Ephrin-A2 Reverse Signaling Negatively Regulates Neural Progenitor Proliferation and Neurogenesis

Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Ephrin-A2 reverse signaling negatively regulates neural progenitor proliferation and neurogenesis Johan Holmberg,1 Annika Armulik,1 Kirsten-André Senti,1,3 Karin Edoff,1 Kirsty Spalding,1 Stefan Momma,1 Rob Cassidy,1 John G. Flanagan,2 and Jonas Frisén1,4 1Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, SE-171 77 Stockholm, Sweden; 2Department of Cell Biology and Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115, USA The number of cells in an organ is regulated by mitogens and trophic factors that impinge on intrinsic determinants of proliferation and apoptosis. We here report the identification of an additional mechanism to control cell number in the brain: EphA7 induces ephrin-A2 reverse signaling, which negatively regulates neural progenitor cell proliferation. Cells in the neural stem cell niche in the adult brain proliferate more and have a shorter cell cycle in mice lacking ephrin-A2. The increased progenitor proliferation is accompanied by a higher number of cells in the olfactory bulb. Disrupting the interaction between ephrin-A2 and EphA7 in the adult brain of wild-type mice disinhibits proliferation and results in increased neurogenesis. The identification of ephrin-A2 and EphA7 as negative regulators of progenitor cell proliferation reveals a novel mechanism to control cell numbers in the brain. [Keywords: SVZ; adult; neuronal progenitors; proliferation; ephrins; reverse signaling] Supplemental material is available at http://www.genesdev.org. Received October 1, 2004; revised version accepted December 16, 2004. Most neurons are generated before birth and neurogen- ing proliferation and neurogenesis within the stem cell esis in the adult brain is limited to a few regions. Cells niche. with stem cell properties can be propagated in vitro from The expression of ephrins and their Eph tyrosine ki- many regions of the adult brain and spinal cord, but the nase receptors in neural, intestinal, epidermal, and he- majority of these cells appear to be largely quiescent or matopoietic stem/progenitor cell populations in the only give rise to glial cells (Weiss et al. 1996; Johansson adult (Conover et al. 2000; Batlle et al. 2002; Ivanova et et al. 1999; Palmer et al. 1999; Horner et al. 2000; Yama- al. 2002; Ramalho-Santos et al. 2002; Tumbar et al. 2004) moto et al. 2001; Martens et al. 2002). High proliferative suggests that they may modulate tissue homeostasis. activity is limited to the dentate gyrus of the hippocam- Ephrins and Ephs are pivotal regulators of developmental pus and the lateral walls of the lateral ventricles. Stem processes such as axon guidance, cell migration, synapse cells in these niches give rise to neurons that migrate formation, and vascular development (Wilkinson 2001; locally within the dentate and from the lateral ventricle Holmberg and Frisén 2002; Palmer and Klein 2003), but wall to the olfactory bulb to integrate into the synaptic much less is known about potential roles in the adult circuitry in the target area (Carlén et al. 2002; Gage 2002; organism. Ephrins fall into two classes based on their van Praag et al. 2002; Carleton et al. 2003). mode of membrane attachment, where A-class ephrins The number of new neurons that integrate in the brain are linked to the cell membrane by a GPI anchor and is regulated both at the level of proliferation of stem/ B-class ephrins have a transmembrane and a cytoplas- progenitor cells as well as the survival of newborn neu- matic domain. Eph receptors are also divided in two rons. Although several different growth factors and cy- classes, A and B, based on structural homology. Ephrin- tokines can increase neural stem/progenitor cell prolif- As were thought to exclusively interact with A-class eration when administered exogenously (Lie et al. 2004), Ephs and B ephrins mainly with B-class Ephs (Gale et al. little is known about the endogenous molecules regulat- 1996), but it is now clear that there is a higher degree of promiscuity between the classes than previously 3Present address: Department of Developmental Biology, Stockholm anticipated (Himanen et al. 2004). Ephrins and Ephs me- University, SE-10961 Stockholm, Sweden. diate bidirectional signaling, where receptor signaling 4Corresponding author. is referred to as forward signaling and ephrin signaling E-MAIL [email protected]; FAX 46-8-324927. