109838864 @default.
• W2969245735 cites W2110301548 @default.
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• W2969245735 cites W2138069365 @default.
• W2969245735 cites W2138078272 @default.
• W2969245735 cites W2153371646 @default.
• W2969245735 cites W2252787563 @default.
• W2969245735 cites W2319605272 @default.
• W2969245735 cites W2416524868 @default.
• W2969245735 cites W2547724800 @default.
• W2969245735 cites W2606745140 @default.
• W2969245735 cites W2737893453 @default.
• W2969245735 cites W2769238697 @default.
• W2969245735 cites W2803007323 @default.
• W2969245735 cites W2901254075 @default.
• W2969245735 cites W2916811543 @default.
• W2969245735 cites W2939870752 @default.
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