The co-expression of Neogenin with SOX2 in hippocampal neurons

Accepted Manuscript The co-expression of Neogenin with SOX2 in hippocampal neurons Namgue Hong, Mi-Hye Kim, Churl K. Min, Hee Jung Kim, Jae Ho Lee PII:

S0006-291X(17)31194-4

DOI:

10.1016/j.bbrc.2017.06.062

Reference:

YBBRC 37968

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 7 June 2017 Accepted Date: 13 June 2017

Please cite this article as: N. Hong, M.-H. Kim, C.K. Min, H.J. Kim, J.H. Lee, The co-expression of Neogenin with SOX2 in hippocampal neurons, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.06.062. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT BBRC

The co-expression of Neogenin with SOX2 in hippocampal neurons

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Namgue Hong1,2 , Mi-Hye Kim1,2, Churl K. Min3, Hee Jung Kim1,*, Jae Ho Lee4, 5,*

Department of Physiology, College of Medicine, Dankook University, Cheonan 31116

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Department of Medical Laser, Graduate School, Dankook University, Cheonan 31116,

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Department of Biological Sciences, Ajou University, Suwon, 443-749 S. Korea,

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Department of Biomedical Sciences, College of Life Sciences, CHA University, Pochen, Gyounggi-do, S. Korea,

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CHA Fertility Seoul Center, CHA Medical Group, Seoul, S. Korea.

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* Corresponding author

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E-mail: [email protected] (HJK)

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[email protected] (JHL)

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Abstract Dementia has been shown to be closely related with neuronal degeneration and/or a decrease in the activity of neural stem cells in many brain regions, including the hippocampus. It has been recently established that Neogenin is involved

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in the cell fate determination by regulating Oct3/4, SOX and Nanog, notable embryonic cell markers, expressions in pre-implantation mouse embryos. Further, Neogenin expression at both mRNA and protein levels is manifest in many brain regions in mice, but it remains unclear whether Neogenin expression is prerequisite for the maintenance of neural stem cells, particularly, playing a critical role in the hippocampus, a brain region known to be involved in memory

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generation and consolidation. Here, we provide evidence that supports that Neogenin is implicated in the maintenance of neural stem cells in the hippocampus by enhancing PCNA expressions. We have performed RT-PCR analysis,

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Western blotting, and immunohistochemistry with fetal rat brain tissues at E18 for Neogenin mRNA and protein profiling. Neuronal cells obtained from the hippocampus were subjected to FACS analysis for the identification of Neogenin-positive and/or neuronal stem cell marker-positive cells. Western blotting results showed that Neogenin expression was higher in the hippocampal region compared to the cortical region. FACS analysis results indicated that a significant population of fetal rat neuronal cells exhibiting Neogenin expression also displayed SOX2 expression,

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implying co-expression of Neogenin and SOX2 in the hippocampus. Next, we investigated the role of Neogenin through gain- and loss-of-function studies with cultured rat hippocampal neurons. Neogenin down-regulation by small hairpin RNAs led to a dramatic decrease in SOX2 expression while its up-regulation by overexpression caused an increase in PCNA expression, a cell proliferation marker, compared with none-transfected cells. From this study, we propose a

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model whereby Neogenin could maintain neural stem cell population and control cell proliferation.

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Keywords: Neogenin, SOX2, Neuronal stem cell, Hippocampal neurons, Dementia

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Highlights Neogenin colocalizes with SOX2 in dissociated hippocampal neurons.

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PCNA is upregulated by Neogenin overexpression.

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Neural stem cells reside in the hippocampus expressing Neogenin in situ.

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Introduction Neuronal degeneration-derived brain diseases cause a severe public health care problem affecting both young and elderly generation. Dementia as a neuronal degeneration disorder is one of the biggest issue, and the number of patients

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is dramatically increasing recently worldwide following an increase in elderly population[1]. There has been, however, no successful therapeutic means by which dementia could be completely overcome or at least reversed once it is triggered to progress. Recently, many therapeutic tools even resort to neural stem cell replacement/transplantation and neuro-detoxification [2]. The main reason for the lack of ordinary remedy may stem from the fact that the underlying

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mechanism for the onset of dementia is complicated and redundant such that a single target therapy may not be an appropriate approach.

