β‐catenin signaling modulates the tempo of dendritic growth of adult‐born hippocampal neurons

Article15 September 2020Open Access Transparent process β-catenin signaling modulates the tempo of dendritic growth of adult-born hippocampal neurons Jana Heppt Jana Heppt orcid.org/0000-0002-6703-4921 Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Marie-Theres Wittmann Marie-Theres Wittmann orcid.org/0000-0003-3924-7572 Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany Institute of Human Genetics, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Iris Schäffner Iris Schäffner orcid.org/0000-0002-7045-4601 Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Charlotte Billmann Charlotte Billmann Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Jingzhong Zhang Jingzhong Zhang Institute of Developmental Genetics, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany Suzhou Institute of Biomedical Engineering and Technology (SIBET), Chinese Academy of Sciences, Suzhou, China Search for more papers by this author Daniela Vogt-Weisenhorn Daniela Vogt-Weisenhorn Institute of Developmental Genetics, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany Search for more papers by this author Nilima Prakash Nilima Prakash Institute of Developmental Genetics, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany Hamm-Lippstadt University of Applied Sciences, Hamm, Germany Search for more papers by this author Wolfgang Wurst Wolfgang Wurst Institute of Developmental Genetics, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany Search for more papers by this author Makoto Mark Taketo Makoto Mark Taketo Division of Experimental Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Dieter Chichung Lie Corresponding Author Dieter Chichung Lie [email protected] orcid.org/0000-0002-6035-6442 Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Jana Heppt Jana Heppt orcid.org/0000-0002-6703-4921 Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Marie-Theres Wittmann Marie-Theres Wittmann orcid.org/0000-0003-3924-7572 Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany Institute of Human Genetics, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Iris Schäffner Iris Schäffner orcid.org/0000-0002-7045-4601 Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Charlotte Billmann Charlotte Billmann Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Jingzhong Zhang Jingzhong Zhang Institute of Developmental Genetics, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany Suzhou Institute of Biomedical Engineering and Technology (SIBET), Chinese Academy of Sciences, Suzhou, China Search for more papers by this author Daniela Vogt-Weisenhorn Daniela Vogt-Weisenhorn Institute of Developmental Genetics, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany Search for more papers by this author Nilima Prakash Nilima Prakash Institute of Developmental Genetics, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany Hamm-Lippstadt University of Applied Sciences, Hamm, Germany Search for more papers by this author Wolfgang Wurst Wolfgang Wurst Institute of Developmental Genetics, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany Search for more papers by this author Makoto Mark Taketo Makoto Mark Taketo Division of Experimental Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan Search for more papers by this author Dieter Chichung Lie Corresponding Author Dieter Chichung Lie [email protected] orcid.org/0000-0002-6035-6442 Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Author Information Jana Heppt1, Marie-Theres Wittmann1,2, Iris Schäffner1, Charlotte Billmann1, Jingzhong Zhang3,4, Daniela Vogt-Weisenhorn3, Nilima Prakash3,5, Wolfgang Wurst3, Makoto Mark Taketo6 and Dieter Chichung Lie *,1 1Institute of Biochemistry, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg, Erlangen, Germany 2Institute of Human Genetics, Universitätsklinikum Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany 3Institute of Developmental Genetics, Helmholtz Center Munich, German Research Center for Environmental Health, Neuherberg, Germany 4Suzhou Institute of Biomedical Engineering and Technology (SIBET), Chinese Academy of Sciences, Suzhou, China 5Hamm-Lippstadt University of Applied Sciences, Hamm, Germany 6Division of Experimental Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan *Corresponding author. Tel: +49 9131 85 24622; E-mail: [email protected] The EMBO Journal (2020)39:e104472https://doi.org/10.15252/embj.2020104472 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 In adult hippocampal neurogenesis, stem/progenitor cells generate dentate granule neurons that