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ as reverse signaling (Cowan and Henkemeyer 2002). gad.326905. Whereas the mechanisms for ephrin-B signaling are be- 462 GENES & DEVELOPMENT 19:462–471 © 2005 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/05; www.genesdev.org Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Ephrin-A2 regulates neural progenitor proliferation ginning to be elucidated (Cowan and Henkemeyer 2002), stem cell niche to modulate the number of cells that are mechanisms for ephrin-A reverse signaling, for which generated in the adult brain. there is convincing biochemical and genetic evidence (Davy and Robbins 2000; Huai and Drescher 2001; Knoll Results et al. 2001; Cutforth et al. 2003), are largely unknown. Complementary expression of ephrin-A2 and EphA7 Identification of signaling pathways used by ephrins and in a neural stem cell niche Ephs is often difficult due to a lack of simple models for the morphogenic events these molecules participate in, Analysis of the expression of all A-type ephrins and their and the discovery of other effects of ephrin or Eph sig- EphA receptors in the mouse brain revealed promi- naling would aid in the characterization of signaling nent expression of ephrin-A2 and EphA7 in cells of the pathways. lateral ventricle wall (Fig. 1; Supplementary Fig. 1; data We have studied the role of A-class ephrins and Eph not shown). In addition, low levels of EphA4 mRNA receptors in a neural stem cell niche in the adult brain. were detected by RT–PCR and in situ hybridization and We report that ephrin-A2 and EphA7 negatively regulate very low levels of protein were seen in the lateral ven- neural progenitor proliferation, and that this effect is me- tricle wall by immunohistochemistry with an antibody diated by ephrin-A2 reverse signaling. Inhibition of the against EphA4 (data not shown). Neural stem cells reside ephrin-A2/EphA7 interaction by infusion of soluble pro- in proximity to the lumen of the ventricular system both teins or in mutant mice results in increased progenitor during embryogenesis and in the adult brain. EphA7 is cell proliferation and neurogenesis in the adult brain. We expressed in the ventricular zone already at embryonic propose that these molecules provide a feedback system day 12.5, but expression of A ephrins in this region can- between cells of different maturational states in the not be detected until later in embryonic development Figure 1. Ephrin-A2 and EphA7 in a neural stem cell niche in the adult brain. (a,b) Immunohistochemical localization of EphA7 and ephrin-A2 in the walls of the lateral ventricles (LV) in the adult mouse brain. (c–e) EphA7 and ephrin-A2 are expressed in complementary and mutually exclusive patterns. (f–j) EphA7 is seen in S100␤-immunoreactive ependymal (E) cells and in a subset of GFAP-positive subventricular zone (SVZ) cells (arrowhead in h) but not in transit amplifying progenitor cells (Mash1+) or migrating neuroblasts (PSA-NCAM+). (k–o) In contrast, ephrin-A2-immunoreactivity is absent in ependymal cells and astrocytes and restricted to Mash1+/Dlx+ progenitor cells and PSA-NCAM+ neuroblasts. Bars: a,b, 100 µm; c–o, 10 µm. D (dorsal) and L (lateral) indicate the orientation of all images. GENES & DEVELOPMENT 463 Downloaded from genesdev.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press Holmberg et al. (Zhang et al. 1996; Rogers et al. 1999). This expression fect of clustered and unclustered proteins prompted us to pattern appears to be evolutionarily conserved, with ex- infuse unclustered ephrin-B2-Fc, which also proved to pression of EphA receptors in the ventricular zone pre- increase proliferation to a similar extent as reported with ceding ephrin-A2 expression, which starts late in em- clustered ephrin-B2 (Conover et al. 2000). Although bryogenesis in macaque monkeys (Donoghue and Rakic these data indicate that both A- and B-class ephrins and 1999). Eph receptors can modulate proliferation, it is ambigu- EphA7 and ephrin-A2 are expressed in a complemen- ous whether both clustered and unclustered proteins act tary and mutually exclusive pattern in the adult mouse as agonists or antagonists, and thus whether ephrins and brain lateral ventricle wall (Fig. 1), with cells displaying Eph receptors act as positive or negative regulators of the respective protein residing in direct physical proxim- proliferation. Moreover, it is unclear from the infusion of ity (Fig. 1c–e). EphA7-immunoreactive cells express nes- recombinant proteins whether ephrins have a physi- tin (Fig. 1g), a marker associated with neural stem cells ological function in regulating cell proliferation. (Lendahl et al. 1990). EphA7 is ubiquitously expressed by ependymal cells as well as by a subset of astrocytes in Ephrin-A2 and EphA7 are negative regulators the subventricular zone (Fig. 1f–h), both being candidate of proliferation neural stem cells (Doetsch et al. 1999; Johansson et al. This prompted us to analyze ephrin-A2−/− and EphA7−/− 1999). EphA7 is not detectable in neural progenitor cells mice (Feldheim et al. 2000; Holmberg et al. 2000). BrdU or PSA-NCAM-immunoreactive migratory neuroblasts labeling of dividing cells in ephrin-A2−/− and EphA7−/− in the lateral ventricle wall or the rostral migratory mice revealed an increase in the number of labeled cells stream (Fig.

Load more