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It is widely believed that adult stem cells are implicated with the maintenance and the repair of adult tissues and body system, including the brain [2,3]. Not surprisingly, endogenous neural stem cell therapy has been proposed as an option for therapy against various degenerative brain disorders [4,5]. In brain, endogenous neuronal stem cells are known to exist in many regions to replenish and replace neurons when neurons are degenerated or in demand [1,5]. The key to the success of endogenous stem cell therapy is to maintain the existing stem cell population or stimulate any dormant, if any, endogenous stem cells enough to counteract their intrinsic degeneration, which could be attributed to a major cause

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of dementia[6,7]. In other words, endogenous neuronal supply could be stimulated when needed by administrating ligands for a specific receptor(s) whose activation could lead to proliferation of endogenous neural stem cells while maintaining self-renewal and differentiation capacity[8]. This approach prompted us to identify a neuronal receptor

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whose activation could lead to cellular proliferation. So far, as many as ten neuronal stem cell markers have been identified; Pax6, Notch1, PCNA, Hes5, SOX-1 -2, and Nestin are among them[9,10,11,12,13,14]. None of them has

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been described as a receptor. Rather, most of neuronal stem cell markers are transcription factors. Neogenin is a multi-functional receptor belonging to IgG superfamily with a plethora of functionalities assigned ranging from neuropath finder in neural tissues, ion function mediator in the liver, to morphogenetic factor in the mammary gland[15,16,17,18]. Early studies indicated that Neogenin is found in most tissues, especially abundant in organs where adult stem cell-rich population resides such as the testis and the ovary[19]. Neogenin expression in the brain and its role as a guiding molecule in axonal growth and path-finding in neural circuit formation are well established [20]. At the same, this finding opens up a possibility that Neogenin may be involved in the regulation of neural stem cells proliferation or differentiation[21]. Control of stem cell behavior would be very useful for the

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development of a therapy against neuronal degeneration diseases. Therefore, one branch of neural stem cell biology focuses on the identification of a signaling receptor molecule on stem cells in order to gain access to manipulating stem cell proliferation and differentiation. A receptor of neural stem cells that could relay and transduce cues from outside

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into intracellular signaling for cellular proliferation and differentiation, however, has not been elucidated. Toward this end, we have attempted to answer some of these questions: (i) Does Neogenin exist in neural stem cells obtained from rat fetal brain? (ii) Is Neogenin a receptor that relays chemical cues, including its ligand, into cellular proliferation? This

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knowledge could provide a basis for the development of a therapeutic means against degenerative brain disorders.

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Materials and methods Primary cell culture of hippocampal neurons Rat fetuses at the embryonic day 18 (E18) were surgically removed from the wombs of maternal rats after anesthetized

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with 16.5% urethane. Hippocampi were dissected surgically with fine forceps from the fetus brain under a stereomicroscope and placed in HEPES-buffered Hanks’ salt solution (HHSS) composed of 20 mM HEPES, 137 mM NaCl, 1.3 mM CaCl2, 0.4 mM MgSO4, 0.5 mM MgCl2, 5.0 mM KCl, 0.4 mM KH2PO4, 0.6 mM Na2HPO4, 3.0 mM NaHCO3, and 5.6 mM glucose, pH 7.4. Cells were dissociated by trituration through a 5-ml pipette and a flame-

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narrowed Pasteur pipette, pelleted by centrifugation, and resuspended in neurobasal medium, pH 7.4, without Lglutamine containing 2% B27 supplement, 0.25% Glutamax I and penicillin/streptomycin/amphotericin B at 100 U/ml,

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100 µg/ml and 0.025 µg/mL, respectively. Hippocampal neurons were grown as described previously [22] with some modifications. Dissociated cells were then plated at a density of 110,000 cells/glass onto a 25-mm round cover glass that was pre-coated with Matrigel at 0.2 mg/ml (BD Bioscience, Bedford, MA, USA) and grown in a humidified atmosphere of 10% CO2 and 90% air at 37°C. Fresh media were fed on days 3, 7 and 10 by exchange of 75% of the

Flow cytometric sorting

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media. Cells used in these experiments were cultured without a mitotic inhibitor for a minimum of 12 days.