contribute to hippocampal plasticity. The establishment of a morphologically defined dendritic arbor is central to the functional integration of adult-born neurons. We investigated the role of canonical Wnt/β-catenin signaling in dendritogenesis of adult-born neurons. We show that canonical Wnt signaling follows a biphasic pattern, with high activity in stem/progenitor cells, attenuation in immature neurons, and reactivation during maturation, and demonstrate that this activity pattern is required for proper dendrite development. Increasing β-catenin signaling in maturing neurons of young adult mice transiently accelerated dendritic growth, but eventually produced dendritic defects and excessive spine numbers. In middle-aged mice, in which protracted dendrite and spine development were paralleled by lower canonical Wnt signaling activity, enhancement of β-catenin signaling restored dendritic growth and spine formation to levels observed in young adult animals. Our data indicate that precise timing and strength of β-catenin signaling are essential for the correct functional integration of adult-born neurons and suggest Wnt/β-catenin signaling as a pathway to ameliorate deficits in adult neurogenesis during aging. Synopsis Canonical Wnt-signaling has been identified as a regulator of adult hippocampal neurogenesis. Here, we show that precise timing and strength of β-catenin dependent signaling is essential for dendritogenesis and the correct functional integration of adult-born neurons and suggest Wnt/β-catenin signaling as a pathway to ameliorate age-associated deficits in adult neurogenesis. Canonical Wnt signaling follows a biphasic pattern during adult neurogenesis with high activity in stem/progenitor cells and during maturation. Tight regulation of timing and strength of β-catenin signaling is necessary for proper dendrite and spine development. Aging reduces canonical Wnt signaling activity and decelerates dendritic growth. Enhancement of β-catenin signaling in the aging dentate gyrus restores dendritic growth and spine formation to levels observed in young adult animals. Introduction In the adult mammalian hippocampus, neural stem/progenitor cells in the subgranular zone of the dentate gyrus (DG) undergo a complex sequence of proliferation, differentiation, and maturation steps to add dentate granule neurons to the hippocampal network. A key step for the functional integration of adult-born dentate granule neurons is the development of a morphologically highly stereotypic dendritic arbor to receive afferents in the molecular layer (Goncalves et al, 2016b). Disruption of dendritic arbor development of adult-born neurons is thought to contribute to cognitive and emotional deficits in aging, neurodegenerative, and neuropsychiatric diseases and to the development of an aberrant circuitry in epilepsy (Li et al, 2009; Sun et al, 2009; Winner et al, 2011; Kim et al, 2012; Murphy et al, 2012; Fitzsimons et al, 2013; Cho et al, 2015; Jessberger & Parent, 2015; Llorens-Martin et al, 2015; Trinchero et al, 2017; Kerloch et al, 2018). In young adult mice, the dendritic arbor of adult-born dentate granule neurons is largely established within the first 3–4 weeks of development. Around 10 days after their birth, new neurons feature a basic dentate granule neuron architecture with an apical dendrite spanning the dentate granule cell layer and initial branching in the inner molecular layer. Dendritic growth with further branching and dendritic extension into the outer molecular layer is maximal during the first 2–3 weeks and is followed by a period of pruning of excessive dendritic branches to attain the highly stereotypic dentate granule neuron morphology (Zhao et al, 2006; Kleine Borgmann et al, 2013; Sun et al, 2013; Goncalves et al, 2016a). Several factors including hippocampal network activity, transcription factors, cytoskeletal regulators, neurotransmitters, and signaling molecules were found to modulate dendrite morphology of adult-born neurons (Ge et al, 2006; Bergami et al, 2006; Gao et al, 2009; Jagasia et al, 2009; Ma et al, 2009; Piatti et al, 2011; Llorens-Martin et al, 2013; Vadodaria et al, 2013; He et al, 2014; Trinchero et al, 2017). A complete understanding of the central pathways controlling dendrite development in adult hippocampal neurogenesis is, however, still missing. Wnt proteins are key regulators of adult hippocampal neurogenesis (Lie et al, 2005; Qu et al, 2010, 2013; Jang et al, 2013; Seib et al, 2013; Arredondo et al, 2019). Current data indicate that Wnts control different stages of adult neurogenesis via distinct pathways: While early developmental steps such