Freshly dissociated hippocampal cells isolated from embryonic day 18 (E18) rat embryos were washed twice with PBS. Then, 1 ml of prepared fixation/permeabilization working solution (eBioscience, San Diego, CA, USA) was added to

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each tube, and incubated at 4°C for 30 min in the dark [23]. Cells were washed twice with permeabilization buffer (eBioscience, San Diego, CA, USA). Then, cells were incubated with mouse anti-SOX2 antibody (Abcam, Cambridge,

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UK), mouse anti-Nestin antibody, or rabbit anti-Neogenin antibody (Santa Cruz Biotech, Santa Cruz, CA) at 1:200 dilution for 30 min in the dark. After washing twice in permeabilization buffer, cells were then further incubated for 30 min with either Alexa 555-conjugated goat anti-mouse IgG antibodies (Santa Cruz Biotech) or FITC-conjugated donkey anti-rabbit IgG antibody (Santa Cruz Biotech). After washing in permeabilization buffer, flow cytometry was conducted with FACS Caliber flow cytometer (BD Biosciences, San Jose, CA) and analyzed using the Cell Quest-Pro software (BD Biosciences).

Immunohistochemistry

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E18 fetus rat brains were transcardially perfused with cold saline, followed by cold 4% paraformaldehyde (pH 7.4) in PBS and embedded within OCT component. Brains were cryo-sectioned at a thickness of 25µm with a microtome (CM3050S, Leica, Germany). The immunostaining procedure was as described previously[24]. The primary antibodies

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used for immunostaining were: polyclonal rabbit anti-Neogenin (Santa Cruz Biotech); polyclonal goat anti-SOX2 (Santa Cruz Biotech). The brain sections were incubated with each primary antibody at 1:50 dilution at 4°C for 16 hrs in a staining chamber with gentle shaking. After washing twice with PBS, the brain sections were further incubated with FITC-conjugated anti-goat IgG antibodies or Alexa 555-conjugated anti rabbit IgG secondary antibodies. The brain

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sections were subjected to DAPI (6-diamidino-2-phenylindole) staining for nuclear counting before mounted on a cover glass with mounting solution (Vector Laboratories, Burlingame, CA, USA), and observed with a confocal microscope

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(LSM 700, Carl Zeiss, Germany). The brain sections were imaged by tile section and Z-stack scanning at the hippocampal region. After scanning, confocal images were packed up and analyzed by ZEISS 200 software.

Immunocytochemistry

Hippocampal neurons cultured on a Matrigel-coated cover glass for at least 12 days were fixed with 4% paraformaldehyde, washed 3 times in PBS and smeared on a glass slide. Goat anti-SOX2 antibody or rabbit anti-

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Neogenin antibody (Santa Cruz Biotech) was added to the glass slides in a staining solution at 1:100 dilution supplemented with 0.2% BSA and 0.01% Na-azide at 4°C overnight. After washing in PBS, FITC-conjugated donkey anti-goat antibodies for SOX2 (Santa Cruz Biotech), or Alexa 555-conjugated donkey anti-rabbit antibody for Neogenin

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(Invitrogen, Grand Island, NY) were added at 1:500 dilution in the same diluent and incubated for 1 hr at room temperature. Then, the slides were counter-stained with DAPI for nuclear staining before observed under the confocal

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microscope (LSM 700, Carl Zeiss, Germany).

Western blotting

Neural stem cell marker protein and Neogenin protein levels of the hippocampal tissues were analyzed by Western blotting with anti-stem cell marker antibody and anti-Neogenin antibody, respectively. About 80 µl of cell lysates was boiled for 5 min with 20 µl of 5x sample buffer. Twenty microliters of boiled protein samples was loaded into each well of a gradient polyacrylamide gel (10%, Bio-Rad, Hercules, CA), and then transblotted to a nitrocellulose membrane. Transblotted membrane was blocked in 5% fat-free milk-containing Tris buffer with 0.5% Tween-20 (TBST) for 1 hr at

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room temperature, and then incubated overnight at 4°C with mouse anti-Oct3/4 antibody, mouse anti-SOX2 antibody, mouse anti-Nestin antibody or mouse anti-Neogenin antibody (Santa Cruz Biotech) that had been previously diluted at 1:1000 in the 2% fat-free milk TBST solution. The blotted membrane was washed in 0.5% TBT and incubated with

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horseradish peroxidase-conjugated anti mouse IgG for1 hr. The immunoreactive protein bands were detected by using Western Bright ECL detection reagent (K-12045, Advansta, Monroe Park, CA). This experiment was repeated three times with three different prepared brain samples. Then numerical analysis of each target band intensity ratios.

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Transfection of hippocampal neurons with Neogenin cDNAs and small hairpin RNAs

Rat hippocampal neurons were transfected in vitro using a modification of the calcium phosphate procedure

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described previously [22]. Briefly, hippocampal neurons were placed in serum-free DMEM supplemented with 1 mM kynurenic acid, 10 mM MgCl2, and 5 mM HEPES, pH 7.5 for 30 min, to reduce neurotoxicity. A DNA/calcium phosphate precipitate was prepared by 2 M CaCl2, 1 µg plasmid DNA and H2O with an equal volume of 2× HEPESbuffered saline per each well. The precipitate was allowed to form for 30 min at room temperature and added drop-wise to each well. After a 90 min incubation, cells were washed twice with DMEM supplemented with MgCl2 and HEPES and then returned to conditioned media, saved at the beginning of the procedure. The transfection efficiency was 10.7 ±

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1.9% based on the percentage of cells stained with DAPI. Data were averaged from three independent experiments for each condition, with 3 fields per glass coverslips in each experiment.

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Statistical analysis

All values were expressed as means ± SEM. Statistical analysis for the values was carried out using Student’s t-test with

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the significance level set at P < 0.05 and a one-way analysis of variance (ANOVA) followed by Bonferroni’s test.

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Results Neogenin expression in hippocampal neurons Neogenin is a member of cell-surface receptor proteins of the immunoglobulin superfamily [25], plays a key role in

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early cell fate determination relaying extracellular stimuli by up-regulating Oct3/4, Sox2, and Nanog, key transcriptional regulators for stem cell differentiation/maintenance in preimplantation mouse embryos [26]. As a next step toward exploring the possibility that Neogenin might be implicated in neural stem cell maintenance, we examined Neogenin expression patterns in hippocampal neurons in comparison with SOX2 and Nestin, key transcription factors

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implicated in various stem cells, including neural stem cells. Hippocampal neurons were dissected from the fetal rat brain at embryonic day 18 and dissociated prior to immunostaining with polyclonal anti-Neogenin antibodies, anti-

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Nestin antibodies and/or anti-SOX2 antibodies. Immunofluorescence microscopic observation revealed a co-localization of Neogenin, Nestin, and SOX2 expressions (Fig. 1A) among hippocampal neurons. In addition, FACS results were consistent with an earlier result showing co-localization of Neogenin, Nestin, and SOX2 expressions, accounting for 98.54% of hippocampal neuron population to be Nestin+/SOX2+ and 99.64% to be Neogenin+/SOX2+ (Fig. 1B and C), implying that neural stem cells, represented by either Nestin-positive or SOX2-positive, are indeed Neogenin-positive. Neogenin expression as revealed by immunofluorescence was further confirmed by immunoblotting: Neogenin and

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SOX2 expressions are higher in hippocampus from the fetus brain than from the adult brain, and the differences are statistically significant (Fig. 2A and B). However, Nestin expression from the fetus brain was significantly lower than