as proliferation and fate determination of precursors are regulated by canonical Wnt/β-catenin signaling (Lie et al, 2005; Kuwabara et al, 2009; Karalay et al, 2011; Qu et al, 2013), late developmental steps such as neuronal maturation and morphogenesis are thought to be primarily regulated by non-canonical Wnt signaling pathways (Schafer et al, 2015; Arredondo et al, 2019). Supporting a sequential action of distinct Wnt pathways is the finding that early adult-born neuron development and the initiation of neuronal morphogenesis are accompanied by the attenuation of canonical Wnt/β-catenin signaling activity and the increased activity of the non-canonical Wnt/planar cell polarity (PCP) signaling pathway (Schafer et al, 2015). However, the observations i) that pathologies associated with aberrant Wnt/β-catenin activity are paralleled by dendritic growth defects of adult-born dentate granule neurons (Duan et al, 2007; Singh et al, 2011; Murphy et al, 2012; Llorens-Martin et al, 2013; De Ferrari et al, 2014; Qu et al, 2017; Martin et al, 2018) and ii) that ablation of β-catenin from developing dentate granule neurons in juvenile mice causes massive dendritic defects and neuronal death (Gao et al, 2007), raise the possibility that Wnt/β-catenin signaling fulfills important functions during late steps of adult hippocampal neurogenesis. We here report that attenuation of canonical Wnt signaling in early immature neurons is followed by reactivation of the pathway during maturation, resulting in a biphasic pattern of canonical Wnt signaling activity in the adult neurogenic lineage. We also show that this biphasic activity pattern is essential to ensure correct dendrite development and that β-catenin signaling in maturing neurons modulates the tempo of dendritic growth and spine formation. Finally, we demonstrate that countering the age-associated decrease in canonical Wnt/β-catenin signaling reverses dendritic growth and spine formation deficits of adult-born neurons in middle-aged mice. Thus, our data reveal a new function of β-catenin signaling in maturation of adult-born neurons and suggest canonical Wnt/β-catenin signaling as a candidate pathway to counteract age-related deficits in hippocampal neurogenesis-dependent plasticity. Results Canonical Wnt signaling exhibits biphasic activity during adult hippocampal neurogenesis Analyses of different reporter mouse lines consistently revealed high activity of canonical Wnt signaling in the adult DG (O'Brien et al, 2004; Lie et al, 2005; Garbe & Ring, 2012) but were inconclusive regarding canonical Wnt signaling activity during different stages of adult-born neuron development (Garbe & Ring, 2012). To shed light on the activity pattern of canonical Wnt signaling in adult neurogenesis, we analyzed two different reporter mouse lines: BATGAL mice harbor the LacZ reporter gene downstream of seven TCF/LEF-binding sites and the minimal promoter-TATA box of the siamois gene (Maretto et al, 2003); Axin2LacZ/+ mice heterozygously harbor the LacZ reporter in the endogenous locus of the bona fide canonical Wnt signaling target Axin2 (Lustig et al, 2002) (Fig 1A). We first analyzed the percentage of reporter-positive cells as a proxy for canonical Wnt signaling activity. Both reporter lines showed a qualitatively comparable pattern of canonical Wnt signaling activity (Fig 1B and C). In both lines, around one-fourth of the Nestin+ radial glia-like stem/progenitor cells were reporter positive. Immature neurons that were identified by the expression of DCX rarely displayed reporter gene expression, whereas a high percentage of mature Calbindin+ dentate granule neurons was positive for the reporter. The two reporter lines differed with regard to reporter activity in Tbr2+ precursor cells. In BATGAL animals, the fraction of reporter-positive Tbr2+ cells and Nestin+ cells were comparable, suggesting that canonical Wnt signaling activity was sustained during lineage progression of radial glia-like stem/progenitor cells toward fast-proliferating precursor cells. In contrast, the reporter pattern in Axin2LacZ/+ mice suggested that canonical Wnt signaling activity was readily attenuated in Tbr2+ cells (Fig 1B and C). We further determined signal intensity of the reporter in BATGAL mice as an additional measure of canonical Wnt signaling activity (Fig 1D). Tbr2+ cells had on average lower reporter expression than Nestin+ cells, which supports the notion that attenuation of canonical Wnt signaling activity is initiated at the level of Tbr2+ precursor cells. Calbindin+ cells showed on average the highest level of reporter signal, strongly indicating reactivation of canonical Wnt signaling in mature neurons. Figure 1. Canonical Wnt signaling activity in the adult hippocampus Schematic representation of the LacZ alleles of the BATGAL and Axin2LacZ/+ reporter mice for canonical Wnt signaling activity. Representative images showing co-expression of the stage-specific markers Nestin, Tbr2, Doublecortin (DCX), and Calbindin (all in green), with the β-galactosidase (β-Gal, red) reporter in 8-week-old BATGAL and Axin2LacZ/+ mice. Nuclei are counterstained with DAPI (in blue). Scale bar = 10 μm. Insets show a 1.5× magnification of selected cells (position indicated by dashed box). Scale bar = 5 μm. Fraction of Nestin-, Tbr2-, DCX- and Calbindin-positive cells expressing β-Gal in BATGAL and Axin2LacZ/+ animals show stage-specific canonical Wnt signaling during adult hippocampal neurogenesis (BATGAL: Tbr2 vs. DCX P = 0.0056, DCX vs. Calbindin P < 0.0001; Axin2LacZ: Nestin vs. Tbr2 P = 0.0003, Tbr2 vs. DCX P = 0.0115, DCX vs. Calbindin P < 0.0001; n = 3 animals per mouse model and marker). Detection of the corrected total cell fluorescence (CTCF) of the β-Gal reporter in Nestin-, Tbr2-, DCX-, and Calbindin-positive cells in BATGAL mice corroborate stage-specific activity pattern of canonical Wnt signaling during lineage progression (Nestin vs. Tbr2 P < 0.0001, Tbr2 vs. DCX P = 0.0029, DCX vs. Calbindin P < 0.0001; Nestin: n = 236 cells, Tbr2: n = 134 cells, DCX: n = 200 cells, Calbindin: n = 300 cells). BrdU pulse chase scheme. BrdU was injected intraperitoneally (i.p.) three times: (i) every 2 h for 30-min time point (0 days post-injection [dpi]) (ii) every 24 h for all other time points. For the 30-min time point animals were sacrificed 30 min after the final BrdU injection. Quantification of BrdU+ cells expressing β-Gal reveals biphasic activity of canonical Wnt signaling during adult hippocampal neurogenesis (0 vs. 7 dpi P = 0.0036, 7 vs. 28 dpi P = 0.0115, 7 vs. 42 dpi P = 0.0007; 0 dpi time point; n = 4 animals, 3 dpi n = 3 animals, 7 dpi n = 3 animals, 14 dpi n = 4 animals, 28 dpi n = 6 animals, and 42 dpi n = 6 animals). Total cell fluorescence of β-Gal in BrdU+ cells indicate a biphasic pattern in canonical Wnt signaling strength during lineage progression (0 vs. 3 dpi P < 0.0001, 7 vs. 14 dpi P < 0.0001, 14 vs. 28 dpi P < 0.0001, 28 vs. 42 dpi P = 0.0333; 0 dpi n = 194 cells, 3 dpi n = 245 cells, 7 dpi n = 224 cells, 14 dpi n = 72 cells, 28 dpi n = 103 cells, and 42 dpi n = 114 cells). Data information: Data represented as mean ± SEM. Significance was determined using Kruskal–Wallis test followed by Dunn′s multiple comparisons test, and significance levels are displayed in GP style (*P < 0.0332, **P < 0.0021, and ****P < 0.0001). Download figure Download PowerPoint To further assess the time course of canonical Wnt signaling activity, newborn cells in 8-week-old reporter mice were birthdated with Bromodeoxyuridine (BrdU, Fig 1E). For this analysis, we focused on the BATGAL reporter strain, because Axin2 functions in a feedback loop as a negative regulator of canonical Wnt signaling (Lustig et al, 2002); consequently, heterozygous loss of Axin2 may affect the physiological time line of adult-born neuron development. BATGAL reporter activity was analyzed at time points that correspond approximately to the proliferating progenitor cell stage (30 min post-injection), the neuroblast stage (3 days post-injection [dpi]), early and mid-immature neuron stage (7 and 14 dpi, respectively), and the early and late mature neuron stage (28 and 42 dpi, respectively) (Jagasia et al, 2009; Snyder et al, 2009). Thirty minutes after BrdU injection, 42% of BrdU+ cells showed reporter activity. This fraction dropped to 25 and 7% at the 3 and 7 dpi time points, respectively, to subsequently increase to 14% at 14 dpi, 38% at 28 dpi, and 52% at 42 dpi (Fig 1F). Furthermore, analysis of β-galactosidase signal intensities in BrdU+ cells showed that the average reporter expression was strongly increased at 28 and 42 dpi (Fig 1G). Collectively, these data indicate that canonical Wnt signaling activity in the adult neurogenic lineage is attenuated in Tbr2+ precursor cells and immature DCX+ neurons during the first week of development and is up-regulated around the second week during the maturation of DCX+ neurons into Calbindin+ neurons. Correct dendrite development of adult-born neurons is dependent on the timing and dosage of β-catenin signaling activity We first asked whether the reactivation of canonical Wnt/β-catenin signaling during maturation was required for adult-born neuron development. We focused in particular on dendrite development given that reactivation of