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the adult brain,

Neogenin up and down regulation in hippocampal neural stem cells

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In view to further demonstrate co-localization of Neogenin and stem cell marker proteins Nestin and SOX2, we next investigated the immunohistological localization of SOX2-positive and Neogenin-positive cells from a horizontal rat brain section that revealed the hippocampus along with the overlying cortex. In the cortex, there was little immunoreactivity, if any, for either Neogenin or SOX2; By contrast, Neogenin and SOX2 immunoreactivity are mainly concentrated in the hippocampus with one overlapping another (Fig. 4A), indicating that neural stem cells localized in the hippocampus express Neogenin in vivo. Co-localization of Neogenin and stem cell markers are indirectly strengthened by immunoblotting results showing that the hippocampus expressed more Neogenin, Oct4, SOX2, and Nestin compared to the cortex (Fig. 3B and C).

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Role of Neogenin in neuronal stem cells We next investigated whether and how Neogenin was implicated in neural stem cell maintenance by introducing either Neogenin expression vectors (Neogenin overexpression, OE) or vectors harboring shRNAs targeting Neogenin

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(Neogenin knock-down, KD) into primary hippocampal neuronal cells whereby Neogenin expression level was manipulated. To visually differentiate Neogenin overexpression from Neogenin knock-down, red fluorescence protein (RFP) and green fluorescence protein (GFP) were co-expressed in pairs, respectively, as an indicator (Fig. 4), and the resulting Neogenin expression level was confirmed both by immunofluorescence and by immunoblotting (Fig. 4). To

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our surprise, however, SOX2 expression was less pronounced both in Neogenin KD by 11.7 ± 2.4% (n=8) and Neogenin OE transfectants by 38.2 ± 13.8% (n=8) compared to control (Fig. 4A~C). Furthermore, PCNA expression

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was significantly enhanced in Neogenin OE transfectants (253.2 ± 62.6% (n=5) compared to control (Fig. 4D~F).

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Discussion Our results provide evidence that Neogenin may act as a receptor in neural stem cells for the maintenance of undifferentiation and/or pluripotency. Supporting evidence includes (i) co-localization of Neogenin and Nestin and

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SOX2 expression, key transcription regulators needed to maintain stem cells, in the dissociated fetal hippocampal stem cells, (ii) co-localization of Neogenin and SOX2 in the hippocampus but not the cortex in in situ brain sections, and (iii) manipulation of Neogenin expression, down-regulation or up-regulation, yields differential consequences to neural stem cell proliferation as evidenced by PCNA activation.

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Being identified as a receptor for neuronal axon guidance cues, Neogenin has been implicated in diverse developmental processes as diverse as neuronal development, endochondrial bone formation, mammary gland

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development, and cell cycle progression [18,25,27,28,29]. It is interesting to note that the immerging view from these studies is that the Neogenin signaling may be serving as a potential convergent point for cross talks among different extracellular cues, regardless of their nature of being chemical or physical, at which cellular differentiation and development are subjected to fine tuning to maintain developmental integrity in a permissive milieu. This line of view leaves open an intriguing possibility that the Neogenin signaling may be of broad relevance for the establishment and

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maintenance of neural stem cells, particularly, in respect to its receiving temporal and spatial input from various extracellular stimuli. To this regard, it is, therefore, highly likely that the Neogenin signaling pathway is richly endowed with versatility needed to allow development- and growth-triggering signal entry to effectively couple specific extracellular stimuli to appropriate cellular responses. As found for a stem cell growth-triggering role, Neogenin

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activation by extracellular stimuli act as a cellular switch to turn on and off a signaling thereby leading early stem cell to commitment to their cell fate specification [29]. Although Neogenin is known to be a multifunctional receptor, yet