Wnt/β-catenin signaling activity overlapped with the period of maximal dendritic growth speed (Sun et al, 2013). In canonical Wnt signaling, stabilized β-catenin activates transcription through members of the TCF/LEF transcription factor family (Moon, 2004). To inhibit β-catenin-induced transcription, we transduced fast-proliferating precursor cells in the DG of young adult mice with the CAG-dnLEF-IRES-GFP Moloney Murine Leukemia retrovirus (MMLV), which bi-cistronically encodes for a dominant-negative LEF-mutant protein (dnLEF) and GFP (Karalay et al, 2011). To validate the ability of dnLEF to inhibit β-catenin-induced transcription in adult-born neurons, the CAG-dnLEF-IRES-GFP MMLV was injected into the DG of BATGAL reporter mice. BATGAL mice injected with an MMLV encoding for GFP (CAG-GFP) served as control (Fig EV1A). Comparison of the reporter signal on day 17 post-viral injection between dnLEF-transduced cells and control transduced cells demonstrated that expression of dnLEF inhibited canonical Wnt signaling-induced transcriptional activity (Fig EV1B and C). Click here to expand this figure. Figure EV1. Expression of dnLEF reduces canonical Wnt signaling activity in adult-born granule neurons Experimental scheme of retroviral injection paradigm. Adult BATGAL mice were stereotactically injected with either the CAG-dnLEF-IRES-GFP (dnLEF) or CAG-GFP (control) MMLV and were sacrificed 17 dpi. Representative images of β-galactosidase reporter expression (red) in transduced neurons (green). Arrows and arrowheads point to transduced neurons with and without reporter signal, respectively. Scale bar = 10 μm. Insets show magnifications of transduced cells. Scale bar = 5 μm. Measurements of the corrected total cell fluorescence of the β-galactosidase signal in control and dnLEF neurons show reduced canonical Wnt signaling activity in neurons expressing dnLEF (P < 0.0001; control: n = 47 cells from three animals, dnLEF: n = 35 cells from three animals). Data information: Data represented as mean ± SEM, significance was determined using two-tailed Mann–Whitney U-test, and significance levels are displayed in GP style (****P < 0.0001). Download figure Download PowerPoint We next co-injected young adult (i.e., 8-week-old) mice with CAG-dnLEF-IRES-GFP and a MMLV encoding for RFP (CAG-RFP). Animals were analyzed 17 dpi (Fig 2A). Compared to RFP+ GFP− control neurons, CAG-dnLEF-IRES-GFP-transduced neurons displayed a more immature morphology, with shorter total dendritic length likely caused by shorter terminal dendrites as indicated by the analysis of branch point number and Sholl analysis. In addition, dnLEF-expressing neurons almost invariably exhibited basal dendrites, a transient feature of developing dentate granule neurons and morphological indicator of immaturity (Ribak et al, 2004) (Fig 2B–E). Hence, dnLEF-expressing neurons lagged behind control neurons with regard to dendrite development, indicating that canonical β-catenin-dependent signaling is required for the timely execution of the dendrite development program. Figure 2. Loss of canonical Wnt signaling and sustained canonical Wnt signaling impair dendritogenesis of adult-born neurons Experimental scheme of retroviral injection paradigm. Adult mice were stereotactically co-injected with the MMLV CAG-dnLEF-IRES-GFP (dnLEF) and CAG-RFP (control) and were sacrificed 17 dpi. Representative images of transduced adult-born neurons at 17 dpi. CAG-dnLEF-IRES-GFP (dnLEF, green) and CAG-RFP (control, red) double transduced cells (arrows) and CAG-RFP single transduced cells (arrowheads). Scale bar = 20 μm. Representative reconstructions of control and dnLEF neurons. Scale bar = 20 μm. Analysis of morphology showed a reduction in dendritic length in dnLEF-transduced neurons (P < 0.0001), while the number of branch points remained comparable (P = 0.7914). Sholl analysis displayed a reduction in dendritic complexity in dnLEF-transduced neurons and indicated decreased growth of terminal dendritic branches (P < 0.0001; control: n = 20 cells from three animals, dnLEF: n = 38 cells from six animals). dnLEF neurons displayed basal dendrites (P < 0.0001; control: n = 20 cells from three animals, dnLEF: n = 38 cells from six animals). Schematic representation of the conditional alleles of the Ctnnb1(ex3)fl (β-catex3) mouse model and the retroviral paradigm used to analyze canonical Wnt signaling gain of function in neural progenitors. Control animals harbor the wild-type allele for Ctnnb1. Representative images depicting CAG-GFP-IRES-Cre transduced adult-born neurons in control and β-catex3 mice at 17 dpi. Scale bar = 20 μm. Representative reconstructions of control and β-catex3 neurons. Scale bar = 20 μm. Quantification showed decreased dendritic length of β-catex3 neurons (P < 0.0001); no difference was apparent in number of branch points (P = 0.3256). Sholl analysis displayed a less complex dendritic tree of β-catex3 neurons (P < 0.0001; control: n = 20 cells from five animals, β-catex3: n = 20 cells from five animals). The number of basal dendrites was increased in β-catex3 neurons (P = 0.0003; control: n = 20 cells from five animals, β-catex3: n = 20 cells from five animals). Data information: Data represented as mean ± SEM, significance was determined using two-way ANOVA for Sholl analysis and two-tailed Mann–Whitney U-test for all other analyses, and significance levels were displayed in GP style (***P < 0.0002 and ****P < 0.0001). Download figure Download PowerPoint Dendritic arborization in the molecular layer provides the structural basis for formation of glutamatergic synaptic input from the entorhinal cortex. Previous studies identified glutamatergic input as a critical signal for survival of adult-born neurons (Tashiro et al, 2006a). To determine the long-term survival of dnLEF-transduced neurons, mice were co-injected with CAG-dnLEF-IRES-GFP and CAG-RFP and analyzed at 17 and 42 dpi (Fig EV2A). At 42 dpi, the number of dnLEF-transduced neurons was dramatically reduced and the ratio of GFP+ to RFP+ cells dropped from approximately 1.5 at 17 dpi to 0.5 at 42 dpi, indicating that dnLEF expression strongly decreased survival of adult-born neurons. Moreover, dnLEF-expressing neurons featured a dendritic morphology with subtle alterations in the Sholl analysis (Fig EV2B–D). Click here to expand this figure. Figure EV2. Survival of neurons with loss and gain of β-catenin signaling is impaired Experimental scheme of retroviral injection paradigm. Adult mice were stereotactically co-injected with the CAG-dnLEF-IRES-GFP (dnLEF) and CAG-RFP (control) MMLVs and were sacrificed 17 and 42 dpi. Representative images of the DG with transduced neurons at 17 and 42 dpi. CAG-dnLEF-IRES-GFP transduced cells in green, and CAG-RFP transduced cells in red. Scale bar = 100 μm. Quantification of RFP-positive and GFP-positive cells show reduced survival of neurons transduced with dnLEF (P = 0.0286; n = 4 animals). Morphology analysis of surviving neurons shows no difference in dendritic length (P = 0.1004) and number of branch points (P = 0.8654) between dnLEF and control neurons, but a reduction in complexity (Sholl analysis; P = 0.0176; control: n = 18 cells from four animals, dnLEF: n = 15 cells from four animals). Schematic representation of the retroviral paradigm used to analyze survival of canonical Wnt signaling gain of function in neural progenitors. Representative images of the dentate gyrus of control and β-catex3 mice with GFP expressing transduced neurons (green) and the immature marker doublecortin (DCX) (gray) at 42 dpi. Scale bar = 100 μm. Quantification of GFP-positive cells in control and β-catex3 animals show reduced survival of neurons with stable expression of β-cat from the stage of fast-proliferating progenitor stage on (P = 0.0286; n = 4 animals). Morphology analysis of surviving GFP-positive neurons shows no difference in dendritic length (P = 0.0503) and number of branch points (P = 0.5051), but differences in complexity in Sholl analysis (P = 0.0240) between genotypes (control: n = 20 cells from four animals, dnLEF: n = 15 cells from four animals). Data information: Data represented as mean ± SEM, significance was determined using two-way ANOVA for Sholl analysis and two-tailed Mann–Whitney U-test for all other analyses, and significance levels were displayed in GP style (*P < 0.0332). Download figure Download PowerPoint Given that inhibition of canonical Wnt/β-catenin signaling retarded dendrite growth, we hypothesized that activation of β-catenin signaling promotes dendritic development. The Ctnnb1(ex3)fl mouse mutant (hereafter called β-catex3) allows for Cre-recombinase-induced expression of a stabilized form of β-catenin (Harada et al, 1999). To enhance β-catenin-dependent signaling, we induced recombination in fast-dividing precursors by stereotactic injection of a MMLV bi-cistronically encoding for GFP and Cre-recombinase (CAG-GFP-IRES-Cre). Ctnnb1(ex3)wt mice injected with the CAG-GFP-IRES-Cre MMLV served as controls. Animals were analyzed 17 days post-viral injection (Fig 2F). Surprisingly, transduced neurons (GFP+; DCX+) in β-catex3 mice featured an immature dendritic arbor that was characterized by a decrease in total dendritic length, irregular short neurites, and a reduced complexity in the Sholl a