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signaling pathway activated by Neogenin poorly understood, this notion is nicely illustrated by a recent study showing that Neogenin facilitates BMP/Smad signaling by providing the physical link between Neogenin and the BMP receptor [28]. Our studies also added to these reports Neogenin implication in the activation of signaling molecules, playing a key role in the maintenance of undifferentiation and pluripotency of stem cells [25]. However, a wealth of information remains to be uncovered with regard to (i) what is the physiological context wherein these extracellular cues make their way to the regulation of stem cell maintenance and differentiation, (ii) what are molecular components relaying the positional/chemical cues. Our results provide that the expression level of Neogenin is attributed the physiological context for the maintenance of stem cells: a moderate to high level of Neogenin

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expression ensures its ligation with ligands thereby triggers undifferentiation and pluripotency. However, a lower Neogenin expression has stem cells begin to lose its pluripotency, tilting toward cell differentiation. An implication of these findings is that a novel means to generate neural stem cells is made possible, e.g., by selectively enhancing its

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proliferation as a result of Neogenin overexpression and/or its ligand treatment. These studies have also highlighted how the activity of Neogenin integrates with other molecular machinery to implement and stabilize cellular undifferentiation and differentiation created by the localized extracellular milieu.

Neogenin has been shown to exist in a multiple form in a cell context-dependent manner, which may account for a

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variety of Neogenin signaling implicated in, for example, axon guidance, cell adhesion, migration, differentiation, and organ morphogenesis [30]. Neogenin has multiple combinations with other receptor such as DCC, UNC series and

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frizzled. In case of neuronal pathfinding, Neogenin and DCC heterodimer led to axon repulsive behavior while Neogenin homodimer presented axon attractive behavior[31,32]. Recently, Neogenin expression was identified in precursor cells in the embryonic and adult central nervous system [33]. Therefore, it is little surprising that loss of Neogenin function led to a dramatic decrease in SOX2 expression while gain of Neogenin function resulted in an increase in PCNA expression. Further work is required to explore the functional impact of Neogenin in neural stem cells

Conclusion

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and in endogenous neurons that triggers signaling cascades whose downstream effectors are SOX2, Oct3/4 or PCNA.

Neogenin is identified as a putative receptor whose activation is required to induce and maintain neural stem cells.

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governed by Neogenin.

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Neogenin also may contribute to cell proliferation. Our data support this link and extend similar studies in native CNS

Conflict of interest

This study and all authors of the manuscript have no potential conflicts of interest and no financial interests to declare.

Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (2013R1A1A2061491, 2017R1D1A1B03032473).

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Figure legends Figure 1 FACS analysis for Neogenin-, SOX2-, and Nestin-positive cells in primary hippocampal cells dissociated from the E18 rat hippocampus. (A) Representative immunofluorescence images of primary neural cells dissociated

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from the rat fetus hippocampus at embryonic day 18 following immunofluorescence staining for SOX2, Nestin, and Neogenin. DAPI was used for nuclear staining. (B) FACS analysis of dissociated hippocampal cells showing SOX2positive, Nestin-positive, Neogenin-positive, SOX2/Nestin double-positive, and SOX2/Neogenin double-positive cells. (C) Quantification of SOX2-positive (SOX2+), Nestin-positive (Nestin+), SOX2/Nestin-double positive (SOX2+/

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Nestin+), and SOX2/Neogenin double-positive (SOX-2+/Neogenin+) cells in FACS analysis.

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Figure 2 Neogenin expression of rat hippocampus. (A) Hippocampus was isolated from either the E18 rat (fetal) brain or the adult brain. Total soluble proteins in hippocampal lysate were subjected to Western blot ting and probed with antibodies against Neogenin, SOX2, and Nestin, respectively. β-actin was for internal loading control. (B) Densitometric quantification of the immunoreactive protein bands in (A). *p<0.05 against control.

Figure 3 Localization of Neogenin-positive cells in the hippocampus. (A) Immunohistological localization of

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SOX2-positive and Neogenin-positive cells in the fetal rat hippocampus and cortex. Fetal brain at E18 was thinsectioned to visualize the hippocampal and cortical regions and subjected to immunostaining with anti-SOX2 or antiNeogenin antibodies. (B) Western blotting analysis of Neogenin, Oct4, SOX2, and Nestin expression in rat fetal (E18)

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hippocampus and cortex. GAPDH was for internal loading control. (C) Densitometric quantification of immunoreactive

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band intensities shown in (B). *p<0.05 against control.

Figure 4 Effects of up-regulation and down-regulation of Neogenin on SOX2 and PCNA expression in cultured rat hippocampal neurons. (A) Cultured rat hippocampal neurons were transfected with shRNA targeting Neogenin (Neogenin KD). To visually differentiate Neogenin KD, GFP were co-expressed. Confocal microscopic images of transfected cells following immunofluorescence staining for SOX2 were seen. Blue, DAPI; Green, GFP; Red, SOX2. (B) Cultured rat hippocampal neurons were transfected with Neogenin cDNA vector (Neogenin OE). To visually differentiate Neogenin OE, RFP were co-expressed. Confocal microscopic images of transfected cells following immunofluorescence staining for SOX2 were seen. Blue, DAPI; Red, RFP; Green, SOX2. (C) Graphic quantification of

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fluorescence intensity of SOX2. Fluorescence intensities from SOX2 (red or green fluorescence) were quantified. *p<0.01 against control (ANOVA with Bonferroni’s test). (D) Cultured rat hippocampal neurons were transfected with shRNA targeting Neogenin (Neogenin KD). To visually differentiate Neogenin KD, GFP were co-expressed. Confocal

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microscopic images of transfected cells following immunofluorescence staining for PCNA were seen. Blue, DAPI; Green, GFP; Red, PCNA. (E) Cultured rat hippocampal neurons were transfected with Neogenin cDNA vector (Neogenin OE). To visually differentiate Neogenin OE, RFP were co-expressed. Confocal microscopic images of transfected cells following immunofluorescence staining for PCNA were seen. Blue, DAPI; Red, RFP; Green, PCNA.

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(F) Quantification of fluorescence intensity of PCNA. Fluorescence intensities from PCNA (red or green fluorescence)

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were quantified. (*p<0.01 against control (ANOVA with Bonferroni’s test))

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[26] J.H. Lee, S.S. Choi, H.W. Kim, W.C. Xiong, C.K. Min, S.J. Lee, Neogenin as a receptor for early cell fate determination in preimplantation mouse embryos, PLoS One 9 (2014) e101989.

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transcriptional targets and their regulatory enhancers, EMBO J 26 (2007) 5093-5108.

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developmental processes, Int J Biochem Cell Biol 39 (2007) 1569-1575.

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[35] C.E. Turner, Paxillin and focal adhesion signalling, Nat Cell Biol 2 (2000) E231-236.

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Nestin

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merge neo gen in

SOX2 Counts

DAPI

M AN U

SOX2

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merge

1.31

0.01

99.64

0.02

0.33 SOX2

Counts

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C

0.02

Nestin

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neogenin

98.54

SOX2

Neogenin

nestin

0.12

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n e s ti n

B Counts

SOX2 C o u n t s

DAPI

C o u n t s

A

Neogenin

Figure 1

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Fetus

A

Adult

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neogenin

SC

SOX2

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nestin

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B

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β-actin

Figure 2

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A DAPI

SOX2

neogenin

merge

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DAPI/SOX2/neogenin

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Cortex

Hippocampus

Figure 3

HIP

CORT C

neogenin

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120

GAPDH

HIP

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100

CORT

80

*

60 40

*

20

*

0 Neogenin

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nestin

band intensity (%)

Oct4

SOX2

SC

B

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Figure 3 (cont’d)

Oct4

SOX2

Nestin

ACCEPTED MANUSCRIPT

SC

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B

DAPI/neogenin shRNA-GFP/SOX2

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A

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C

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DAPI/neogenin-RFP/SOX2

Figure 4

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DAPI/neogeni shRNA-GFP/PCNA

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E

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F

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DAPI/neogenin-RFP/PCNA

Figure 4 (cont’d)